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SELEZIONE ARTICOLI SU APPLICAZIONI CLINICHE

MONITORAGGIO PAZIENTI CIRROTICI

1. Pontisso P, Quarta S, Caberlotto C, Beneduce L, Marino M, Bernardinello E, Boscato N, Fassina G, Cavalletto L, Gatta A, Chemello L. Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma. Int J Cancer. 119:735-40. 2006

2. Biasiolo A, Chemello L, Quarta S, Cavalletto L, Bortolotti F, Caberlotto C, Beneduce L, Bernardinello E, Tono N, Fassina G, Gatta A, Pontisso P. Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis. J Viral Hepat. 15:246-9. 2008.

3. Biasiolo A, Tono N, Ruvoletto M, Quarta S, Turato C, Villano G, Beneduce L, Fassina G, Merkel C, Gatta A, Pontisso P. IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease. PLoS ONE. 7:e40658. 2012.

4. Buccione D, Fatti G, Gallotta A, Loggi E, Di Donato R, Testa L, Saitta C, Santi V, Di Micoli A, Erroi V, Fazio V, Picciotto A, Biasiolo A, Degos F, Pontisso P, Raimondo G, Trevisani F. SCCA-IgM as predictor of hepatocellular carcinoma in patients with liver cirrhosis. OJGas. 2:56-61. 2012.

5. Biasiolo A, Trotta E, Fasolato S, Tono N, Ruvoletto MG, Martini A, Terrin L, Fassina G, Angeli P, Gatta A, Pontisso, P. SCCA-IgM is predictive of hepatocellular carcinoma development in patients with HCV cirrhosis-A prospective study. Dig Liver Dis. 46:e61. 2014

VALUTAZIONE DELLA RISPOSTA TERAPEUTICA

1. Giannini EG, Basso M, Bazzica M, Contini P, Marenco S, Savarino V, Picciotto A. Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen-associated (SCCA) variants' serum levels in anti-HCV positive cirrhotic patients. J Viral Hepat. 17:563-568. 2010.

2. Fransvea E, Trerotoli P, Sacco R, Bernabucci V, Milella M, Napoli N, Mazzocca A, Renna E, Quaranta M, Angarano G, Villa E, Antonaci S, Giannelli G. SCCA-IC serum levels are predictive of clinical response in HCV chronic hepatitis to antiviral therapy: a multicentric prospective study. J Viral Hepat. 19:704-710. 2012

3. Morisco F, Di Costanzo G G, Guarino M, Tortora R, Loperto I, Auriemma F, Ferraiuoli C, Tuccillo C, Beneduce L, Caporaso N . SCCA-IgM: a biomarker to monitor the outcome of therapy with sorafenib in advanced HCC. Dig Liver Dis. 45:S217-18. 2013.

4. Morisco F, Di Costanzo G G, Guarino M, Tortora R, Loperto I, Clery E, Auriemma F, Tuccillo C, Beneduce L, Caporaso N. Circulating SCCA-IgM complex is able to monitor the success of loco-regional therapy in HCC patients. Dig Liver Dis. 45:S218. 2013.

5. Pozzan C, Cardin R, Piciocchi M, Cazzagon N, Maddalo G, Vanin V, Giacomin A, Pontisso P, Cillo U, Farinati F. Diagnostic and Prognostic Role of Scca-Igm Serum Levels in Hepatocellular Carcinoma (Hcc). J Gastroenterol Hepatol. [Epub ahead of print]. 2014.

FAST TRACK

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients

is associated with development of hepatocellular carcinoma

Patrizia Pontisso1*, Santina Quarta1, Cristina Caberlotto1, Luca Beneduce2, Maria Marino2, Elisabetta Bernardinello1,Natascia Tono

1,3, Giorgio Fassina

2, Luisa Cavalletto

1, Angelo Gatta

1and Liliana Chemello

1

1Clinica Medica 5, Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy2Xeptagen, Pozzuoli (Na), Italy3Istituto Oncologico Veneto (IOV), Padova, Italy

About 3–4% of cirrhotic patients develop primary liver cancerevery year. Specific serologic markers have not yet been identifiedfor screening of high risk patients. The serpin squamous cell carci-noma antigen (SCCA) is overexpressed in liver cancer and circu-lating SCCA-IgM complexes have been described in patients withhepatocellular carcinoma (HCC). The aim of the present studywas to assess the behavior of SCCA-IgM in relation to HCC devel-opment in patients with cirrhosis. A retrospective, longitudinalstudy was conducted in a cohort of prospectively followed cir-rhotic patients. Two groups with similar clinical profile at presen-tation were studied : group A included 16 patients who developedHCC during a median follow up of 4 years; group B included 17patients who did not develop HCC during the same time interval.Circulating SCCA-IgM immune complexes were determined usinga recently standardized ELISA assay. At presentation similar lev-els of SCCA-IgM complexes [mean 6 SD: 267.40 6 382.25 U/mlvs. 249.10 6 446.90 U/ml, p 5 0.9006] and of alpha-fetoprotein[AFP; 24.11 6 59.04 IU/ml vs. 10.91 6 23.34 IU/ml, p 5 0.3995]were detected in group A and in group B. The increase over time(/) of SCCA-IgM, assessed within at least one year before clinicaldiagnosis of HCC, was remarkably higher in group A than ingroup B (mean 6 SD 5 280.05 6 606.71 (U/ml)/year vs. 237.92 695.94 (U/ml)/year, p 5 0.0408), while AFP increase was not signifi-cantly different (11.89 6 23.27 (IU/ml)/year vs. 3.67 6 11.46 (IU/ml)/year, p 5 0.2179). Receiver operating characteristic (ROC)curves were plotted for the rate of change in the levels of bothmarkers and the diagnostic accuracy measured as AUROC washigher for SCCA-IgM / (0.821) than for AFP / (0.654). In conclu-sion, the progressive increase of SCCA-IgM over time was associ-ated with liver tumor development, suggesting that monitoring thebehavior of SCCA-IgM might become useful to identify cirrhoticpatients at higher risk of HCC development.' 2006 Wiley-Liss, Inc.

Key words: SCCA-IgM; hepatocellular carcinoma; serologic prognosticmarker; cirrhosis

Hepatocellular carcinoma (HCC) is one of the major healthproblems worldwide, due to its high incidence and severe progno-sis. Figures depicting half a million new cases per year have beenreported, and projection studies have estimated an increase of tu-mor development within the next decade in developed countries.1

The reasons advocated to explain this phenomenon are theincreased rate of HCV infection and an improvement in clinicalmanagement of liver cirrhosis, identified as the major risk factorfor HCC development.2 About 3–4% of cirrhotic patients developprimary liver cancer every year and this justifies surveillance pro-grams to detect HCC at an early stage.3 The prognosis of thepatients depends mainly on the evolutionary stage of the neo-plasm, ranging from 5-years survival higher than 70% in surgicalpatients to less than 3 months in very advanced tumors.4 Tumorsize is one of the main factors influencing the possibility of bothsurgical or ablative interventions, and small tumors have a betterchance to be cured. The best strategy to monitor cirrhotic patientsis echographic follow-up, which is able to reveal hepatic lesionsof about 1 cm size, whereas alpha-fetoprotein (AFP) is currentlyused in clinical practice but its poor specificity and sensitivityarise concerns on its use as a screening tool.5 Till date, no specificserologic markers have been identified to predict tumor develop-

ment in cirrhotic patients to better focus surveillance programsand address health related resources. Recent findings have identi-fied the occurrence of the serine protease inhibitor squamous cellcarcinoma antigen (SCCA) hyper-expression in liver cancer tis-sue6,7 and a new serologic assay has been developed to detect cir-culating SCCA.8 The best results were obtained when SCCA com-plexed with IgM was determined, and significantly higher valueswere observed in HCC, compared to patients with chronic liverdisease and cirrhosis in a cross-sectional study.8 The aim ofthe present study was to assess the behavior of SCCA-IgM im-mune complexes in relation to HCC development in patients withcirrhosis.

A retrospective, longitudinal study was conducted in a cohort ofcirrhotic patients with HCV infection, regularly followed up inour institution with serum alpha-fetoprotein (AFP) testing and he-patic ultrasonography every 6 months. The patients were dividedinto the following groups: group A included 16 cirrhotic patientswho developed HCC during a median follow up of 4 years (range2–8 years). The diagnosis of liver cancer was formulated on thebasis of ultrasound results, confirmed by CT scan, by magneticresonance when indicated and by ultrasound-guided fine needle bi-opsy. Group B included 17 control patients with cirrhosis, who didnot develop HCC during the same time interval. Both groups hadsimilar clinical profile at presentation (Table I) and none of thepatients received antiviral therapy during the previous 8–10 yearsbefore and at the time of the study. Serum samples were collected,under informed consent, at the time of clinical visits and stored at220�C for further analysis. All the patients were histologicallyproven, Child A cirrhosis at the time of the first serum test (T1),being the grade of inflammatory activity 6.33 6 1.86 for thepatients of group A and 5.55 6 1.13 for the patients of group B(p 5 0.38). A second serum test (T2) was performed after a medianinterval of 3 years, corresponding to a median period of 2 years(range 1–4 years) before HCC diagnosis for the patients of groupA. None of the patients was coinfected with HBV, and alcohol ordrug abuse were excluded as potential liver disease cofactors.

Circulating SCCA-IgM immune complexes levels were deter-mined using an ELISA assay kit (Hepa-IC, Xeptagen SpA, Italy)according to the manufacturer’s instructions. Briefly, plates pre-coated with anti-human SCCA antibody were incubated with ei-ther serially diluted standards or serum samples, and the presenceof SCCA-IgM complexes were revealed by the addition ofenzyme-conjugated anti-human IgM. The plate was then washedand the substrate solution was incubated for 20 min. Subsequently,the plate was read on a microtiter plate reader at 405 nm.8 The

Contract grant sponsor: Regione Veneto (Regional Program on Surveil-lance and Control of Chronic Liver Disease and HCV-correlated Compli-cations: Liver Cirrhosis and HCC); Grant numbers: 4383/1999, 1417/2001,2920/2002; Grant sponsor: Foundation Citta della Speranza Onlus.*Correspondence to: Clinica Medica 5, Dipartimento di Medicina

Clinica e Sperimentale, Via Giustiniani, 2 35123 Padova, Italy.E-mail: [email protected] 22 August 2005; Accepted 25 January 2006DOI 10.1002/ijc.21908Published online 20 March 2006 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 119, 735–740 (2006)' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

amount of SCCA-IgM complexes were expressed in arbitraryUnits/ml (U/ml). In the same serum sample alpha-fetoprotein(AFP) was also assessed using a solid phase ELISA assay (DRGInternational, USA).

The increase of SCCA-IgM and of AFP over time (U) was cal-culated using the following formula:

U ¼ ½X� IgM�ðT2Þ � ½X� IgM�ðT1Þ½ðT2Þ � ðT1Þ�

Statistical analysis was carried out using the Student’s t-test, theFisher exact test, the Spearman correlation coefficient and the me-dian test. The level of significance was set as p < 0.05. All analy-ses were performed using Analyse-it1 software (England). Thearea under the receiver operating characteristic (ROC) curves werecalculated and compared using the MedCalc software (Belgium).

At presentation, similar SCCA-IgM complexes reactivity wasdetectable in cirrhotic patients who developed HCC (group A) andin the group without HCC development during the same length offollow-up (group B) [mean 6 SD: 267.40 6 382.25 U/ml vs.249.10 6 446.90 U/ml, p 5 0.9006]. Alpha-fetoprotein did notcorrelate with the presence of SCCA-IgM in the same serum sam-ple (r 5 20.0010), AFP values being similar in both groups atpresentation in terms of mean level and of incidence of AFP levels

>20 IU/ml, as described in Table I. Figure 1 shows T1 and T2 fig-ures for both SCCA-IgM and AFP in individual patients.

The increase of SCCA-IgM over time (U) was remarkably higherin cirrhotic patients who eventually developed HCC compared tothose who did not progress to liver cancer (Fig. 2a). The distributionof U values in group A (U mean 6 SD 5 280.05 6 606.71 (U/ml)/year) reflected both an increase of the initial SCCA-IgM value at T1

(in 6/8 patients) and the occurrence of SCCA-IgM neo-reactivity atT2 in 6/8 patients who were undetectable at T1, an event that didnot occur in any of the 7 patients of group B who were undetectableat presentation (T1) (group B U mean6 SD5237.926 95.94 (U/ml)/year, p5 0.0408). The median U values in the 2 groups showeda significant difference (group A 5 42.11 (U/ml)/year, group B 50 (U/ml)/year, p 5 0.0081 Median test) as displayed in Figure 3.Figure 4 depicts the behavior of SCCA-IgM over time in the 2groups of cirrhotic patients: in group A patients, the increase ofSCCA-IgM over time (/) was >20 (U/ml)/year in 75% of cases(12/16), while no change or slight decrement was observed in 25%of the patients (4/16). An opposite behavior was observed inpatients of group B, where only 6% of the patients (1/17) presentedU >20 (U/ml)/year, while in 94% of the cases (16/17) / valuesremained almost unchanged or decreased.

AFP increase in individual cases was not significantly different inboth groups (11.89 6 23.27 (IU/ml)/year vs. 3.67 6 11.46 (IU/ml)/

FIGURE 1 – Individual T1 and T2 data for patients who did not develop HCC during follow up (group B) and in the group of cirrhotic patientswho developed HCC during the same interval of observation (group A) showing SCCA-IgM levels (upper bars) and AFP levels (lower bars).

TABLE I – CLINICAL FEATURES AT PRESENTATION OF THE CIRRHOTIC PATIENTS WHO DEVELOPED HCCDURING FOLLOW-UP (GROUP A) AND OF THE CIRRHOTIC PATIENTS WHO DID NOT DEVELOP HCC DURING

THE SAME TIME OF OBSERVATION (GROUP B)

Group A (16 patients) Group B (17 patients) p

Age (years)1 69 6 9 63 6 11 0.060M/F 11/5 10/7 0.818Platelets (109/L)1 116.736 56.84 117.75 6 60.51 0.964INR1 1.07 6 0.3 1.13 6 0.11 0.550Bilirubin (lmol/L)1 21.246 8.96 18.71 6 6.3 0.367Albumin (g/L)1 35.276 6.42 38.98 6 4.89 0.109AFP(IU/mL )1 24.116 59.04 10.91 6 23.34 0.399>20 IU/mL (%) 31% 18% 0.438

1Mean 6 SD.

736 PONTISSO ET AL.

year, p 5 0.2179), although it was correlated with poor clinical out-come (Table II). Indeed, patients with shorter survival who died dur-ing follow-up, showed a trend towards higher levels of AFP increaseover time, compared to patients still alive in group A, confirming

the aggressive biological behavior previously associated with AFPelevation.9

Figure 5 depicts ROC curves of the increase over time (U) ofSCCA-IgM and AFP and the rate of change in the levels of both

FIGURE 2 – Determination of the increase over time ((U) of SCCA-IgM (a) and of AFP (b) for each component of the group of cirrhoticpatients who developed HCC during follow up (group A, ruled bars) and in the group of cirrhotic patients who did not develop HCC during thesame interval of observation (group B, black bars).

737SCCA-IGM IMMUNE COMPLEXES IN CIRRHOTIC PATIENTS AND DEVELOPMENT OF HEPATOCELLULAR CARCINOMA

biomarkers indicates that the prognostic accuracy measured as thearea under the ROC curves (AUROC) was higher for U SCCA-IgM (0.821) than for U AFP (0.654).

In clinical practice, one of the main unresolved issues for themanagement of patients with cirrhosis is that the individual risk ofHCC development has not yet been clearly defined. Predictive fac-tors have been considered in different studies, and scores withclinical and biological variables, including age, sex, HCV infec-tion and genotype, prothrombin activity, platelet count and symp-toms of portal hypertension have been proposed, allowing theidentification of groups of patients with low or high risk of livercancer development.10–12 At the histological level, liver cell dys-plasia13,14 and hepatocyte proliferation rate15,16 have been pro-posed as predictive factors of increased risk of liver cancer. These

methods, however, are limited by the fact that they require liverbiopsy and in daily practice this invasive procedure is not fre-quently performed in cirrhotic patients, where the diagnosis,excluding the early stage, is usually based on clinical findings. Todate, no serological biomarkers are available to be used in surveil-lance programs. In the present study, we have assessed the behav-ior of the serpin SCCA, initially described in tumors of epithelialorigin.17 This biomarker has been recently detected also in the ma-jority of cases of primary liver cancer where high amounts wereobserved at transcription and protein levels in neoplastic cells butnot in normal liver.6,7 Further studies have revealed that SCCAreactivity is also present in the liver in chronic hepatitis and in cir-rhosis, although the extent of expression is usually lower than thatobserved in neoplastic cells, likely reflecting the regenerative ac-

FIGURE 3 – Box plot for SCCA-IgM increase over time (U) in thegroup of cirrhotic patients who didnot develop HCC during follow up(group B) and in the group of cir-rhotic patients who developed HCCduring the same interval of obser-vation (group A). The box indi-cates the lower and upper quartileand the middle line indicates themedian. Boxes are notched at themedian with the lengths of thenotches representing the 95% con-fidence interval. A dotted-line con-nects the observations within 1.5inter-quartile ranges (IQRs) of thelower and upper quartile. Crossesrepresent the observations between1.5 and 3.0 IQRs from the quar-tiles and circles represent pointsbeyond this.

FIGURE 4 – Distribution of thecirrhotic patients in relation todifferent interval of SCCA in-crease (U). White bars refer tocirrhotic patients without occur-rence of HCC (group B) and blackbars indicate cirrhotic patients,having similar clinical characteris-tics and follow-up who developedHCC after at least 1 year from theend of the study (group A).

738 PONTISSO ET AL.

tivity of the damaged liver.8 Due to the availability of a standar-dized assay to measure circulating SCCA associated with IgMimmunoglobulins, the main form of SCCA found in serum,8 wehave evaluated its behavior over time in 2 groups of cirrhotic

patients with HCV infection, regularly attending the surveillanceprogram for HCC development in our institution and presentingwith similar clinical features and AFP levels. The results of thestudy indicate that the absolute value of the immune complexes atpresentation was similar in cirrhotic patients with HCC progres-sion and in nonprogressive patients. However, a remarkable differ-ence between the 2 groups was found in the behavior of theSCCA-IgM complexes over time, since a progressive increaseoccurred in the majority of the patients who developed HCC afterat least one year from the end of the study, while figures remainedunchanged or slightly decreased in the majority of the cirrhoticpatients without evidence of HCC during the same time interval.This behavior was observed at least one year before clinical diag-nosis of HCC, suggesting that this preclinical phase might becomea suitable window to specifically address new potentially effectivetherapies. The increase of AFP, measured in parallel in the sameserum samples, was not significantly different in patients whoeventually developed HCC and in those without liver tumor pro-gression, although individual patients with shorter survivalshowed a trend towards higher AFP increase. The findingsobserved in this small series of cirrhotic patients recall similarresults obtained when another serologic biomarker was used in arecently published study in men with localized prostate cancer,18

where watchful waiting is one of the managing options.19 The rateof rise in prostate specific antigen (PSA) levels was preoperativelyassessed and the increase of PSA by more than 2.0 ng/ml duringthe year before diagnosis was found significantly associated withthe risk of dying for prostate cancer.18

In conclusion, if the findings reported in the present study willbe further confirmed in larger studies, monitoring SCCA-IgMcomplexes’ behavior over time could became a useful prognosticparameter in cirrhotic patients, to support clinical decisions.

References

1. El Serag HB, Mason AC. Rising incidence of hepatocellular carci-noma in the United States. N Engl J Med 1999;340:745–50.

2. Llovet JM, Beaugrand M. Hepatocellular carcinoma: present statusand future prospects. J Hepatol 2003;38:S136–49.

3. Prorok PC. Epidemiologic approach for cancer screening. Problems indesign and analysis of trials. Am J Pediatr Hematol Oncol 1992;14:117–28.

4. Bruix L, Llovet JM. Prognostic prediction and treatment strategy inhepatocellular carcinoma. Hepatology 2002;35:519–24.

5. Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Bur-roughs AK, Christensen E, Pagliaro L, Colombo M, Rodes J; EASLPanel of Experts on HCC. Clinical management of hepatocellular car-cinoma. Conclusions of the Barcelona–2000 EASL Conference. JHepatol 2001;35:421–30.

6. Pontisso P, Calabrese F, Benvegnu L, Lise M, Belluco C, Ruvoletto MG,Marino M, Valente M, Nitti D, Gatta A, Fassina G. Overexpression ofsquamous cell carcinoma antigen variants in hepatocellular carcinoma.Br J Cancer 2004;90:833–7.

7. Giannelli G, Marinosci F, Sgarra C, Lupo L, Dentico P, Antonaci S.Clinical role of tissue and serum levels of SCCA antigen in hepatocel-lular carcinoma. Int J Cancer 2005;116:579–83.

8. Beneduce L, Castaldi F, Marino M, Quarta S, Ruvoletto M, Benvegnu L,Calabrese F, Gatta A, Pontisso P, Fassina G. Squamous cell carcinomaantigen-IgM complexes as novel biomarkers for hepatocellular carci-noma. Cancer 2005;103:2558–65.

9. Matsumoto Y, Suzuki T, Asada I, Ozawa K, Tobe T, Honjo I.Clinical classification of hepatoma in Japan according to serialchanges in serum alpha-fetoprotein levels. Cancer 1982;49:354–60.

10. Ganne-Carri�e N, Chastang C, Chapel F, Munz C, Pateron D, Sibony M,Deny P, Trinchet JC, Callard P, Guettier C, Beaugrand M. Predictivescore for the development of hepatocellular carcinoma and additionalvalue of liver large cell dysplasia in Western patients with cirrhosis.Hepatology 1996;23:1112–8.

11. Degos F, Christidis C, Ganne-Carri�e N, Farmachidi JP, Degott C,Guettier C, Trinchet JC, Beaugrand M, Chevret S. Hepatitis C virusrelated cirrhosis: time to occurrence of hepatocellular carcinoma anddeath. Gut 2000;47:131–6.

12. Velazquez RF, Rodriguez M, Navascues CA, Linares A, Perez R,Sotorrios NG, Martinez I, Rodrigo L. Prospective analysis of risk fac-tors for hepatocellular carcinoma in patients with liver cirrhosis. He-patology 2003;37:520–7.

TABLE II – INCREASE OVER TIME (U) OF SCCA AND AFP IN PATIENTS OF GROUP AIN RELATION TO CLINICAL OUTCOME

Alive (n 5 12) Dead (n5 4) p

Median follow-up 3 years 1.5 years 0.036AgeMean 6 SD 70.086 9.20 68.756 3.40 0.692Median 73.5 69.5M/F 9/3 3/1 1.00U [SCCA (U/mL)/year]Mean 6 SD 278.0906 628.494 3516 703.545 0.849Median (range) 52 (0–2137) 32.5 (265 to 1404)

U [AFP (IU/mL)/year]Mean 6 SD 0.86 6 13.22 29.266 36.76 0.055Median (range) 2.2 (234.8 to 19) 17 (0.2–70.6)

FIGURE 5 – ROC curves comparing the distribution of the increaseof SCCA-IgM over time (U SCCA-IgM) and the increase of AFP overtime (UAFP) in the group of cirrhotic patients who developed HCCduring follow up (group A) versus the group of cirrhotic patients whodid not develop HCC during the same interval of observation (group B).

739SCCA-IGM IMMUNE COMPLEXES IN CIRRHOTIC PATIENTS AND DEVELOPMENT OF HEPATOCELLULAR CARCINOMA

13. Antony PP, Vogel CL, Barker LF. Liver cell dysplasia: a premalig-nant condition. J Clin Pathol 1973;26:217–23.

14. Borzio M, Bruno S, Roncalli M, Mels GC, Ramella G, Borzio F,Leandro G, Servida E, Podda M. Liver cell dysplasia is a major riskfactor for hepatocellular carcinoma in cirrhosis: a prospective study.Gastroenterology 1995;08:812–7.

15. Sangiovanni A, Colombo E, Radaelli F, Bortoli A, Bovo G,Casiraghi MA, Ceriani R, Roffi L, Redaelli A, Rossini A, Spinzi G,Minoli G. Hepatocyte proliferation and risk of hepatocellularcarcinoma in cirrhotic patients. Am J Gastroenterol 2001;96:575–80.

16. Donato MF, Arosio E, Del Ninno E, Ronchi G, Lampertico P, Mora-bito A, Balestrieri MR, Colombo M. High rates of hepatocellular car-cinoma in cirrhotic patients with high liver cell proliferative activity.Hepatology 2001;34:523–8.

17. Kato H. Expression and function of squamous cell carcinoma antigen.Anticancer Res 1998;16:2149–54.

18. D’Amico AV, Chen MH, Roehl KA, Catalona WJ. Preoperative PSAvelocity and the risk of death from prostate cancer after radical prosta-tectomy. N Engl J Med 2004;351:125–35.

19. Kessler B, Albertsen P. The natural history of prostate cancer. UrolClin North Am 2003;30:219–26.

740 PONTISSO ET AL.

Monitoring SCCA-IgM complexes in serum predicts liverdisease progression in patients with chronic hepatitisA. Biasiolo,1 L. Chemello,1 S. Quarta,1 L. Cavalletto,1 F. Bortolotti,1 C. Caberlotto,1

L. Beneduce,2 E. Bernardinello,1 N. Tono,1,3 G. Fassina,2 A. Gatta1 and P. Pontisso1

1Clinica Medica 5, Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy; 2Xeptagen S.p.A., VE.GA Science Park,

Marghera (VE), Italy; and 3Istituto Oncologico Veneto – I.O.V. (IRCCS), Padova, Italy

Received April 2007; accepted for publication June 2007

SUMMARY. About 30% of the patients with chronic hepatitis

develop a progressive liver disease and one of the most

intriguing issues is the detection of noninvasive markers for

fibrosis stage and disease progression. High levels of squa-

mous cell carcinoma antigen (SCCA)-immunoglobulin M

(IgM) are detectable in hepatocellular carcinoma and their

increase in cirrhotic patients can predict tumour develop-

ment. As SCCA-IgM can also be detectable at low percent-

ages in patients with chronic hepatitis, the aim of this study

was to assess SCCA-IgM complexes in relation to disease

outcome in this group of patients. An ELISA assay was used

to determine the presence of SCCA-IgM in 188 patients with

chronic hepatitis and in 100 controls. An additional serum

sample was available after a median period of 6 years in 57

untreated patients: these patients were subdivided in group

A, including eight patients with a fibrosis score increase ‡2

in a second liver biopsy and group B, including 49 patients

without fibrosis progression during a similar follow up.

SCCA-IgM complexes were detectable in 63 of 188 (33%)

patients but in none of the controls. A significant increase of

SCCA-IgM levels over time was observed in patients with

fibrosis progression (mean ± SD: 117 ± 200 U ⁄ mL ⁄ year),

but not in those without histologic deterioration

(mean ± SD: –8.8 ± 31 U ⁄ mL ⁄ year, P < 0.0001). In con-

clusion, monitoring SCCA-IgM levels over time appears a

useful approach to identify patients with chronic hepatitis at

higher risk for cirrhosis development.

Keywords: chronic hepatitis outcome, fibrosis progression,

serpin, squamous cell carcinoma antigen.

INTRODUCTION

Chronic infection by hepatitis B (HBV) and hepatitis C (HCV)

viruses is one of the main causes of chronic hepatitis, rep-

resenting a relevant health problem worldwide. Natural

history studies indicate that advanced fibrosis and cirrhosis

arise in about 20–40% of the patients with chronic HBV or

HCV [1], usually decades after virus infection [2]. In chron-

ically infected patients, the precise definition of the hepatic

fibrosis stage is considered one of the most important

parameters to assess the risk of disease progression. At pres-

ent, liver biopsy represents the gold standard to assess

presence, type and stage of liver fibrosis [3,4]. Despite its

primary role, this procedure has a number of widely recog-

nized limitations, such as invasiveness, difficult standardi-

zation and high cost [5,6]. For these reasons, the last decade

has been focused on direct and indirect noninvasive markers

able to provide accurate information about liver fibrogenetic

activity and fibrosis stage in patients with potentially pro-

gressive hepatic disease [7]. When chronic infection has

reached the stage of cirrhosis, in most of the cases a

well-compensated phase occurs for a long time, before

development of complications. Epidemiological data have

demonstrated that liver cirrhosis, regardless of its aetiology,

is the most important risk factor for the development of

primary liver cancer [8]. Recent findings indicate that

monitoring the behaviour of the immune complex squamous

cell carcinoma antigen (SCCA)-immunoglobulin M (IgM) in

serum can predict HCC development in patients with

cirrhosis [9]. Since it has been shown that SCCA-IgM

complexes are also detectable in a low percentage of patients

with chronic hepatitis [10], the aim of the present study was

to assess the behaviour of this marker in serum, in relation

to disease outcome in patients with chronic hepatitis.

PATIENTS AND METHODS

Patients

The study was conducted in 188 patients with histologically

proven chronic hepatitis (M ⁄ F: 105 ⁄ 83; mean age ± SD:

Correspondence: P. Pontisso, Clinica Medica 5, Department of Clin-

ical and Experimental Medicine, Via Giustiniani, 2 35128 Padova,

Italy. E-mail: [email protected]

Journal of Viral Hepatitis, 2008, 15, 246–249 doi:10.1111/j.1365-2893.2007.00935.x

� 2008 The AuthorsJournal compilation � 2008 Blackwell Publishing Ltd

44 ± 13 years) followed up in our Institution. Serum sam-

ples from 100 blood donors (M ⁄ F: 66 ⁄ 34; mean age ± SD:

36 ± 9 years) were collected as controls and stored at

–20 �C until use. Table 1 summarizes clinical and epidemi-

ological characteristics of the studied population, where

most of the patients were infected with HCV. Serum samples

were obtained at presentation, when no antiviral treatment

was carried out by any of the patients. An additional serum

sample was available after a median period of 6 years

(range: 2–21 years) in only 57 untreated patients (because

of nonclinical indication to treatment or patient refusal).

These patients underwent a second liver biopsy during fol-

low up and were divided, on the basis of their histologic

findings, in the following two groups: group A, which in-

cluded eight patients with disease progression defined by a

fibrosis score increase ‡2 during follow-up, and group B,

which included 49 patients without histologic evidence of

disease progression during the same time interval (fibrosis

score increase <2). Both groups had similar clinical profile at

presentation, as shown in Table 2. Informed consent was

obtained from all patients before liver biopsy and blood

sample collection.

Virologic markers

HBsAg, HBeAg and the presence of serum anti-HBc, anti-

HBe and anti-HBs antibodies were evaluated by ELISA using

commercially available kits (Abbott Diagnostics, North Chi-

cago, IL, USA). Anti-HCV antibody reactivity was deter-

mined by a third generation ELISA (Ortho Diagnostics,

Raritan, NJ, USA) and confirmed by recombinant immuno-

blot assay (Ortho Diagnostics). Serum HCV RNA was

determined by the Amplicor HCV monitor assay (Roche

Diagnostics, Branchburg, NJ, USA).

SCCA-IgM detection in serum

SCCA-IgM immune complexes levels were determined in

serum using an ELISA assay (Hepa-IC, Xeptagen, Italy),

according to the manufacturer� s instructions.

In patients� follow-ups the increase of SCCA-IgM over time

(/) was evaluated using the formula:

u ¼ SCCA-IgMðT2Þ � SCCA-IgMðT1ÞðT2� T1Þ years

where T1 refers to the time at presentation and T2 refers to

the second time point.

Statistics

Statistical analysis was carried out using the Student�s t-test,

the Fisher exact test, the nonparametric Mann–Whitney test

and the chi-square test, when appropriate. The level of sig-

nificance was set as a P < 0.05. All analyses were performed

using GraphPad InStat software (San Diego, CA, USA).

RESULTS

At presentation, circulating SCCA-IgM immune complexes

were detectable in 63 of 188 (33%) patients with chronic

hepatitis, but in none of the control group. The number of

SCCA-IgM positive patients was not significantly different

between patients infected with HBV (27.5%, 11 ⁄ 40) and

HCV (34%, 50 ⁄ 148). Mean age (47 ± 13 years vs

42 ± 14 years, P = 0.05) and sex distribution (M ⁄ F: 1.18 vs

1.63, P = 0.750) were similar in patients with and without

the presence of SCCA-IgM complex.

Figure 1 shows the distribution of SCCA-IgM levels at the

first (T1) and second (T2) serum test time in the two groups

of patients followed over time. Levels of the complex were

substantially stable over time in patients without disease

progression (median value T1 = 108 U ⁄ mL, T2 =

94 U ⁄ mL), while an increase was detected in 75% of the

Table 1 Clinical and epidemiological characteristics of the

patients with chronic liver disease and of the control group

Chronic hepatitis Controls

No. 188 100

Age (years, mean ± SD) 44 ± 13 36 ± 9

Sex, M ⁄ F 105 ⁄ 83 66 ⁄ 34

Aetiology (%)

HCV 148 ⁄ 188 (79) –

HBV 40 ⁄ 188 (21) –

Table 2 Clinical and epidemiological features at presenta-

tion of the patients with chronic hepatitis, who were tested

for SCCA-IgM in two different time points during a median

follow-up of 6 years

Group A Group B P

No. of patients 8 49

Age (years, mean ± SD) 43 ± 18 40 ± 15 0.559

Sex, M ⁄ F 7 ⁄ 1 30 ⁄ 19 0.238

Aetiology

HCV 5 27 1.000

HBV 3 22

Histology

Grading

Mean ± SD 7 ± 2 6 ± 3 0.271

Median 7 5

Staging

Mean ± SD 3 ± 1 2 ± 1 0.481

Median 3 2

Group A = patients with liver disease progression. Group

B = patients without liver disease progression during the

same interval of observation.


SCCA-IgM behaviour and chronic hepatitis outcome 247

patients with progressive disease (median value

T1 = 313 U ⁄ mL, T2 = 707 U ⁄ mL, P = 0.014).

To better analyse these results, the increase of SCCA-IgM

over time (/) was calculated for each patient. A significant

increase of SCCA-IgM during follow up was detected in pa-

tients with chronic hepatitis and progressive disease, but not

in those without histologic progression (/ mean ± SD:

117 ± 200 U ⁄ year vs –8.8 ± 31 U ⁄ year, P < 0.0001).

As displayed in Figure 2, the distribution of / values in this

latter group of patients, mainly characterized by negative

values, reflects a decrease of this parameter over time in the

majority of the patients without disease progression.

DISCUSSION

Despite a marked decrease in the incidence rate of all types

of viral hepatitis in the past 15 years [11,12], several

thousand deaths from cirrhosis and HCC attributable to

chronic HBV and HCV infection occur each year [13,14].

In clinical practice, one of the most relevant goals is the

early identification of the subgroup of patients with in-

creased risk of histological progression of liver disease [15].

The risk and the speed rate of fibrosis progression can be

influenced by virus-related, host-related and environmental

factors, and the role of co-factors, such as alcohol, metabolic

disorders and viral co-infections, has been widely demon-

strated [16–18]. However, in individual patients �the art of

predicting fibrosis� [19] is only partially based on the pres-

ence of these factors. The prognostic assessment is indeed

more reliably estimated on the basis of serological param-

eters, such as levels of transaminases and of other markers

able to evaluate liver function (i.e. prothrombin activity,

platelet count) [20]. Also liver ultrasound, although less

specific, is useful to assess disease progression during long-

term follow-up [21] and liver stiffness, assessed by the

fibroscan, appears a promising tool [22]. However, liver

biopsy is still the gold standard for liver fibrosis staging,

despite its limitations. With the aim to greatly reduce the

need to apply liver biopsy, many direct and indirect bio-

markers (Fibrotest, APRI, AAR, Forns�index) of liver fibrosis

have been recently proposed [7]. To date, no serological

marker has been identified as an adequate marker to predict

fibrosis progression in individual patients.

Recent findings indicate that immunoglobulins of the IgM

class can form complexes with the SCCA, a serin protease

inhibitor (serpin) [10]. This circulating immune complexes

(SCCA-IgM), has been considered a new biomarker for HCC,

because it has been detected at high levels in the majority of

the patients with primary liver cancer. Further studies have

proposed that the SCCA-IgM complex could be used to

monitor patients with cirrhosis to identify those at higher

risk of HCC development [9]. As it has been shown that

SCCA-IgM complexes are also detectable in a low percentage

of patients with chronic hepatitis [10], we have evaluated its

behaviour in patients with chronic hepatitis followed up in

our Institution. The results of the study indicate that SCCA-

IgM complexes were detectable in about one-third of the

patients, independently of viral aetiology, age, and sex.

Although the number of untreated patients with liver disease

evolution was rather small, monitoring the SCCA-IgM

complexes over time revealed a marked difference between

the two groups of patients, recalling the behaviour recently

found in cirrhotic patients with and without HCC progres-

sion [9]. SCCA-IgM levels increased in the majority of the

patients with histologic deterioration, while values of the

immune complexes remained substantially stable or slightly

decreased in most of the patients without disease progression

during the same interval.

0

1000

T1T1 T2T2

2500

3500

Group A Group B

P = 0.014S

CC

A-I

gM

(U/m

L)

Fig. 1 SCCA-IgM distribution values at the first (T1) and

second (T2) serum test time in patients with chronic hep-

atitis with (group A) or without (group B) disease progres-

sion. Horizontal bars represent median values in different

groups.

–200

–100

0

100

200

300

400

500

600

SC

CA

-Ig

M in

crea

se (

φ)U

/ml/y

ear

Fig. 2 Distribution of the increase of SCCA-IgM over time

(/) in individual patients. Full bars refer to patients with

liver disease progression (group A) and dashed bars refer to

patients without disease progression (group B).


248 A. Biasiolo et al.

In conclusion, monitoring SCCA-IgM complex behaviour

over time could become a useful approach to predict disease

outcome in individual patients with chronic hepatitis.

Further studies are needed to define adequate frequency

of testing and ⁄ or clinically relevant values to better address

optimal treatment in individual patients.

ACKNOWLEDGEMENT

This work was supported in part by a research grant from

the Foundation �Citta della Speranza�, Padova.

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SCCA-IgM behaviour and chronic hepatitis outcome 249

IgM-Linked SerpinB3 and SerpinB4 in Sera of Patientswith Chronic Liver DiseaseAlessandra Biasiolo1, Natascia Tono2, Mariagrazia Ruvoletto1, Santina Quarta1, Cristian Turato2,

Gianmarco Villano1, Luca Beneduce3, Giorgio Fassina3, Carlo Merkel1, Angelo Gatta1, Patrizia Pontisso1*

1 Department of Medicine, University of Padova, Padova, Italy, 2 Istituto Oncologico Veneto (IOV), IRCCS, Padova, Italy, 3 Xeptagen S.p.A., Venice, Italy

Abstract

Background: Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellularcarcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1)and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. Asreported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated withHCC development.

Aim: To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to totalSCCA-IgM levels.

Methodology and Findings: 79 patients with chronic liver disease were studied, including 17 patients with chronichepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies againstserpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgMimmunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnosticsensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median valuesobtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values:0.168 (IQR 0.140–0.427) vs. 0.140 (IQR 0.140–0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgMmedian ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients withcirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079).

Conclusions: IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time.Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpinisoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4levels.

Citation: Biasiolo A, Tono N, Ruvoletto M, Quarta S, Turato C, et al. (2012) IgM-Linked SerpinB3 and SerpinB4 in Sera of Patients with Chronic Liver Disease. PLoSONE 7(7): e40658. doi:10.1371/journal.pone.0040658

Editor: Henrik Einwaechter, Klinikum rechts der Isar der TU Munchen, Germany

Received January 18, 2012; Accepted June 11, 2012; Published July 13, 2012

Copyright: � 2012 Biasiolo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is supported in part by a grant from the National Ministry of Education, University and Research (FIRB Project Prot. RBLA03S4SP_005) and bya research grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC). The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript. No additional external funding was received for this study.

Competing Interests: The authors have read the journal’s policy and have the following conflicts of interest to declare: LB and GF are employees of XeptagenS.p.A. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

* E-mail: [email protected]

Introduction

SerpinB3 and serpinB4 isoforms, also known as squamous cell

carcinoma antigen 1 and 2 (SCCA1 and SCCA2) belong to ov-

serpin/clade B serpin family [1]. Over 1500 serpin members have

been identified in humans, plants, bacteria, archea and poxviruses

to date [2,3].

Genomic cloning of these two isoforms revealed that they are

highly homologous, 91% identical at the amino acid level [4,5],

share conserved tertiary structure, and use a unique conforma-

tional rearrangement for their inhibitory activity [6,7]. However,

serpinB3 and serpinB4 show distinct properties and substrates:

serpinB3 is a papain-like cysteine proteinase inhibitor, while

serpinB4 is a chymotrypsin-like serine proteinase inhibitor [8,9].

Little is known concerning the regulation of their gene expression.

Both isoforms are broadly co-expressed in the spinous and

granular layers of normal squamous epithelium, in several organs

including tongue, tonsil, oesophagus, uterine cervix, vagina, the

conducting airways, Hassall’s corpuscles of the thymus and some

areas of the skin [10]. Regarding their role in normal epithelia, it

has been suggested that SCCA isoforms may protect from

bacterial and viral cystein proteases [11], mast cell chymase [12]

and may also prevent cellular apoptosis of the cornified layer.

It has been demonstrated that SCCA isoforms are often

overexpressed in neoplastic cells of epithelial origin [13], although

their biological role in cancer cell is still unclear. It has been

reported that both serpinB3 and serpinB4 protect neoplastic cells

from apoptosis [14] and that serpinB3 promotes tumour growth

[5,15–16], epithelial to mesenchymal transition and cell prolifer-

ation [17].

PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40658

Overexpression of SCCA isoforms has been also described in

HCC and in highly displastic liver nodules, but not in normal liver

[18–20]. In addition, high levels of SCCA-IgM linked complexes,

but not of the free SCCA protein, have been described in serum of

patient with HCC [21].

To date, little information is available about the profile of

expression of SCCA isoforms in patients with cancer. Some

authors have demonstrated a selective expression of serpinB4

mRNA in squamous cell carcinoma (SCC) tissues from uterine

cervix when compared to normal tissue or SCC tissues from

oesophagus or skin [22–24]. Serological studies have reported

elevated serum levels of serpinB4 isoform, ascribed to direct

release from tumour cells [25,26]. However, there is still

conflicting information about the prevalent circulating SCCA

isoform and additional studies have not confirmed these data

[27].

According to the new theory about cancer immunosurveillance,

now updated as immunoediting [28–30], natural IgMs seem to

play an important role in the innate immune response, not only

against infectious agents, but also in the immunosurveillance

against tumour cell growth. Multivalent IgMs bear a characteristic

capacity to bind a wide range of post-transcriptionally modified

tumour antigens and they all induce cancer-specific apoptosis, by

triggering the intrinsic apoptotic pathway [31]. Several studies

have demonstrated that tumour released antigens can react with

the natural IgM class of immunoglobulins and form circulating

immune complexes in different human tumours. The described

immunocomplexed antigens include CEA in colorectal cancer

[32], PSA in prostate cancer [33], AFP, SCCA and DPC in liver

cancer [21,34,35]. It has been also shown that these circulating

immunocomplexes provide a better diagnostic performance than

the corresponding free biomarker.

The purpose of this study was to evaluate the occurrence of

serum immunoreactivity of IgM linked serpinB3 and serpinB4

isoforms in patients with different extent of chronic liver disease,

compared to total SCCA-IgM levels.

Results

The results obtained in the study, expressed as descriptive

parameters, are summarized in Table 1. OD median value of

serpinB3-IgM was 0.130 in each group of patients. The values of

serpinB4-IgM obtained with SCC103 antibody, which recognises

the serpin-protease complex, were slightly lower in patients with

HCC, compared to patients with cirrhosis (median values: 0.140

(IQR 0.140–0.278) vs 0.168 (IQR 0.140–0.427 p = 0.177). Similar

and not significantly different values of serpinB4-IgM obtained

with SCC104 antibody were found comparing all groups of

studied patients.

SerpinB3-IgM complex was positive in 2 out of 17 (12%)

patients with chronic hepatitis, in 10 out of 36 (28%) patients with

cirrhosis and in 6 out of 26 (23%) patients with HCC. Similar

values of reactivity were obtained for serpinB4-IgM detected

immobilizing SCC103 antibody on the plate of the dedicated

ELISA. A little gain in positivity rate for the detection of serpinB4-

IgM in the three analyzed groups of patients was obtained using

the other serpinB4 specific monoclonal antibody SCC104Ab.

Using this latter antibody 5 out of 17 (29%) patients with chronic

hepatitis, 15 out of 36 (42%) patients with cirrhosis and 9 out of 26

(35%) patients with HCC were positive. When total SCCA-IgM

levels, regarded as the reference biomarker, were measured, 53%

of the patients with chronic hepatitis, 47% of the cirrhotic patients

and 58% of the patients with HCC were positive, showing a better

sensitivity of this reference test, when compared with the sensitivity

of the three above-listed assays. All healthy subjects were negative

for total SCCA-IgM ELISA, while a 7% of positivity rate was

found using the other ELISA assays.

Circulating levels of AFP were also measured, and were positive

in 4 out of 26 (15%) patients with cirrhosis (median value 8.2 IU/

ml) and in 13 out of 26 (50%) patients with HCC (median value

19.8 IU/ml; p = 0.02). In the group of patients with liver cirrhosis

the distribution pattern of IgM-linked immunocomplexes was

further analyzed in relation to liver tumour progression. Seventeen

patients developed liver cancer during follow-up (group A) and

these patients (Figure 1, panel A) showed higher, but not

statistically significant, OD median values for all circulating

biomarkers, when compared to patients without histological

evolution (n = 19, group B) (Figure 1, panel B).

Several reports indicate that serpinB4, corresponding to the

acidic isoform, is the main circulating isoform in patients with

epithelial cancers [40,41,26] and an elevated serpinB4/serpinB3

mRNA ratio, detectable in different cancer cells [22,23,42], has

been described as a poor prognostic factor for early-stage cervical

cancer. In the present study, we have therefore evaluated the ratio

between the different IgM-linked serpin isoforms in the three

groups of patients.

In patients with chronic hepatitis the serpinB4-IgM(SCC103)/

serpinB3-IgM median ratio was 1.40 (range 1.0–4.5), in patients

with cirrhosis it was 1.10 (range 0.9–6.2), and in patients with

HCC it was 1.08 (range 0.4–4.2). A progressive decrease of

median ratio was observed in patients with more advanced liver

disease, although no statistical difference was reached (Figure 2).

These data are in keeping with the finding that 16 out of 26

patients with HCC (62%) and 15 out of 36 patients with

cirrhosis (42%) were found below the LoD value for SerpinB4-

IgM.

Discussion

In the present study a simple assay to quantify the two isoforms

serpinB3 and serpinB4 circulating as IgM-linked immunocom-

plexes in patients with different extent of chronic liver disease was

set-up. The choice of analyzing the IgM-linked instead of the free

isoforms was derived by the results obtained previously, when the

assay to detect SCCA-IgM reactivity was initially described and

performed better than that for the detection of free SCCA protein

in serum [21]. Isoform-specific immunoenzymatic assays have

been set up, using commercially available monoclonal antibodies.

The aim of this investigation was to assess the pattern of expression

of IgM-linked SCCA isoforms in chronic liver disease and to

define whether they might provide any clinical advantage

compared to total circulating SCCA-IgM levels.

To the best of our knowledge, this is the first study on the

behaviour of circulating SCCA-IgM isoforms in patients with

chronic liver diseases. We have recently shown a progressive

increase over time of total SCCA-IgM immunocomplex in sera

of untreated patients with progressive forms of chronic hepatitis

[43] and in cirrhotic patients who developed liver cancer during

follow-up [44]. Although the number of patients was limited, we

have attempted to evaluate whether the reactivity for IgM-linked

serpin isoforms was distributed differently in patients with

chronic hepatitis and in cirrhotic patients with or without liver

cancer progression. The small number of patients did not allow

to identify significant differences between serpinB3-IgM and

serpinB4-IgM behaviour, despite cirrhotic patients who devel-

oped HCC showed a trend to higher reactivity for all circulating

biomarkers.

SerpinB3-IgM and SerpinB4-IgM in Liver Diseases


The majority of previous reports concerning SCCA levels in

serum of patients with epithelial cancers describe serpinB4 as the

dominant serological isoform [37,40,26]. These data were

confirmed by histological studies, showing an elevated expression

of serpinB4 in cancer tissues [22,41], as well as an elevated

serpinB4/serpinB3 mRNA ratio in cervical carcinoma [41,23] and

in head–neck cancer [45]. This effect was explained as a result of a

possible protective role of serpinB4 from inflammation and

apoptosis of tumour cells, probably due to direct inhibition of

cathepsin G [23].

As for SCCA-IgM isoforms and liver cancer, our findings

clearly document a graduate decrease of serpinB4-IgM/ser-

pinB3-IgM ratio when comparing patients with different extent

of liver disease, despite not reaching statistically significant

differences due to the limited number of the patients included in

the study. This trend was mainly a consequence of lower serum

levels of serpinB4-IgM in patients with liver tumour, where the

majority of them showed values below the detection limit for this

assay. Despite our findings are not in agreement with published

studies [37,40,26], it should be noted that current literature on

this subject refers to the analysis of SCCA free isoforms in

cancers of epithelial origin, where the corresponding normal

tissues express physiologically this serpin [46,10], while normal

liver does not [18]. Since SerpinB3 has been found to induce

epithelial to mesenchymal transition and increased proliferative

and invasive potential (17), while no parallel information is

available for SerpinB4, further studies to define the precise

biological activities of the two isoforms, beside their antiprotease

activity, are warranted.

In conclusion, the relative balance of the two serpin isoforms

seems to be altered in HCC and characterized by a lower

serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4

levels.

Materials and Methods

PatientsSerum samples from 28 blood donors and from 79 patients with

different extent of liver disease, including 17 patients with

histologically proven chronic hepatitis, 36 patients with histolog-

ically proven liver cirrhosis and 26 patients with HCC, were

analyzed. None of the patients underwent antiviral treatment at

least 12 months before serum sample collection. Patients with

cirrhosis underwent regular liver ultrasound screening and were

classified as Child A at the time of serum collection. The diagnosis

of HCC was based on the presence of hepatic focal lesion .2 cm

detected by liver ultrasound and confirmed by computed

tomography and/or magnetic resonance as imaging techniques

[36]. The final diagnosis was confirmed by histopathological

analysis on ultrasound-assisted fine needle biopsy, when indicated.

In patients with cirrhosis, two groups were identified on the

basis of HCC progression during follow-up: group A included 17

patients who developed HCC during a follow-up median period of

4 years, while the remaining 19 patients (group B) showed no

histologic evidence of disease progression during the same time

interval. Table 2 summarizes the main epidemiological and

clinical characteristics of the study population: both groups of

cirrhotic patients showed similar clinical profiles in terms of mean

age, sex distribution and aetiology. Overall, patients were

prevalently male, with mean age ranging from 53 to 65 years

and most of them were HCV infected (85%).

The study was performed according to the principles expressed

in the Declaration of Helsinki. Serum samples were collected after

obtaining a signed informed consent from the patients, as

approved by our institutional Ethics Committee. Serum samples

were obtained from whole blood collected into Vacutainer tubes

Table 1. IgM linked SerpinB3 and SerpinB4 isoforms, compared to total SCCA-IgM in patients with different extent of chronic liverdisease.

Chronic hepatitis Cirrhosis HCC Healthy Subjects

n = 17 n = 36 n = 26 n = 28

SerpinB3-IgM (SCC111) Median (OD405 nm) 0.130 0.130 0.130 0.130

IQR 0.130–0.172 0.130–0.221 0.130–0.175 0.130–0.130

95th percentile 0.200

% positivity 12 28 23 7


IQR 0.140–0.275 0.140–0.427 0.140–0.278 0.140–0.249




IQR 0.125–0.227 0.125–0.285 0.125–0.319 0.125–0.125



Total SCCA-IgM Median (AU/mL) 166 134 209 115

IQR 80–511 80–552 118–287 109–129

95th percentile 156


IQR = interquartile range (25th–75th percentile).The percentage of positivity for each immunocomplex was determined as the fraction number of patients with immunocomplex levels above the defined cut-off value(95th percentile).doi:10.1371/journal.pone.0040658.t001



(BD Diagnostics, USA) after centrifugation for 15 min at 20006g.

Serum was aliquoted into cryovials and stored at 280uC until use.

ELISA Assay for IgM-linked serpinB3 and serpinB4Isoforms

Three available monoclonal antibodies (CanAg Diagnostics,

Gothenburg Sweden) specific for the two different isoforms of this

serpin were utilized: SCC111 antibody, recognizing serpinB3 by

Western blot, although a slight cross-reactivity for SerpinB4 in ‘‘in

solution’’ assays has been also described recently [37], SCC103

and SCC104 antibodies which reacted only with serpinB4. In

particular, the SCC103 monoclonal antibody reacted with

serpinB4 complexed with specific proteinase: cathepsinG, while

SCC104 did not recognize the serpinB4 complex, but only the free

form of the serpin. Polystyrene high binding immunoplates (Sigma

Aldrich, Milano, Italy) were coated with 100 ml/well of each

monoclonal anti-human serpin antibody diluted in phosphate-

buffered saline (PBS) at a concentration of 10 mg/ml and

incubated overnight at 4uC. The wells were blocked with 1%

bovine serum albumin (BSA)/PBS and incubated at room

temperature for 2 h. After washing with PBS-0.05%Tween,

100 ml of serum samples were added at 1:8 dilution in PBS-

0.05%Tween containing 1% BSA and incubated at room

temperature for 1 h. The presence of serpinB3-IgM or serpinB4-

IgM complexes were revealed by the addition of 100 ml/well of

peroxidase-conjugated anti-human IgM (Sigma Aldrich, Milano,

Italy) at a 1:1000 dilution for 1 h. After washing, the enzyme

reaction was developed with 100 ml/well of an 2,29-azinobis(3-

ethylbenzthiazoline-6-sulfonic acid-diammonium salt)(ABTS) and

hydrogen peroxidase as chromogenic substrates. Plates were

analyzed by measuring the optical density at 405 nm on a

microtiter plate reader (Tecan, USA). Each sample was tested in

duplicate and each run was performed including positive and

negative controls. Intra-assay coefficient of variation, calculated by

repeated analysis (n = 5) of 3 samples, was 4.6% for serpinB3-IgM

assay, 6.2% for serpinB4-IgM (SCC104) assay and 3.8% for

serpinB4-IgM (SCC103) assay. The inter-assay coefficients of

variation, estimated from five independent runs with 3 samples

tested in duplicate, were less than 15% for each assay. Cut-off OD

values, calculated as 95thpercentile on the distribution curve of the

specific assay in 28 healthy subjects, were: OD 0.200 for serpinB3-

IgM; OD 0.199 for serpinB4-IgM (SCC104Ab) and OD 0.384 for

Figure 1. Serpin-IgM isoforms in cirrhotic patients with (panel A) and without (panel B) HCC evolution. Data in the box-plot graphsrepresent median, upper and lower OD values for serpinB4-IgM (SCC 103), serpinB4-IgM (SCC 104) and serpinB3-IgM.doi:10.1371/journal.pone.0040658.g001



serpinB4-IgM (SCC103Ab). The percentage of positivity for each

immunocomplex was determined as the fraction number of

patients with immunocomplex levels above the defined cut-off

value. The detection limit values (LoD), for each assay, were

calculated following the indications of the EP17-A guidelines [38].

LoD was determined by the formula:

LoD = LoB +1.645 (SD low concentration sample).

LoB (limit of blank) values were assayed with 10 replicates of the

blank reagent (PBS) that contains no analyte. LoD values,

calculated with five replicates of a sample known to contain a

low concentration of analyte, were OD 0.130 for serpinB3-IgM

assay, OD 0.125 for serpinB4-IgM (SCC104Ab) assay, and OD

0.140 for serpinB4-IgM (SCC103Ab) assay. These figures were

used for all the calculations when the patients value was below the

detection limit.

ELISA Assay for total SCCA-IgMTotal SCCA-IgM immunocomplexes were also detected in the

corresponding serum samples by commercial ELISA Kit (Hepa-

IC, generous gift of Xeptagen S.p.A., Venice, Italy) according to

manufacturer’s instructions. In this assay a polyclonal anti-human

SCCA antibody was used as capture antibody assuring the

detection of all SCCA isoforms [21,39]. The amount of total

SCCA-IgM complex was expressed in arbitrary Units/ml (AU/

ml). Cut-off value, calculated as 95th percentile, was 156 AU/ml.

The assay displayed intra-assay and inter-assay coefficients of

variation lower than 10% [21].

Statistical AnalysisConsidering that most variables were not normally distributed,

quantitative data were summarized as median and interquartile

range (IQR). Comparisons between groups were performed using

the non-parametric Mann Whitney U-test and, when more than

two groups had to be compared at the same time, the Kruskal-

Wallis analysis of variance was performed. Qualitative data were

summarized as percentages and the Fisher’s exact test was used for

differences in frequencies. A 2 tailed p-value of ,0.05 was

considered statistically significant. All analyses were performed

using GraphPad InStat 3.0 software (San Diego, CA, USA).

Figure 2. Distribution of serpinB4-IgM(SCC103)/serpinB3-IgM ratios. Scatter plot displays the distribution of serpinB4-IgM(SCC103)/serpinB3-IgM ratios detected in patients with chronic hepatitis, cirrhosis and HCC. Horizontal bars represent median value for each group.doi:10.1371/journal.pone.0040658.g002

Table 2. Epidemiological and clinical characteristics of the patients included in the study.

Chronic hepatitis Cirrhosis HCC Healthy subjects p

Group A Group B

No. of patients 17 17 19 26 28

Age (years, mean6SD) 53613 63614 65612 64612 3969 ,0.0001*

Sex M/F 11/6 12/5 11/8 19/7 17/11 ns

Aetiology

viral 100% 100% 100% 83% ns

non viral 17%

p values according to Kruskall-Wallis ANOVA.*p = 0.98 (according to Mann-Whitney U test) when patients with cirrhosis and with HCC were compared.doi:10.1371/journal.pone.0040658.t002



Acknowledgments

We deeply thank Dr. Rafael Ramirez Morales for his precious support in

editing and critically reviewing the manuscript.

Author Contributions

Conceived and designed the experiments: AB PP. Performed the

experiments: AB NT MGR SQ CT GV. Analyzed the data: LB GF CM

AG. Contributed reagents/materials/analysis tools: LB GF. Wrote the

paper: AB PP.

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Open Journal of Gastroenterology, 2012, 2, 56-61 OJGas doi:10.4236/ojgas.2012.22012 Published Online May 2012 (http://www.SciRP.org/journal/ojgas/)

Serum Scca-IgM as a predictor of hepatocellular carcinoma in patients with liver cirrhosis

Daniela Buccione1, Gianluca Fatti1, Andrea Gallotta2, Elisabetta Loggi1, Roberto Di Donato1, Lilia Testa1, Carlo Saitta3, Valentina Santi1, Antonio Di Micoli1, Virginia Erroi1, Marta Frigerio1, Valentina Fazio4, Antonino Picciotto4, Alessandra Biasiolo5, Francoise Degos6, Patrizia Pontisso5, Giovanni Raimondo3, Franco Trevisani1*

1Dipartimento di Medicina Clinica, Alma Mater Studiorum-Università di Bologna, Bologna, Italia 2Xeptagen S.p.A., Venezia, Italia 3Dipartimento di Medicina Interna, Policlinico Universitario di Messina, Messina, Italia 4Dipartimento di Medicina Interna, Università di Genova, Genova, Italia 5Dipartimento di Medicina Clinica e Sperimentale “G. Patrassi”, Università di Padova, Padova, Italia 6APHP, Hopital Beaujon, INSERM U 773, Clichy, France Email: *[email protected] Received 11 January 2012; revised 2 February 2012; accepted 19 February 2012

ABSTRACT

Aberrant Squamous Cell Carcinoma Antigen (SCCA) expression is an early hepatocarcinogenetic event and circulating SCCA-IgM complexes are elevated in most HCC patients. We evaluated whether serum SCCA- IgM levels can identify HCV +ve cirrhotic patients at low HCC risk. In this retrospective study we enrolled 29 cirrhotic patients in whom serum SCCA-IgM was measured 8 - 69 months (median 31) before HCC diagnosis, and 28 cirrhotic patients who remained HCC- free, with SCCA-IgM measured 15 - 68 months (median 48) before the study end. The best discriminating value of SCCA-IgM was calculated and tested in pre-dicting HCC diagnosis within 12, 24 and 36 months. Sensitivity analysis, considering different HCC incidence, was conducted to identify the patient subgroup with an annual cancer risk below the threshold of a cost-effective semiannual surveillance with ultrasound. Cumulative HCC incidence at 12, 24 and 36 months was 7.0%, 15.7% and 26.3%, respectively. SCCA-IgM levels were higher in HCC than in cirrhotic patients [median: 381 (95% C.I.: 50 - 5289) vs. 100 (70 - 493) AU/mL, P = 0.005]. The SCCA-IgM value ≤ 200 AU/mL accurately identified patients at low risk of HCC development in the subsequent year (sensitivity 75%, specificity 62%, positive predictive value 13% and negative predictive value 97%). Considering an annual HCC incidence ≤ 3%, patients with SCCA-IgM ≤ 200 AU/mL (60% of the whole patients) had an HCC risk below the accepted threshold of a cost-effective surveillance (1.5%). In conclusion, provided that our provocative results are confirmed in larger

studies, SCCA-IgM serum measurement could permit implementation of a two step (with different costs) surveillance: an initial serological surveillance, based on the annual monitoring of this biomarker, and the conventional surveillance by semiannual US when SCCA-IgM becomes >200 AU/mL. This could improve the cost/effectiveness of surveillance of HCV infected patients at risk of HCC. Keywords: SCCA-IgM; HCC Risk Assessment; Surveillance Cost/Effectiveness

1. INTRODUCTION

Hepatocellular carcinoma (HCC) is the fifth most com- mon cancer, the third cause of cancer-related death worldwide and the first cause of mortality in cirrhotic patients [1]. Chronic hepatitis C virus (HCV) infection represents the most important risk factor for HCC in Western countries and in Japan [2]. In HCV infected cirrhotic patients the annual incidence of HCC ranges from 1% to 8%, being higher in Japan (4% - 8%), intermediate in Italy (2% - 4%) and lower in the USA (1.4%) [3]. The prognosis of HCC patients still remains unsatis- factory, the 5-year survival rate being less than 10% both in Europe [4] and the USA [5]. Surveillance of patients at risk of developing HCC, based on the periodic repeti- tion of liver ultrasound (US), makes it possible to detect most cancers at an early stage, still amenable to curative treatments which can greatly improve the prognosis of these individuals [6-10]. Therefore, regular surveillance of patients at risk of HCC is currently recommended by both Western [11] and Eastern [12,13] practical guide- *Corresponding author.

Published Online May 2012 in SciRes. http://www.scirp.org/journal/ojgas

mailto:[email protected]

D. Buccione et al. / Open Journal of Gastroenterology 2 (2012) 56-61 57

lines for HCC management. The cost/effectiveness of surveillance is highly de-

pendent on the incidence of HCC and, in the case of cirrhotic patients, decision analysis studies suggest that surveillance becomes cost-effective when the HCC risk is 1.5% per year or greater [14,15]. Nonetheless, a prospective study in Western patients with cirrhosis did not report a satisfactory cost/effectiveness of a semiannual program based on US and alpha-fetoprotein (AFP) determination [7]. This is due to the fact that the risk of HCC development has not yet been clearly defined in an individual basis, and patients with an insufficient risk are included in surveillance. It would therefore be important to exclude these patients in order to save costs. On this prospect, composite systems based on demographic and clinical factors have been proposed to stratify patients according to the HCC risk [16-19] but they have not received external validation and have not entered into clinical practice. To date, no serological marker able to accurately predict tumour development in cirrhotic patients has been found.

Squamous Cell Carcinoma Antigen (SCCA) is a serine protease inhibitor detectable in the spinous and granular layers of normal squamous epithelium but also expressed by neoplastic epithelial cells [20]. An overexpression of SCCA variants (SCCA-1, SCCA-2 and SCCA-PD) has been described in HCC tissue but not in normal liver [21], and a recent study has shown that SCCA is more expressed in cirrhotic tissue adjacent to high grade dysplastic nodules or HCC than in cirrhotic tissue proximal to large regenerative nodules or low grade dysplastic nodules. Thus, SCCA overexpression has been claimed to be an early event in hepatocarcinogenesis [22]. Noteworthy, the serum concentration of circulating immune complexes composed of SCCA and Immunoglobulin M (SCCA- IgM IC) paralleled the extent of SCCA overexpression in liver specimens [23,24], and in a small group of HCV infected patients with cirrhosis progressively increasing levels of serum SCCA-IgM complex were able to identify subjects at higher risk of HCC development [25]. Exploiting such a predictive ability in an opposite way, this biomarker could be utilized to identify inappropriate candidates for surveillance.

Our study aimed at assessing the ability of serum SCCA-IgM levels to segregate, according to the risk of HCC development, HCV infected patients with liver cirrhosis to identify those with a risk below the threshold of a cost-effective US surveillance.

2. PATIENTS AND METHODS

2.1. Patients

Among cirrhotic patients followed up from April 1994 to January 2010 in 5 Centres, we retrospectively selected

those meeting the following inclusion criteria: 1) infected by HCV ( other aetiological factors), 2) aged ≥ 50 years, 3) maintained under semiannual surveillance, based on US (± alpha-fetoprotein determination), 4) availability of one blood sample stored at –20˚C, collected at least 6 months before HCC occurrence (cases) or the date (January 2009) of study end (controls), 5) control patients were also required to remain tumour-free during the subsequent year, 6) no interferon treatment in the 6 months prior to the collection of the index blood sample.

The 57 patients fulfilling these criteria were divided into 2 groups: HCC group encompassing 29 patients developing HCC during the study period, and control group including 28 patients who remained tumour-free.

2.2. HCC Diagnosis and Staging

HCC was diagnosed by histology or according to the non-invasive criteria proposed by the EASL and thereafter the AASLD guidelines [11]. Prior to the availability of these criteria, the non-invasive diagnosis was based on an AFP value > 200 ng/mL coupled with an imaging work-up suggestive of HCC and definitely confirmed by the patient follow-up. The tumour was staged as “non- advanced” if its burden met the Milano criteria or “advanced” if beyond them [26].

2.3. Serologic Testing

Circulating SCCA-IgM levels were determined in frozen serum samples, stored at –20˚C, using an ELISA assay kit (Hepa-IC, Xeptagen SpA, Marghera, Venezia, Italy).

2.4. Statistical Analysis

Continuous variables were expressed as mean SD or median and 95% C.I. and discrete variables as absolute and relative frequencies.

Continuous non-parametric variables were compared with the Mann-Whitney U test, and discrete variables with the 2 test or Fisher’s exact test, as appropriate.

The Receiver Operating Characteristic (ROC) curve and the corresponding area under the curve (AUC) were calculated to assess the accuracy of the seromarker in distinguishing HCCs from cirrhosis. The best cut-off value was chosen as the value with the highest Youden index ([sensitivity + specificity] – 1) and, in the event of equality, the value with higher sensitivity was chosen.

To assess the ability of SCCA-IgM to predict HCC occurrence in clinical practice, we simulated a prospective study protocol with patient assessment every 12 months. The simulation is depicted in Figure 1. In practice, patients who developed HCC during the 12, 24 and 36 months following the SCCA-IgM measurement were shifted to the HCC group, leaving the HCC-free indi-

Copyright © 2012 SciRes. OJGas


Figure 1. Annual occurrence of hepatocellular carcinoma (HCC) in the studied population at 12, 24 and 36 months from SCCA- IgM determination.

viduals in the cirrhotic group. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of the SCCA-IgM were calculated. PPV and NPV were also calculated simulating HCC incidences expected in clinical practice.

A sensitivity analysis was then conducted to test the ability of SCCA-IgM to identify, according to HCC incidence in the whole patient population, the subgroup with an HCC risk below 1.5%/year, which is considered the threshold of a cost-effective surveillance [11].

A 2-tailed P value < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS 17.0 statistical package (Chicago, IL), Microsoft Excel program and MedCalc 11 software.

2.5. Ethics

The database management conforms to current Italian legislation on privacy and the study conforms to the ethical guidelines of the Declaration of Helsinki. All patients had provided informed consent to register and manage their data in an anonymous way in our database. The study was approved by the ethics committee of the participating Institutions.

3. RESULTS

3.1. Patients’ Characteristics (Table 1)

The HCC group and the control group did not differ for age and gender. In both groups, a few individuals were co-infected with HBV or declared a heavy alcohol intake as cofactors of liver disease. No significant differences were found in the distribution of Child-Pugh classes, with a predominance of patients with well preserved liver function. Most patients did not receive any antiviral therapy, without differences between groups. Nearly 70% of cases had a non-advanced HCC (within the Mi- lano criteria) at the time of diagnosis.

Table 1. Demographic a clinical characteristics of patients.

Variable HCC patients (n = 29)

Cirrhotic patients(n = 28)

P

Age (years)

Gender (males)

Aetiology

HCV

HCV + HBV

HCV + alcohol

Child-Pugh class

A

B

C

Antiviral therapy

no therapy

lamivudine + adefovir

Median follow-up (months)

Tumour size (cm)

Milano criteria

within

beyond

66.5 ± 8.2

18 (62.1%)

25 (86.2%)

2 (6.9%)

2 (6.9%)

27 (93.1%)

2 (6.9%)

0 (0%)

28 (96.6%)

1 (3.4%)

31 (69 - 8)

2.5 ± 1.3

20 (69.0%)

9 (31.0%)

65.8 ± 10.3

16 (57.1%)

23 (82.1%)

3 (10.7%)

2 (7.1%)

22 (78.6%)

6 (21.4%)

0 (0%)

26 (92.9%)

2 (7.1%)

48 (68 - 15)

-

-

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

HCV: hepatitis C virus; HBV: hepatitis B virus; SCCA-IgM: Serpin Squamous Cell Carcinoma Antigen-IgM complexes; n.s.: not statistically significant.

3.2. SCCA-IgM Performance

Blood samples used for SCCA-IgM determinations were collected from 8 to 69 months (median 31 months) before HCC detection in the HCC group, and from 15 to 68 months (median 48 months) prior to the study end in the control group. Median SCCA-IgM values were higher in the HCC group than in the control group [381 AU/ml (95% C.I. 117 - 615) vs. 100 AU/ml (95% C.I. 80 - 146); P = 0.004].

The ROC curve of SCCA-IgM levels found from 8 to 69 month prior to the cancer diagnosis (HCC patients) or the end of the study (HCC-free patients) is reported in Figure 2. The best discriminating value of 200 AU/ml (Youden Index 46.4%) had an overall sensitivity of 57.1%, an overall specificity of 89.3% and, considering the HCC prevalence in our population (51%), a PPV of 84.2% and an NPV of 67.6%. The PPV and NPV were also calculated for the cumulative incidence of HCC observed at 12 (7.0%), 24 (15.7%) and 36 (26.3%) months after SCCA-IgM measurement (Table 2). Notably, in this model the NPV for HCCs occurring within the year following the SCCA-IgM determination rose to 97%, and it was still 91% for HCCs occurring within 24 months. When the HCC prevalence was set at 3% to simulate the annual incidence expected in clinical prac- ice, the NPV for HCC occurrence within 12 and 24 t


D. Buccione et al. / Open Journal of Gastroenterology 2 (2012) 56-61

Copyright © 2012 SciRes.

59

Figure 2. Receiver Operating Characteristic (ROC) curve and corresponding area under the curve (AUC) of SCCA-IgM values measured 8 - 69 months prior to cancer diagnosis (HCC patients) or end of the study (HCC-free patients). The dot indicates the best cut-off value (200 AUA/mL).

Table 3. Sensitivity analysis according to different annual incidences of HCC. With an overall incidence ≤ 3%, cirrhotic patients with SCCA-IgM ≤ 200 AU/ml have a risk of developing hepatocellular carcinoma (HCC) below the threshold (1.5%) of a cost-effective semiannual surveillance with liver ultrasound.

Table 2. Sensitivity (Se), Specificity (Sp), Positive (PPV) and Negative (NPV) Predictive Values of the best discriminating value of SCCA-IgM measured 12, 24 and 36 months prior to the HCC occurrence (cases) or the study end (controls).

Timing (months)

HCC cumulative incidence (%)

Se (%) Sp (%) PPV (%) NPV (%)

–12

–24

–36

7.0

15.7

26.3

75

67

60

62

65

67

13

26

39

97

91

82

Annual incidence of HCC (%)

Whole population (N = 57)

Patients with SCCA-IgM ≤ 200 AU/ml (N = 34)

Patients with SCCA-IgM > 200 AU/ml (N = 23)

2

3

4

0.8

1.2

1.6

3.7

5.5

7.4 SCCA-IgM: Serpin Squamous Cell Carcinoma Antigen-IgM complexes.

months further increased to 98.8% and 98.5%, respectively. 4. DISCUSSION

The sensitivity analysis showed that, when annual incidence of HCC in the whole population was 3%, cirrhotic patients with SCCA-IgM ≤ 200 AU/ml had a tumour incidence of 1.2%, not enough for implementing a cost-effective semiannual surveillance with US [11] (Table 3). The accepted threshold of 1.5% was indeed crossed by patients with SCCA-IgM ≤ 200 AU/ml only when the tumour incidence was at least 4% in the whole population.

Several studies indicate that surveillance of individuals at risk of HCC increases the chance of detecting tumours at a stage amenable to curative or effective treatments, thus improving the prognosis of these patients [6-9,27]. Since the cost/effectiveness of this procedure is highly dependent on the incidence of HCC [14,28], the decision to enter a patient into a surveillance program should be determined by his/her level of HCC risk. Nowadays, the individual risk of HCC development in patients infected

OJGas


by HCV (the main cause of this tumour in the Western world and in Japan) has not yet been clearly defined. Therefore, although a Markov model would indicate that semiannual surveillance with US and AFP is cost-effective [28], this assumption has not been confirmed by a prospective observational study, principally including anti-HCV positive patients, where the estimated cost of each year of life gained exceeded 100,000 $ [7]. To optimize the cost-effectiveness of surveillance, we should be able to identify patients in whom surveillance is not worthwhile due to a very low HCC risk. The NPV becomes a crucial indicator for this purpose.

Some predictive indices have been proposed in HCV infected patients, but these studies do not report the NPV and only considered patients after [29] or during antiviral therapy [30]. The availability of inexpensive, easy to measure serological markers, able to accurately define this risk over a given—and relatively long-time span would be instrumental for this purpose. Since the aberrant expression of SCCA is an early event in hepatocarcinogenesis [22] and the circulating levels of the IgM immune-complexes parallel the extent of its tissue expression [23], monitoring SCCA-IgM is a promising tool for this perspective.

The prospective approach we simulated made it possible to test the performance of SCCA-IgM measured 12 months prior to HCC detection. The value of 200 AU/ml showed a valuable NPV of 97% one year before cancer diagnosis, despite the high HCC prevalence (7%) observed in our model. Moreover, reducing the HCC prevalence to a value simulating the tumour incidence expected in clinical practice (3%), the NPV reached 98.8%, and the sensitivity analysis revealed that, at this cancer prevalence, the SCCA-IgM can confidently identify HCV- infected cirrhotic patients with a too low risk of develop- ping HCC in the subsequent year to enter a semiannual surveillance based on US. This ability, however, disap- pears when the HCC incidence in the whole population is 4% or higher. Therefore, provided that the expected annual risk of HCC is ≤3% in a given population of HCV infected patients (as can be anticipated in patients with advanced fibrosis/early cirrhosis), SCCA-IgM serum measurement could permit implementation of two step (with different costs) surveillance: an initial serological surveillance, based on the annual monitoring of this biomarker, and the conventional surveillance by semiannual US when SCCA-IgM becomes >200 AU/mL (Figure 3). Using this model, in our series, 34/57 (60%) cases would have been initially excluded from a non cost-effective semiannual surveillance, and annually postponed until this practice becomes cost-effective, without incurring an unacceptable risk of HCC underdiagnosis (as suggested by an NPV 99%).

This pilot study, however, has several limitations: 1) it

Figure 3. Surveillance algorithm of patients at risk of developing HCC: possible practical application of SCCA-IgM annual monitoring to improve the cost-effectiveness of surveillance of HCV-infected patients with an expected annual HCC incidence ≤ 3%.

is a retrospective investigation, suffering therefore of the typical methodological biases of these studies; 2) it is based on a small sample size; 3) our population included nine patients (16%) with HBV infection or heavy alcohol intake as cofactors of liver disease. The small sample size prevented the possibility to obtain reliable results on SCCA-IgM performances in the subgroups. On the other hand, the aetiological panel of our HCV-infected patients reproduces that into which daily clinical practice runs; 4) our results cannot be generalized to patients with non- HCV related cirrhosis or younger than 50 years (excluded from the study).

Therefore, the results of our seminal study, overtur- ning the ordinary use of a new and promising onco- marker from an indicator of a high risk condition to a guide to identify low risk patients, should be considered as a starting point for further prospective investigations aimed at confirming whether SCCA-IgM annual moni-toring can improve the cost/effectiveness of surveillance for HCC with an acceptable number of missed early diagnoses.

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Volume 46, Supplement 1, Page e61, February 2014

SCCA-IGM ARE PREDICTIVE OF HEPATOCELLULAR CARCINOMA DEVELOPMENT IN PATIENTS WITH HCV CIRRHOSIS. A PROSPECTIVE

STUDY. Biasiolo A1, Trotta E1, Fasolato S1, Tono N1, Ruvoletto MG1, Terrin L1, Fassina G2, Angeli P1, Gatta A1, Pontisso P1.

1Department of Medicine, University of Padua, Italy, 2Xeptagen, VEGA Park, Venice, Italy Introduction and aims. In chronic hepatitis C increasing SCCA-IgM levels were found predictive of fibrosis progression and in HCV cirrhosis high levels of this biomarker were associated with an increased risk of HCC development in a recent multicenter cross-sectional study. Aim of the study was to assess the clinical significance of SCCA-IgM in cirrhotic patients in a prospective study. Materials and Methods. 71 patients with cirrhosis (M/F: 53/18) were consecutively enrolled and followed up for a median period of 53 months at our Institution. Etiology was HCV in 37%, HBV in 17%, alcohol in 44% and metabolic or unknown in 2% of the cases. The majority of the patients (69%) were Child A. SCCA-IgM was measured in serum at presentation by ELISA (Hepa-IC, Xeptagen). Results. SCCA-IgM was more frequently detected in HCV cirrhosis than in the remaining patients (38% vs 13%, p= 0.02). During follow up 11/26 HCV patients developed HCC (median time: 11 months). In this group the positivity of the biomarker at presentation was significantly associated with HCC development (70% vs 25%, p=0.04), but not with other cirrhosis complications. Kaplan–Meier curves confirmed a lower HCC-free survival in HCV cirrhotic patients positive for SCCA-IgM, compared to negative cases (p=0.03).The relative risk of HCC development was 2.7 in HCV cirrhosis (95% CI=1.6-4.6) and increased up to 3.2 (95% CI= 1.1-9.6) in the subset of SCCA-IgM positive patients. Conclusions. In patients with HCV cirrhosis SCCA-IgM was highly predictive of HCC development and may be considered as a prognostic tool for the subclassification of cirrhotic patients.

Successful antiviral therapy determines a significant decreasein squamous cell carcinoma antigen-associated (SCCA)variants� serum levels in anti-HCV positive cirrhotic patients*E. G. Giannini,1 M. Basso,2 M. Bazzica,2 P. Contini,3 S. Marenco,1 V. Savarino1

and A. Picciotto2 1Gastroenterology Unit, Department of Internal Medicine, University of Genova, Genova, Italy; 2Viral Hepatitis Unit,

Department of Internal Medicine, University of Genova, Genova, Italy; and 3Clinical Immunology Unit, Department of Internal Medicine, University of

Genova, Genova, Italy

Received July 2009; accepted for publication September 2009

SUMMARY. Aberrant squamous cell carcinoma antigen

(SCCA) expression is an early event in hepatocarcinogenesis,

and increasing serum levels of SCCA variants IgM immune

complexes (SCCA-IgM IC) have been found in cirrhotic

patients developing hepatocellular carcinoma (HCC). We

longitudinally evaluated a cohort of cirrhotic patients with

hepatitis C virus infection (HCV) who underwent pegylated

interferon (PEG-IFN) and ribavirin treatment. SCCA-IgM IC

levels were assessed in the sera of 33 cirrhotic patients with

HCV (21 males, median age 57 years) before, at the end and

at 6-month and 1-year follow-up after treatment with PEG-

IFN and ribavirin. SCCA-IgM IC serum levels (arbitrary

units/mL, AU/mL) were evaluated according to treatment

outcome: sustained virological response (SVR) vs nonre-

sponse (NR). Overall, 15 patients obtained a SVR to antiviral

therapy (45%). There was no significant difference in base-

line SCCA-IgM IC serum levels between SVR and NR

patients. When compared to baseline (451.2 AU/mL), SVR

patients showed a significant decrease in median SCCA-IgM

IC serum levels at the end of treatment (186.8 AU/mL,

P = 0.013) and at both 6-month (96.8 AU/mL, P < 0.001)

and 1-year follow-up (52.4 AU/mL, P < 0.001), while no

significant modification was observed in NR patients. In

patients with HCV-related liver cirrhosis, successful antiviral

therapy is associated with a dramatic and significant decrease

in SCCA-IC serum levels. Because of the pathophysiological

correlation between SCCA and liver carcinogenesis, it

is hypothesized that in patients with liver cirrhosis, SVR may

be accompanied by a decreased proliferative stimulation.

Keywords: cirrhosis, hepatitis C virus, peg-interferon, riba-

virin, squamous cell carcinoma antigen.

INTRODUCTION

Chronic hepatitis C virus (HCV) infection is a leading cause

of end-stage liver disease and hepatocellular carcinoma

(HCC) [1] worldwide. In patients chronically infected with

HCV, the presence of liver cirrhosis is the most important

factor for the development of HCC [2]. Longitudinal evalu-

ation of patients with compensated cirrhosis as a result of

HCV infection has shown that the yearly development rate of

HCC ranges between 2% and 4% and that in these patients,

HCC is the most frequent liver-related complication [3,4]. In

patients with cirrhosis and HCV, antiviral therapy with

pegylated interferon (PEG-IFN) and ribavirin is aimed at

halting disease progression and decreasing the incidence of

HCC [5]. Some studies have thus shown that successful

antiviral therapy decreases the rate of HCC development in

these patients.

Squamous cell carcinoma antigen (SCCA) is a serine

protease inhibitor that is physiologically found in the

spinous and granular layers of normal squamous epithelium

and is also typically expressed by neoplastic cells of epithelial

origin [6]. Recently, overexpression of SCCA variants (SCCA-

1, SCCA-2 and SCCA-PD), as detected by immunohisto-

chemistry, has been described in resected specimen of HCC

tissue but not in normal liver [7]. Furthermore, SCCA

Abbreviations: HCV, hepatitis C virus; HCC, hepatocellular

carcinoma; SCCA, squamous cell carcinoma antigen; SCCA-IgM IC,

squamous cell carcinoma antigen immunoglobulin M immune

complexes; SVR, Sustained virological response; NR, Nonresponse;

ALT, alanine aminotransferase; AST, aspartate aminotransferase;

AU, arbitrary units; PEG-IFN, pegylated interferon.

Correspondence: Edoardo G. Giannini, MD, PhD, FACG,

Gastroenterology Unit, Department of Internal Medicine, University

of Genova, Viale Benedetto XV, no.6, 16132, Genova, Italy. E-mail:

[email protected]

*Preliminary results of this study have been submitted to the annual

scientific meeting of the American Association for the Study of Liver

Disease (Boston, MA, October 30–November 3, 2009).

Journal of Viral Hepatitis, 2010, 17, 563–568 doi:10.1111/j.1365-2893.2009.01217.x

� 2009 Blackwell Publishing Ltd

overexpression seems to be an early event in hepatocarci-

nogenesis, as in cirrhotic patients, both the prevalence and

the intensity of immunostaining for SCCA were significantly

greater in dysplastic nodules than in large regenerative

nodules [8]. Noteworthy, the serum concentration of

circulating immune complexes composed by SCCA and

immunoglobulin M (SCCA-IgM IC) paralleled the extent of

SCCA overexpression in liver specimens, thus making

of serum SCCA-IgM IC an interesting marker for diagnosis of

HCC [9,10]. More recently, it has been shown that pro-

gressive increase over time in serum SCCA-IgM IC can be

observed in patients with chronic hepatitis C with worsening

disease stage and in cirrhotic patients who develop HCC

[11,12]. There are no data in the literature on the possible

modification of serum SCCA-IgM IC in patients with HCV

infection undergoing antiviral therapy. In this setting,

monitoring of serum SCCA-IgM IC may improve our

understanding of the antiproliferative action of successful

antiviral treatment and patients� outcome.

In this study, our aim was to evaluate the behaviour of

serum SCCA-IgM IC in a cohort of cirrhotic patients with

HCV who were treated with PEG-IFN and ribavirin so as to

evaluate whether successful antiviral therapy may be asso-

ciated with a modification in SCCA-IgM IC serum levels.

PATIENTS AND METHODS

Study cohort

In this study, we included 33 patients chronically infected

with HCV who underwent PEG-IFN and ribavirin combina-

tion antiviral therapy at our Institution between March

2002 and April 2007 and who fulfilled the following clinical

and biochemical criteria: age between 18 and 70 years,

clinical or histological diagnosis of compensated cirrhosis, no

history of previous decompensation of liver disease, ultra-

sound examination of the abdomen negative for focal liver

lesions, serum bilirubin <2 mg/dL, serum albumin >3.5 g/

dL, prothrombin activity >80%, serum creatinine <1.3 mg/

dL, haemoglobin >12 g/dL in men and >11 g/dL in women,

leukocyte count >3000/mm3 or absolute neutrophil count

>1000/mm3 and platelet count >100 000/mm3.

Patients were excluded if they had other causes of liver

disease, previous organ transplantation, pre-existing

uncontrolled psychiatric disease, seizure disorders, severe

cardiovascular disease, haemoglobinopathies, haemophilia,

poorly controlled diabetes, autoimmune diseases, intrave-

nous drug use and/or alcohol abuse, positivity for human

immunodeficiency virus infection and if they were unable to

use contraception.

This study was investigator-driven, was carried out in the

clinical practice and was not supported by any pharma-

ceutical company. Informed consent was obtained from all

patients.

Treatment protocol

Patients were treated with either PEG-IFN a2a (180 lg/week

subcutaneously) or PEG-IFN a2b (1.5 lg/kg/week subcuta-

neously) plus oral weight-based ribavirin in two separated

doses (total dose was 800 mg/day for patients weighing

<65 kg, 1000 mg/day for patients with a body weight

ranging from 65 to 85 kg and 1200 mg/day for patients

weighing 85 kg or more). Selection of PEG-IFN was at the

discretion of the study physicians in charge of the patients,

as this was not a randomized study. Treatment duration was

24 weeks for genotype 2 and genotype 3 patients, and

48 weeks for genotype 1 and 4 patients. At week 12,

treatment was discontinued in patients who did not clear

HCV-RNA or who had a reduction in viral load lower than

2.0 log when compared to baseline, and at week 24 treat-

ment was discontinued in genotype 1 and 4 patients who

were still HCV-RNA positive [13]. Sustained virological

response (SVR) was assessed on the basis of serum HCV-RNA

negativity at 24 weeks of follow-up even if treatment was

discontinued before the assigned schedule as a result of side

effects or noncompliance to therapy. Nonresponse (NR) to

treatment was defined as lack of HCV-RNA clearance during

treatment and at follow-up [13]. Serum HCV-RNA was

measured by Amplicor HCV Monitor (Roche, Milan, Italy.

Cut-off limits, quantitative test: 600 IU/mL; qualitative test:

50 IU/mL). HCV genotype was determined before treatment

in all patients with the INNO-LiPA HCV II kit (Bayer Diag-

nostics, Emeryville, CA, USA).

Clinical evaluation

Clinical and biochemical evaluations were performed at

baseline, week 2, week 4 and then monthly thereafter while

on treatment, at 24 weeks follow-up and then every

6 months thereafter. Ultrasonographic examination of the

liver- and serum a-fetoprotein determination was carried out

at baseline and every 6 months while on treatment.

SCCA-IgM IC assay

Serum samples for SCCA-IgM IC were obtained at baseline,

at the end of treatment and at 6-month and 12-month

follow-up and stored at )80 �C. Circulating SCCA-IgM IC

levels were determined using an ELISA assay kit (Hepa-IC,

Xeptagen SpA, Italy) according to the manufacturer�sinstructions. Briefly, plates precoated with anti-human SCCA

antibody were incubated with either serially diluted stan-

dards or serum samples, and the presence of SCCA-IgM IC

were revealed by the addition of enzyme-conjugated anti-

human IgM. The plate was then washed, and the substrate

solution was incubated for 20 min. Subsequently, the plate

was read on a microtiter plate reader at 405 nm [9–11]. The

amount of SCCA-IgM IC were expressed in arbitrary units/


564 E. G. Giannini et al.

mL (AU/mL), as previously reported by other authors

[9–11].

Statistical analysis

Continuous variables are shown as median and 95% confi-

dence interval for the median, and discrete variables are

shown as absolute value and percentage. Comparison of

continuous variables has been carried out with nonpara-

metric tests for paired (Wilcoxon test) or nonpaired (Mann–

Whitey U-test) samples. Discrete variables were compared

using the Fisher�s exact test. Correlation between variables

was carried out with Spearman�s rank correlation test. A

two-tailed P < 0.05 was considered statistically significant.

Statistical analysis was carried out with MedCalc software

version 5.00.019 (MedCalc Software, Mariakerke, Belgium).

RESULTS

Table 1 shows the main characteristics of the study popu-

lation. Patients were prevalently males (64%) and infected

with HCV genotype 1 (58%). Serum SCCA-IgM IC levels

were similar in males (n = 21, 253.2 AU/mL, 93.9–

571 AU/mL) and females (n = 12, 203.1 AU/mL, 67.5–

687.7 AU/mL) and showed no statistically significant cor-

relation with age and biochemical variables (aspartate

aminotransferase, alanine aminotransferase, serum albumin

serum bilirubin, serum prothrombin activity and serum

a-fetoprotein, Table 2).

In the whole population, we observed a significant decrease

when compared to baseline (253.2 AU/mL, 98.5–457.4 AU/

mL) in serum SCCA-IgM IC levels at the end of PEG-IFN and

ribavirin therapy (143.5 AU/mL, 85.1–244.6 AU/mL;

P = 0.011) and at both 6-month (137.2 AU/mL, 86.8–

255.4 AU/mL; P = 0.043) and 1-year follow-up (61.2 AU/

mL, 29.0–110.5 AU/mL; P = 0.001, Fig. 1). A SVR to

antiviral treatment was obtained in 15 patients (45%).

Baseline median serum SCCA-IgM IC levels tended to be

higher in SVR patients when compared to NR, although

there was no statistically significant difference between the

two groups. Furthermore, there were no significant differ-

ences in SCCA-IgM IC serum levels between SVR and NR

patients at all the time points of the study (end of treatment,

6-month and 1-year follow-up, Table 3).

Longitudinal evaluation of serum SCCA-IgM IC levels

according to treatment outcome showed that SVR to antiv-

iral therapy was associated with a statistically significant

decrease in serum SCCA-IgM IC levels when compared to

baseline. Among SVR patients, the decrease in serum SCCA-

IgM IC levels when compared to baseline was already

evident at the end of treatment (baseline: 451.2 AU/mL vs

end of treatment: 186.8 AU/mL; P = 0.013) and was more

marked at both 6-month (96.8 AU/mL; P < 0.001) and

1-year follow-up (52.4 AU/mL; P < 0.001). In NR patients,

SCCA-IgM IC serum levels showed no significant modifica-

Table 1 Main characteristics of the study population

Variable Unit Value

Age Years 57 (49–64)

Gender Male/female 21 (64)/12 (36)

ALT IU/L 111 (70–129)

AST IU/L 77 (58–99)

Albumin g/L 44 (42–47)

Total bilirubin lm 13.6 (11.9–15.3)

Prothrombin activity % 98 (92–101)

a-fetoprotein lg/L 8.4 (4.8–22.6)

HCV genotype 1/non-1 19 (58)/14 (42)

SCCA-IgM IC AU/mL 253.2 (98.5–457.4)

Data are shown as median and 95% confidence interval for

the median for continuous variables and as absolute count

and percentage for categorical variables. ALT, alanine

aminotransferase; AST, aspartate aminotransferase; SCCA-

IgM IC, squamous cell carcinoma antigen IgM immune

complexes; AU, arbitrary units; HCV, hepatitis C virus.

Table 2 Correlations between serum squamous cell carci-

noma antigen immunoglobulin M immune complexes levels

and study variables at baseline

Variable rs P

Age 0.075 0.702

ALT )0.137 0.471

AST )0.087 0.648

Albumin 0.149 0.476

Total bilirubin )0.159 0.392

Prothrombin activity 0.194 0.341

a-Fetoprotein 0.013 0.963

ALT, alanine aminotransferase; AST, aspartate amino-

transferase.

Fig. 1 Serum squamous cell carcinoma antigen immuno-

globulin M immune complexes levels at baseline, at the end

of pegylated interferon and ribavirin therapy and at

6-month and 1-year follow-up in the whole population.


SCCA levels and antiviral therapy 565

tion when compared to baseline at both the end of treatment

and during follow-up (Figs 2a,b). In SVR, decrease in serum

SCCA-IgM IC levels was not correlated with modification in

serum aminotransferase and a-fetoprotein levels (data not

shown).

Three patients developed HCC during follow-up (two NR and

one SVR). When compared to baseline, serum SCCA-IgM IC

levels showed an increase in both NR patients (base-

line fi end of treatment fi end of follow-up: patient 1:

12.6 fi 38.6 fi 44.4 AU/mL; patient 2: 391.7 fi853.6 fi 2019.1 AU/mL), while the SVR patient showed a

blunted decrease in SCCA-IgM IC levels (451.2 fi 374.8 fi230.4 AU/mL). Among the NR patients who did develop HCC,

serum a-fetoprotein was consistently within the normal range

(i.e., £20 lg/L) in one and showed a nonsignificant decrease in

the other one (baseline fi end of treatment fi end of fol-

low-up: patient 1: 9.2 fi 13.3 fi 9.0 lg/L; patient 2:

64.5 fi 53.3 fi 20.2 lg/L), while in the SVR patient who

developed HCC it was consistently normal (10.3 fi 7.8 fi9.4 lg/L).

DISCUSSION

Aberrant SCCA expression seems to be an early event in

human hepatocarcinogenesis, and dysplastic nodules of

cirrhotic patients have an increased prevalence and intensity

of immunostaining for SCCA than large regenerative nod-

ules [7,8]. Furthermore, serum levels of SCCA-IgM IC are

correlated with expression of SCCA in liver resected speci-

mens of patients with HCC [9], and increasing SCCA-IgM IC

serum levels have recently been demonstrated in the sera of

untreated chronic hepatitis C patients with progressive

disease as well as in cirrhotic patients who develop HCC

[11,12]. It has been reported that SCCA isoforms protect

neoplastic cells from apoptosis, and SCCA1 seems to promote

tumour growth [14–16].

We assessed serum SCCA-IgM levels in a series of cirrhotic

patients with compensated disease who underwent PEG-IFN

and ribavirin antiviral therapy. Because therapy with PE-

G-IFN – besides its immunomodulatory and antiviral effects

– should have antiproliferative properties [17–19], and

successful antiviral treatment is associated with a decreased

risk of HCC [20,21], we sought to investigate the behaviour

of SCCA-IgM IC during PEG-IFN and ribavirin treatment and

in the course of follow-up in a series of patients with

HCV-related cirrhosis. Our aim was to evaluate whether

successful antiviral therapy was associated with a decrease

Table 3 Squamous cell carcinoma antigen immunoglobulin M immune complexes (SCCA-IgM IC) serum levels in the study

cohort, before and after pegylated interferon and ribavirin therapy, subdivided according to treatment outcome

Serum SCCA-IgM (AU/mL)

Baseline End of treatment 6-month follow-up 12-month follow-up

NR (n = 18) 142.3 (81.9–322.8) 125.7 (64.8–199.4) 156.3 (92.2–407.3) 85.7 (27.5–244.0)

P = 0.138 P = 0.311 P = 0.278 P = 0.235

SVR (n = 15) 451.2 (80.3–803.7) 186.8 (55.9–397.2) 96.8 (57.4–305.6) 52.4 (18.4–108.6)

Data are shown as median and 95% confidence interval for the median. NR, nonresponder; SVR, sustained virological

responder; AU, arbitrary units.

P < 0.001

P < 0.001

P = 0.013

Sustained virological responder patients2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

SCC

A-I

gM I

C s

erum

leve

ls (

AU

/mL

)

0 1 2 3

Non responder patients2100

1900

1700

1500

1300

1100

900

700

500

300

100

SCC

A-I

gM I

C s

erum

leve

ls (

AU

/mL

)

0 1 2 3

(a)

(b)

Fig. 2 Longitudinal behaviour of squamous cell carcinoma

antigen immunoglobulin M immune complexes serum

levels in sustained virological responders (a) and nonre-

sponders (b) at baseline (0), at the end of pegylated inter-

feron and ribavirin treatment (1) and at both 6-month (2)

and 1-year follow-up (3).



in SCCA-IgM IC serum levels. Patients with liver cirrhosis

were selected as it was anticipated that their SCCA-IgM IC

serum levels should have been elevated [11,12], and liver

cirrhosis rather than chronic hepatitis is a risk factor for HCC

in patients with HCV infection.

In this study, we observed that serum SCCA-IgM IC levels

were elevated in the majority of patients, with no statistically

significant difference between baseline values of SVRs and

nonresponders to treatment. We observed that serum SCCA-

IgM IC levels decreased during antiviral therapy and

continued to decrease during both 6-month and 1-year

follow-up. However, the main result of our study was that

the drop in serum SCCA-IgM IC levels was statistically

significant in patients who obtained a SVR to antiviral

therapy alone. Indeed, at the end of treatment, serum SCCA-

IgM IC levels were already significantly reduced when

compared to baseline in SVR patients but not in nonre-

sponders. Furthermore, at both 6- and 12-months follow-up,

SCCA-IgM IC serum levels significantly declined further

when compared to pretreatment values in SVR patients

alone, while in nonresponders there was no significant

modification respect to baseline. This reduction in SCCA-IgM

IC levels was not associated with modification in amino-

transferase and a-fetoprotein, and therefore cannot be

interpreted as a by-product of decreased necroinflammatory

activity. Although SCCA-IgM IC serum levels tended to be

lower both during treatment and follow-up in SVR when

compared to nonresponders, the results of our study show

that its levels cannot be used to predict response to PEG-IFN

and ribavirin therapy, at least in patients with cirrhosis. HCC

was diagnosed in three cirrhotic patients during follow-up,

and SCCA-IgM IC levels showed a constant increase during

both treatment and follow-up in the two NR patients who

developed HCC, while in the SVR patient who did develop

HCC SCCA-IgM IC levels were already high at baseline and

showed a blunted decline during successful antiviral treat-

ment. Noteworthy, in these three patients, serum a-feto-

protein levels did not follow the same behaviour but rather

showed either a decreasing pattern or were consistently

within the normal range. These findings further support the

possible use of serum SCCA-IgM IC levels for the identifica-

tion of cirrhotic patients at risk of developing HCC [9–11]. A

limitation to this study is the fact that SCCA-IgM IC serum

levels at the diagnosis of HCC were not available.

In summary, we have observed that antiviral therapy with

PEG-IFN and ribavirin determines a significant decrease in

SCCA-IgM IC serum levels of cirrhotic patients with HCV,

and most importantly we have shown that this decrease is

significant in patients who obtain a SVR to antiviral treat-

ment alone. Noteworthy, we have found that SCCA-IgM IC

serum levels do not predict response to antiviral therapy, at

least in cirrhotic patients. Due to the relatively small number

of events observed, it was not possible to draw firm conclu-

sions on the association between development of HCC and

behaviour of SCCA-IgM IC serum levels during treatment.

Larger studies are needed so as to better evaluate the

relationship between longitudinal modification of serum

SCCA-IgM IC levels and development of HCC in cirrhotic

patients with HCV infection.

CONFLICT OF INTEREST

No conflicts of interest exist.

FINANCIAL SUPPORT

None.

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SCCA-IC serum levels are predictive of clinical responsein HCV chronic hepatitis to antiviral therapy: a multicentricprospective studyE. Fransvea,1 P. Trerotoli,2 R. Sacco,3 V. Bernabucci,4 M. Milella,5 N. Napoli,1 A. Mazzocca,1

E. Renna,1 M. Quaranta,6 G. Angarano,5 E. Villa,4 S. Antonaci1 and G. Giannelli1 1Department of

Emergency and Organ Transplantation, Section of Internal Medicine Allergology and Immunology; 2Department of Biomedical Science and Human

Oncology, Section of Medical Statistics; University of Bari Medical School, Bari, Italy; 3Department of Gastroenterology, Pisa University Cisanello

Hospital, Pisa, Italy; 4Department of Internal Medicine, Gastroenterology Unit, University of Modena & Reggio Emilia Italy; 5Department of

Interdisciplinarity Medicine, Section of Infectious Diseases; University of Bari Medical School, Bari, Italy; and 6Department of Experimental Oncology,

Laboratory of Analyses; National Cancer Institute, Bari, Italy

Received December 2011; accepted for publication February 2012

SUMMARY. The combination of pegylated interferon (Peg-

IFN) and ribavirin is currently the gold standard therapy in

patients with HCV chronic infection. The duration of ther-

apy, as well as the therapeutic dosage, depend on the

genotype. Identification of the genotype and rapid virological

response (RVR) are widely accepted as the most important

predictors of clinical outcome during antiviral therapy but to

optimize cost-benefits and to reduce possible side effects,

further prognostic factors are needed. Squamous cell carci-

noma antigens immunocomplex (SCCA-IC) has been

reported to be increased in the serum of patients with liver

cancer. In this multicentric prospective study, we investi-

gated the serum levels of SCCA-IC in 103 patients with HCV

chronic infection. Serum HCV-RNA was detected before the

beginning of treatment, after 4, 12, 24 or 48 weeks, and at

week 24 during follow-up. RVR, early virological response

and sustained virological response (SVR) were assessed fol-

lowing the international guidelines. SCCA-IC levels were

higher in responders (238 AU, interquartile difference 130–

556 AU) and decreased significantly to 125 AU (70–

290 AU). The mean baseline value in nonresponders was

149 AU (86.5–306.5 AU), but after 4 weeks of treatment

the serum levels decreased to 115 AU (80–280 AU): the

profile of reduction was different between patients with or

without a positive SVR. Logistic regression with SVR as

dependent variable identified as significant independent

variables: the reduction in SCCA-IC after 1 month (OR =

4.82; 95% CI 1.39–16.67; P = 0.131) and a genotype other

than 1 (OR = 0.094; 95% CI 0.21–0.42; P = 0.002); sex

and age were also significant factors influencing SVR. SCCA-

IC seems to be a reliable independent prognostic marker of

therapeutic effectiveness in anti-HCV positive patients

undergoing antiviral therapy.

Keywords: antiviral therapy, chronic C hepatitis, prognostic

factor, SCCA-IC.

INTRODUCTION

The gold standard treatment for chronic hepatitis C, to

prevent or delay progression to liver cirrhosis and hepato-

cellular carcinoma (HCC), is currently the combination of

pegylated interferon-a (Peg-IFN-a) with ribavirin [1,2]. The

HCV genotype and a rapid virological response (RVR) have

been widely recognized as the two most important prog-

nostic factors for the response to antiviral therapy [3,4]. The

international guidelines for the treatment of HCV patients

have suggested that these prognostic factors may be usefully

applied to tailor the therapeutic regimen and optimize the

cost-benefit ratio [5]. Nevertheless, additional prognostic

factors are urgently needed in clinical practice.

Recently, in a preliminary study, it was reported that serum

levels of squamous cell carcinoma antigen immuno-complex

(SCCA-IC) were decreased in patients with HCV-related cir-

rhosis who responded to antiviral therapy [6]. However, the

limited number of patients so far investigated, and the lack of

studies in patients with chronic HCV-related hepatitis do not

Abbreviations: EVR, early virological response; HCC, hepatocellular

carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency

virus; PCR, polymerase chain reaction; Peg-IFN, pegylated interferon;

RVR, rapid virological response; SCCA, squamous cell carcinoma

antigens; SCCA-IC, squamous cell carcinoma antigens immunocomplex;

SS, systemic sclerosis; SVR, sustained virological response.

Correspondence: Gianluigi Giannelli, MD, Department of Emergency

and Organ Transplantation, Section of Internal Medicine Allergol-

ogy and Immunology. Padiglione Morgagni, Policlinico, Piazza G.

Cesare 11, 70124 Bari, Italy. E-mail: [email protected]

Journal of Viral Hepatitis, 2012 doi:10.1111/j.1365-2893.2012.01604.x


allow definite conclusions to be drawn as regards the validity

of this test in patients undergoing antiviral therapy. In par-

ticular, there are no data available correlating the use of

SCCA-IC with RVR, widely recognized as the gold standard

marker of clinical response to antiviral therapy.

SCCA-IC is an immuno-complex whereby IgM immuno-

globulins link the serin inhibitor protease SCCA [7,8]. SCCA

was first reported to be expressed in the liver of HCC patients

and for this reason, was proposed as a potential biomarker

for the detection of HCC [9]. SCCA-IC serum levels have been

demonstrated to show a better diagnostic capacity than

SCCA and have therefore been proposed for use in combi-

nation with alpha-fetoprotein as an additional biomarker for

HCC detection [10].

Aim of this study was to test the utility of SCCA-IC as a

marker of response in patients with HCV chronic infection

undergoing antiviral therapy. A multicentric prospective

study was designed, in which serum samples were collected

at baseline before starting the therapy, at RVR, early viro-

logical response (EVR) and 6 months after the end of ther-

apy, and a cohort of patients with HCV chronic infection

undergoing antiviral therapy were enrolled.

MATERIALS AND METHODS

Patients

During the period 2007–2010, 103 patients referred to the

following centres: Unit of Internal Medicine �C. Frugoni� and

of Infectious Diseases, University of Bari; Unit of Gastroen-

terology, University of Pisa; and Unit of Gastroentorology,

University of Modena And Reggio Emilia were enrolled in

this study.

Patients with liver cirrhosis, haematological abnormalities

(haemoglobin level <12 g/dL in women and <13 g/dL in

men; neutrophil count <1.5 · 103 cells/mL; platelet count

<90 · 103 cells/mL), pre-existing severe psychiatric condi-

tions (especially depression), severe cardiac disease, haemo-

globinopathies, haemophilia, autoimmune diseases, human

immunodeficiency virus (HIV) co-infection, previous liver

transplantation and other causes of liver disease (hepatitis B

virus infection, alcohol dependence) were excluded. Women

unable or unwilling to practise contraception were also excluded.

Study design

In total, 103 patients were recruited, 61 men and 42

women. Mean age was 53.4 year (±12.7 year) in men and

58 year (±9.5 year) in women, with a significant difference

between the two groups (t-test = 2.05; P = 0.04).

Blood samples were collected at baseline before the

beginning of therapy, after 4, 12, 24 and 48 weeks during

treatment and at 24 weeks of follow-up. The study was not

supported by any pharmaceutical company. Informed con-

sent was obtained from all patients.

Patients were treated with weekly subcutaneous pegylated

interferon (Peg-IFN)-a2a (180 lg/week) or Peg-IFN-a2b

(1.5 lg/kg/week) plus oral ribavirin at a dosage of 800–

1200 mg/day depending on pretreatment body weight

(800 mg/day for weight <60 kg, 1000 mg/day for weight

‡ kg and <75 kg; and 1200 mg/day for weight ‡75 kg).

This study was not randomized, and the physicians in

charge of the patients chose the Peg-IFN at their own dis-

cretion. Patients with genotype 2 and 3 were treated for

24 weeks, and those with genotype 1 and 4 for 48 weeks.

Virological response was evaluated at weeks 4, 12, 24 and

48 during treatment and at 24 weeks of follow-up by

qualitative PCR (Amplicor; Roche Diagnostic System,

France), with a sensitivity of 50 UI/mL. HCV genotype was

determined before treatment in all patients with the INNO-

LiPA HCV II kit (Bayer Diagnostics, Emeryville, CA, USA).

Sustained virological response (SVR) was defined as the

absence of detectable HCV-RNA in serum by qualitative PCR

at the end of therapy and at week 24 of follow-up. Early

virological response was defined as undetectable serum HCV-

RNA or a reduction in HCV-RNA levels by at least 2 logs

from baseline values at week 12 of treatment. Rapid viro-

logical response, was defined as undetectable serum

HCV-RNA at week 4 of treatment. Patients with measurable

HCV-RNA by qualitative PCR at the end of the follow-up

period were considered nonresponders.

SCCA-IC measurement

SCCA-IC serum concentrations were determined in sera

previously collected and stored at )20 �C until use. Serum

concentrations of SCCA-IC were measured as in our previous

works, using ELISA commercial kits purchased from Xept-

agen (Padua, Italy).

Statistical analysis

Categorical variables are summarized as count and per-

centage. Chi-squared test was used to evaluate differences

between independent groups. Comparisons were made by

parametric tests if normally (Gaussian) distributed, non-

parametric tests otherwise. Quantitative variables are sum-

marized as mean and standard deviation. Differences of

SCCA-IC, lacking a Gaussian distribution, are summarized as

median and interquartile range and comparison was per-

formed by Kruskal–Wallis for comparing multiple indepen-

dent groups or Friedman test for nonparametric repeated

measure analysis of variance. Multiple comparisons between

paired or independent groups were performed using Wilco-

xon test and P-values were adjusted taking into account the

number of comparisons according Bonferroni.

The percentage variation, determined as the ratio between

the difference of SCCA-IC at time t0 less SCCA-IC at time t-1,

divided by SCCA-IC at time t-1, was calculated to evaluate

the reduction in SCCA-IC concentrations between two


2 E. Fransvea et al.

consecutive time points. The percentage variation was then

classified in two classes according to the median value. A

logistic regression model was built to evaluate the effect on

SVR of the class of SCCA-IC reduction (more than 12% after

the first month and after the third month, more than 7% at

the last follow-up visit), genotype (1 vs other), adjusted for

age (classified as <58 vs ‡58 year) and sex (M vs F).

To evaluate the concordance of percentage of SVR patient

predicted by SCCAIC classes and prediction of SVR by RVR

and EVR was performed the McNemar test to compare paired

percentage. A P-value <0.05 was considered statistically

significant. All analyses were performed with software SAS

9.2 for PC (SAS Institute, Cary, NC, USA).

RESULTS

There were 63 responders (Table 1); mean age was 53.5

year (±11.9 year) for responders and 58.3 year (±10.7 year)

for nonresponders; this difference resulted statistically

significant (t-test = 2.1; P = 0.038) (Fig. 1). There were no

statistically significant differences in the percentage of

responders between men and women: 62.3% (38/61) vs

59.5% (25/42) (v2 = 0.08; P = 0.7767).

Genotype 1 was observed in 41.3% (26/63) of responders,

vs 90% (36/40) of non responders: the percentage of geno-

type 1 was significantly different between responders and

nonresponders (v2 = 24.24; P < 0.0001). The median value

of SCCA-IC by genotype and response is shown in Table 2

and profile by time of genotype 1 patients is shown in Fig. 2.

Responders with all genotypes had higher levels of SCCA-IC

than nonresponders. Furthermore, in responders, the level of

SCCA-IC showed a tendency to decrease, whereas in non-

responders the SCCA-IC level appeared to remain the same at

each time point. The median level of SCCA-IC at baseline

resulted lower respect to overall median value, but profile for

responders patients resulted similar (like a parallel line) until

the last determination, where values for genotype 1 and

overall sample resulted the same. Nonresponders patients

with genotype 1 had identical value respect to overall sam-

ple, because the most part of nonresponders patients was

genotype 1.

The initial value of SCCA-IC (Fig. 2) in responders was

238 AU (130–556), this decreased to 188 AU (116–400)

after 1 month, to 134 AU (90–385) after 3 months and

reached the value 125 AU (70–290) at the end of follow-up.

In nonresponders, the initial value was 149 AU (86.5–

306.5) and remained substantially unchanged after 1 month,

142.5 AU (82.5–286.5), showing a slight decrease after

3 months 115 AU (80–280) and remaining at this level

until the end of follow-up: 119 AU (82–260). The difference

in concentration between responders and nonresponders at

the first month resulted statistically significant (P = 0.0031),

while at the other time points it was not significant

(P > 0.05). The decrease between consecutive time points

resulted statistically significant (P < 0.001) in the respond-

ers group: the baseline-first month difference was 43 AU

(15–101); first – third month, 38 AU (10–68); third month

– end of the study, 14 AU (3–45). In the nonresponders

group, the decrease at consecutive time points resulted sig-

nificant between baseline-first month (5 AU; 0–14.5;

P < 0.001), but not at subsequent time points, when the

reduction was 0.5 AU (0–12.5) and 0 AU (9 to )8). Median

reduction resulted significantly different between responders

and nonresponders (P < 0.0001 at each comparison) at

every time point. These results allowed us to conclude that

the profile of reduction was different between patients that

would achieve a positive SVR as compared to nonresponders.

To evaluate the effect of SCCA-IC reduction in SVR, a lo-

gistic regression model was built with the presence of SVR as

dependent variable, while the independent variables were

the class of SCCA-IC reduction after 1, 3 months and at

the end of follow-up, genotype 1 vs others, sex (M vs F)

and age (<58 vs ‡58). The model resulted statistically

significant (Table 3; P < 0.0001). Sex was not shown to be

a statistically significant variable, while age younger

than 58 increased the probability of achieving SVR

(OR = 9.66; CI95% 2.21–42.15; P = 0.0025). An OR =

0.094 (CI95% 0.21–0.42; P = 0.002) was shown for

Table 1 Main characteristics of study sample for sustained

virological response (SVR) and nonresponders patients

SVR

N = 63

NO SVR

N = 40 P-value

Age (SD) 53.5 year (11.9) 58.3 year (10.7) 0.0386

Sex

Male 38 (60.3%) 23 (57.5%) 0.07767

Female 25 (39.7%) 17 (42.5%)

Genotype

1 26 (41.3%) 36 (90.0%) <0.0001

2 24 (38.1%) 3 (7.5%)

3 10 (15.9%) –

4 3 (4.7%) 1 (2.5%)

Table 2 Median (interquartile range) of SCCA-IC according

to genotype and response at the end of the study period

Genotype 1 Other Genotype

SVR YES

Baseline 205.5 (126–370) 307 (140–570)

First month 179.5 (118–303) 200 (110–448)

Third month 132 (90–204) 153 (81–406)

End of follow-up 124.5 (72–200) 156 (67–340)

SVR NO

Baseline 149 (85.5–300) 240 (102.5–409)

First month 142.5 (82.5–271.5) 232.5 (91.5–409)

Third month 115 (80–255) 231 (90–399.5)

End of follow-up 114 (82–250) 224 (99–344)


SCCA-IC serum levels are predictive of clinical response 3

genotype 1, suggesting that with this genotype patients are

more likely to be nonresponders. Predictive factors of SVR

were the reduction after first month, after the third month

and at follow-up. No effect of interaction between time points

and genotype resulted statistically significant, and this

parameter was therefore removed from the model. Looking

at the odds ratio, it seems that a reduction of more than 12%

between the baseline value and the first time point increases

by 4.82-fold the probability of SVR (95% CI 1.39–16.67;

P = 0.131). A reduction between the first month and third

month of more than 12% increases the probability of SVR

15-fold (3.44–66.74; P = 0.0003), and a further reduction

of more than 7% until the end of the study period increases

the probability of SVR 9-fold (95% CI 2.22–42.15;

P = 0.043).

Taking into account only genotype 1 patients reduction in

SCCA-IC between first month and baseline resulted with a

borderline significativity (OR = 3.65, CI95% 0.97–13.76,

P = 0.055), while reduction between third month and first

month remain statistically significant (OR = 9.09, CI95%

2.085–39.69, P = 0.003).

Figure 3 shows the probability of response for each value

of percentage reduction in SCCA-IC between baseline and

the first month for the whole sample and for genotype 1

Total sample103

Responders56

Nonresponders

47

Responders53

Nonresponders

33

Responders50

Nonresponders

31

Responders14

Nonresponders

3

Responders11 + 2

Nonresponders

3 + 3 + 3

First month

Third Month

End offollow-up

Connector to responder statusConnector to non responder status

Fig. 1 Patients course from the study

start to the end of follow-up.

500

600 Responders

500

600 Non responders

300

400

300

400

Ove

rall

100

200

100

200

0 0

600 Responders 600 Non responders

300

400

500

300

400

500

Gen

otyp

e 1

100

200

100

200

0 0

Baseline 1 month 3 month 12 month Baseline 1 month 3 month 12 month

Baseline 1 month 3 month 12 month Baseline 1 month 3 month 12 month

Fig. 2 Median and interquartile range

of SCCA-IC concentrations at each

follow-up time point. In responders, the

median level at each time point was less

than the median at the previous time,

while in nonresponders the median level

was approximatively the same at each

time point.



subgroup: the probability of SVR obtained with the univar-

iate logistic model (independent variable SVR Y/N and

dependent percentage reduction in SCCA-IC as continuous

variable) is increased by more than 50% for small levels of

reduction starting from 7% to 8% as compared to the

baseline value. For genotype 1, subgroup instead the prob-

ability of being a responder patient at the end of follow-up

resulted more than 50% for higher value of reduction (at

least 15%). Positive predictive value of SVR of SCCA-IC

reduction was 69.8% (44 with reduction / 63 SVR patients),

while RVR 79.4% (50 RVR/63 SVR) and EVR 96.8% (61

EVR/63 SVR). There was a statistically significant difference

between predictive value of class of SCCA-IC and EVR

(McNemar test 17.0, P < 0.0001), but not respect to RVR

(McNemar test 1.8, P = 0.179). This results are confirmed

in the subgroup of genotype 1 patients: predictive value of

class of SCCA-IC reduction was 65.4% (17 with reduction /

26 SVR), while for RVR was 69.3% (18/26), that resulted

not significantly different (P = 0.76) and for EVR 96.1%

(25/26) significantly different respect to class of SCCA-IC

(P = 0.0047) (Table 4).

DISCUSSION

In this study, we have demonstrated that a decreased serum

concentration of SCCA-IC after 4 weeks of antiviral therapy

is an independent prognostic factor of therapeutic response.

We base this conclusion on the following data: (i) the

reduction in SCCA-IC was significantly different between

patients achieving SVR or not; (ii) the profile of SCCA-IC is

different between responders and nonresponders even with

different genotypes; (iii) If the reduction is evaluated as

percentage, the effect of the decrease between baseline and

the first month value becomes even more noticeable.

The model evaluating the effect of predictors such as

genotype and SCCA-IC reduction at each time point was

shown to be well able to predict the response. The reduction

at the first time point seems to be a good predictor with low

variability, and the probability allows us to conclude that if a

reduction is observed at the first follow-up visit the patient

will achieve a positive clinical response.

The genotype shows a strong influence on obtaining a

SVR, but does not affect the conclusions about SCCA-IC, that

can therefore be considered a useful marker for monitoring

patients when measured at each visit: before starting ther-

apy and after the first and second follow-up visit. Thus, if

Table 3 Odds ratio, 95% confidence intervals and P-value

obtained with the multivariate logistic regression model

Variable OR CI 95% P-value

Genotype 1 vs other 0.09 0.02–0.42 0.002

Reduction at

first month <12%

4.82 1.39–16.67 0.0131

Reduction at

third month<12%

15.16 3.44–66.74 0.0003

Reduction at the end

of study period <7%

4.08 1.04–15.97 0.0432

Age < 58 year vs older 9.66 2.21–42.15 0.0025

1

0.9

0.8

0.6

0.7

0.4

0.5

Prob

abili

ty S

VR

Overall baseline vs 1 month

Genotype 1 baseline vs 1 month

Genotype 1 1 month vs 3 month

0.2

0.3

0

0.1

–0.4 –0.2 0 0.2 0.4 0.6 0.8

Percentage variation of SCCAIC

Fig. 3 Probability of being a responder

as a function of the SCCA-IC reduction.

The x-axis shows the percentage vari-

ation obtained as SCCA-IC value at

baseline less the value at the first

month divided by the baseline value.



SCCA-IC serum levels reduce during therapy, the patient will

achieve SVR. Should be noticed that genotype 1 subgroup

has less probability to be a responder, but even in this group

if a reduction in SCCA-IC level is observed a SVR could be

obtained.

In a previous work by Giannini et al., [6] the authors

reported that a higher SCCA-IC, but not SCCA, serum con-

centrations reduction in patients with liver cirrhosis was

observed in responders to antiviral therapy. Probably

because of the small sample size, they could not make an

appropriate test of the significance of the reduction with

respect to the future response. Moreover, in that work the

genotype was not evaluated together with SCCA-IC in a

multivariate model. In this article, instead, the authors

focused on the definition of a parameter to predict SVR, so

both genotype and repeated reductions of the marker have

been evaluated in a multivariate model. This confirmed the

importance of the reduction of the marker, more than the

value of the marker itself, as an independent variable, spe-

cifically with respect to the genotype. Our data demonstrate

that patients with SVR have a decreasing profile according to

genotype. This suggests that SVR could be predicted by

genotype, as already known, but that important information

on a positive response can be gained if there is a reduction

more than 12% at the first month as compared to the

baseline value.

Therefore, an antigen immuno-complex such as SCCA-IC

can predict the clinical outcome. It remains to be seen what

molecular mechanism is related to the decrease in SCCA-IC,

to explain its significance as a prognostic factor.

In other studies, SCCA-IC was reported as a new bio-

marker for early detection of HCC. However, while the sen-

sitivity performance was very encouraging, the specificity

was disappointing. In particular, patients without HCC but

with underlying chronic liver disease had unexplainably

high serum levels of SCCA-IC [8,11]. We hypothesize that

SCCA-IC could be related to the liver fibrosis/inflammation

status [12]. In fact, we detected increased levels of SCCA-IC

in patients with systemic sclerosis (SS). In particular. among

these patients, we found that those with fibrotic involvement

of the lung displayed the highest serum levels of SCCA-IC, as

compared with the other subsets of SS patients with a dif-

ferent clinical outcome [13]. Therefore, it is likely that SCCA-

IC serum levels reflect the fibrosis-immunity-related status,

which could explain why this parameter is a prognostic

factor independently of the genotype or the RVR.

In conclusion, we report a new prognostic factor that can

predict the therapeutic response in patients with chronic

hepatitis C undergoing antiviral therapy. SCCA-IC can be

easily measured using a commercially available ELISA kit.

Finally, we suggest that being an independent prognostic

factor, SCCA-IC may be useful to refine patients selection,

discriminating those patients that will benefit from antiviral

therapy and thus optimizing the cost-benefit ratio.

ACKNOWLEDGEMENT

This study was supported by the Italian Ministry of Health

(grant to GG number RFPS-2006-2-340979. 08).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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Seeff LB. Diagnosis, management,

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ML, Messinger D, Zeuzem S. Rapid

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Table 4 Comparison of positive predictive value (SVR) and negative predictive values (Not SVR) between reduction in SCCA-IC

respect to rapid virological response (RVR) and early virological response (EVR) patients

Reduction of

SCCA at first

month <12% RVR P-value EVR P-value

Overall sample

SVR (n = 63) 44 69.8% 50 79.4% 0.1797 61 96.8% <0.0001

Not SVR (n = 40) 9 22.5% 6 15.0% 0.3173 6 15.0% 0.1797

Genotype 1 subgroup

SVR (n = 26) 17 65.4% 18 69.2% 0.763 25 96.2% 0.0047

Not SVR (n = 36) 8 22.2% 4 11.1% 0.1573 4 11.1% 0.045



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determines a significant decrease in

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associated (SCCA) variants� serum

levels in anti-HCV positive cirrhotic

patients. J Viral Hepat 2010; 17:

563–568.

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et al. Squamous cell carcinoma

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hepatocellular carcinoma. Cancer

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et al. Progressive increase of SCCA-

IgM immune complexes in cirrhotic

patients is associated with develop-

ment of hepatocellular carcinoma.

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tiers in biomarkers for hepatocellular

carcinoma. Dig Liver Dis 2006; 38:

854–859.

10 Giannelli G, Fransvea E, Trerotoli P

et al. Clinical validation of combined

serological biomarkers for improved

hepatocellular carcinoma diagnosis

in 961 patients. Clin Chim Acta

2007; 383: 147–152.

11 Beale G, Chattopadhyay D, Gray J

et al. AFP, PIVKAII, GP3, SCCA-1

and follisatin as surveillance bio-

markers for hepatocellular cancer in

non-alcoholic and alcoholic fatty

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12 Trerotoli P, Fransvea E, Angelotti U

et al. Tissue expression of Squamous

Cellular Carcinoma Antigen (SCCA)

is inversely correlated to tumor size

in HCC. Mol Cancer 2009; 8: 29.

13 Giannelli G, Iannone F, Fransvea E,

Chiala A, Lapadula G, Antonaci S.

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Abstracts of the 19th National Congress of Digestive Diseases / Digestive and Liver Disease 45S (2013) S55–S218 S217

median overall stiffness was 5.2 kPa (4.35–6.15) [lcSSc=5.8 (4.4–6.5); dcSSc=4.7 (4.3–5.8); p=0.310]. A good correlation between stiffness and durationof disease was found (r=0.49, p=0.01), without differences between the formsof disease. In particular, the stiffness raises of 0.2 kPA every year of disease inboth forms. No correlation was found between TRAIL levels and stiffness orHGF values (p=ns). A good correlation was found between HGF values withduration of disease and stiffness in both forms of SSc (p<0.01), especially indcSSc patients.Conclusions: Increasing of stiffness value in pair with the duration of diseasesuggests a possible evolution of hepatic fibrosis in SSc patients. HGF valuescorrelate with duration of disease and stiffness in both forms of SSc, and inparticular in dcSSc patients.

P.18.21

ENHANCED LIVER FIBROSIS (ELF) TEST: A PROSPECTIVESTUDY OF NON-INVASIVE DIAGNOSTIC METHOD OF LIVERFIBROSIS

R. Catanzaro∗ ,1, M. Milazzo 1 , S. Arona 1, A. Italia 1, M. Cannavò 1,N. Bellavia 1 , D. Vasta 2, D. Arcoria 3

1UOC di Medicina Interna “A. Francaviglia”, AOU “Policlinico-VittorioEmanuele”, Presidio “G. Rodolìco”, Insegnamento di Gastroenterologia,Università degli Studi di Catania, Catania, Italy; 2MedLab - Analisi Cliniche,Catania, Italy; 3Specialista convenzionato sul territorio in Diabetologia eMalattie del Ricambio, Paternò, Catania, Italy

Background and aim: Fibrosis prediction is an essential part of the assess-ment and management of pts with chronic liver disease. The ELF Test consistsof an algorithm of three fibrosis markers: hyaluronic acid, amino-terminalpropeptide-of-type-III-collagen and inhibitor of matrix-metalloproteinase-1.Aim of our study was to evaluate a serological marker method-ELF Test in theassessment of liver fibrosis, comparing with liver biopsy.Material and methods: We included 31 pts (14 M – 45%, 17 F – 55%; mean± SD ages 52±11 yrs) with chronic C hepatitis (n=12/31 – 39%), chronicB hepatitis (n=10/31 – 32%) and non-alcoholic steatohepatitis (n=9/31-29%).A percutaneous liver biopsy specimen was obtained from all pts. Liver fibro-sis stages were evaluated according to the Metavir scoring-system (F0–F4).Serum samples were analysed using the proprietary assays developed forELF Test by Siemens Healthcare Diagnostics Inc. Results were entered intothe established algorithm and expressed as discriminant scores for a com-parison to Metavir histological staging. Liver fibrosis was classified in mild(ELF score <7.7), moderate (ELF score 7.7–9.8) and severe fibrosis (ELFscore ≥9.8). Statistical analysis was performed by evaluating area-under-the-ROC-curves (AUROC), sensitivity (Se), specificity (Sp), positive- (PPV) andnegative-predictive-values (NPV).Results: We found the following distribution by Metavir: F1=7, F2=11, F3=4,F4=9. The ELF Test diagnosed mild fibrosis in 7 pts, moderate fibrosis in17 pts and severe fibrosis in 7 pts. The accuracy according to AUROC forthe diagnosis of significant fibrosis (F ≥2; ELF score ≥7.7) was 0.869 (95%CI 0.69–0.96; cut-off=8.49; p=0.0001), Se=0.87 and Sp=0.56, PPV=0.87 andNPV=0.57. The accuracy for the diagnosis of cirrhosis (F=4; ELF score ≥9.8)was 0.995 (95% CI 0.81–0.99; cut-off=9.03; p=0.0001), Se=0.66 and Sp=0.95,PPV=0.85 and NPV=0.87.Conclusions: The ELF Test showed better efficacy in highlighting the pres-ence of cirrhosis than in discriminating intermediate stages of liver fibrosis.Liver biopsy is still the gold standard in staging fibrosis. The ELF Test couldbe employed for a preliminary selection of patients eligible for biopsy andcould be used during their follow-up.

P.18.22

SERUM FGF19 LEVELS ARE INDEPENDENTLY RELATED TO BMIIN HEALTHY BLOOD DONORS: AN INTERIM ANALYSIS OF ANONGOING STUDY

R. Arena∗ , P. Cecinato, A. Lisotti, F. Azzaroli, M. Montagnani, P. Simoni,A. Porro, S. Belvisi, C. Calvanese, C. Colliva, A. Belardinelli, F. Buonfiglioli,P. Pagliaro, A. Roda, G. Mazzella

University of Bologna, Bologna, Italy

Background and aim: Fibroblast growth factor 19 (FGF19) is an enterokineplaying key roles in enterohepatic signalling, bile acid (BA) synthesis, gall-bladder motility and metabolic homeostasis. Aim of our study was to evaluatethe correlation between serum fasting FGF19 and glucose, lipid metabolismand BMI in healthy subjects.Material and methods: 285 blood donors were prospectively enrolled fromour Transfusion Center, from January 2011 to March 2012. Exclusion criteriawere: increased ALT, γGT or ALP, history of liver, GI or gallstone disease,previous abdominal surgery and treatment with metabolic or GI medications.All patients underwent lab-tests: fasting glucose and insulin, total cholesterol,HDL, LDL, triglycerides, ALT, γGT, ALP, fasting FGF19 serum level (ELISAassay), serum BA levels (HPLC-ESI-MS/MS in 150 subjects). Student t-testwas used for the comparison of groups; multivariate analysis was used toidentify variables independently related.Results: 279 subjects (153M/126F; age 41.6±11.6 years) met the inclu-sion criteria. Mean BMI was 25.1±3.7 (male 25.6±3.1; female 23.9±3.9P=0.0001); mean fasting FGF19 was 124.8±84.2 pg/ml. Fasting serum FGF19levels were significantly higher in subjects with BMI<25 (144.4±106.6) vsoverweight subjects (102.9±76; P<0.001). FGF19 was inversely correlatedwith BMI (r= –0.245; P<0.0001). 52 subjects presented insulin-resistance(HOMA-R ≥2.5). Insulin resistance was not correlated with FGF19 level(131.7±102.9 no IR vs 112.6±89.1 IR subjects). Serum cholesterol andtriglycerides did not correlate with FGF19 serum levels. On multivariateanalysis, FGF19 was independently related to BMI (r=–7.78; P<0.001).Conclusions: Serum FGF19 was significantly lower in otherwise healthyoverweight subjects and a linear inverse correlation between BMI and serumFGF19 was observed. The mechanisms responsible for these findings areprobably related with a different bile acid homeostasis, and deserve furtherinvestigation.

P.18.23

SCCA-IgM: A BIOMARKER TOMONITOR THE OUTCOME OFTHERAPYWITH SORAFENIB IN ADVANCED HCC

F. Morisco ∗ ,1, G.G. Di Costanzo 2 , M. Guarino 1, R. Tortora 1, I. Loperto 1,F. Auriemma 1, C. Ferraiuoli 1 , C. Tuccillo 3 , L. Beneduce 4 , N. Caporaso 1

1Department of Clinical and Experimental Medicine, Gastroenterology Unit,University of Naples “Federico II”, Naples, Italy; 2Liver Unit, AORN“A.Cardarelli”, Naples, Italy; 3Department of Clinical and ExperimentalMedicine “F. Magrassi and A. Lanzara”, Gastroenterology Unit, SecondUniversity of Naples, Naples, Italy; 4Xeptagen SpA, Marghera, Venice, Italy

Background and aim: Circulating Squamous Cell Carcinoma Antigene(SCCA)-IgM complex has been found higher in patients with hepatocellularcarcinoma (HCC) than in patients with chronic liver disease and cirrhosis.This study aimed to evaluate the ability of SCCA-IgM serum levels to monitorthe efficacy of therapy with Sorafenib in patients with HCC.Material and methods: Forty-two patients with a new diagnosis of HCC(stage B and C, according to BCLC score) were enrolled in a prospectivestudy from April 2011 to July 2012 in two referral centres. The diagnosisof HCC was made according to guidelines AASLD 2010. All patients weretreated with Sorafenib (800 mg/day). Response to therapy was evaluated withimaging techniques according to the mRECIST criteria. Serum SCCA-IgMlevels were determined by the Hepa-IC kit (Xeptagen SpA, Marghera, Venezia,Italy) at basal time and after 3 months from the beginning of treatment. Thequantization of the complex SCCA-IgM is expressed in Arbitrary Units(AU)/ml.Results: At basal time SCCA-IgM were detectable (>100 AU/ml) in serum of

Andrea

Rectangle

Andrea

Rectangle

S218 Abstracts of the 19th National Congress of Digestive Diseases / Digestive and Liver Disease 45S (2013) S55–S218

25/42 (59.5%) patients. Successively, the patients were divided in two groups:Group A (16 patients) who responded to therapy and Group B (9 patients)who did not respond to therapy. In Group A the mean value decreased from276.75 AU/ml at T0 to 190.3 AU/ml at T3 (p<0.001). In Group B mean valueremained stable during the follow-up (252.4 AU/ml at T0 and 285.2 AU/ml atT3, p not significant).Conclusions: These results suggest that the assessment of SCCA-IgM may behelpful in monitoring the outcome of HCC treatment with Sorafenib.

P.18.24

CIRCULATING SCCA-IgM COMPLEX IS ABLE TOMONITORTHE SUCCESS OF LOCO-REGIONAL THERAPY IN HCCPATIENTS

F. Morisco ∗ ,1, G.G. Di Costanzo 2 , M. Guarino 1, R. Tortora 2, I. Loperto 1,E. Clery 1, F. Auriemma 1, C. Tuccillo 3 , L. Beneduce 4 , N. Caporaso 1

1Department of Clinical and Experimental Medicine, Gastroenterology Unit,University of Naples “Federico II”, Napoli, Italy; 2Liver Unit, AORN“A.Cardarelli”, Napoli, Italy; 3Department of Clinical and ExperimentalMedicine “F. Magrassi and A. Lanzara”, Gastroenterology Unit, SecondUniversity of Naples, Napoli, Italy; 4Xeptagen SpA, Marghera, Venezia, Italy

Background and aim: About 3–4% of cirrhotic patients develops primaryliver cancer every year. The serpin squamous cell carcinoma antigen (SCCA)

is elevated in liver cancer specimens and circulating SCCA-IgM complexeshave been described in patients with hepatocellular carcinoma (HCC). Thisstudy aimed to evaluate the ability of SCCA-IgM serum levels to monitor theefficacy of loco-regional treatments.Material and methods: In two referral centres of South Italy, sixty-fourpatients with a new diagnosis of HCC at stage A and B, according to BCLCscore, were enrolled in a prospective study from April 2011 to July 2012. Thediagnosis of HCC was made according to the AASLD 2010 guidelines.All patients underwent loco-regional treatments such as Laser Thermal Ab-lation (LTA), Radio-Frequency (RF), Percutaneous Ethanol Injection (PEI),Transarterial Chemoembolization (TACE). Response to therapy was evaluatedwith imaging techniques according to the mRECIST criteria.Serum SCCA-IgM levels were determined by the Hepa-IC kit (Xeptagen SpA,Marghera Venezia, Italy) at basal time and after 1 month from the beginningof treatment. The quantization of the complex SCCA-IgM is expressed inArbitrary Units (AU)/ml.Results: At basal time SCCA-IgM were detectable (>100 AU/ml) in serumof 34/64 (53%) patients. These patients were divided in two groups: Group A(29 patients) who responded to therapy and Group B (5 patients) who didn’trespond to therapy. In Group A the mean value decreased from 263.83 AU/mlat T0 to 229.7 AU/ml at T1 (p<0.05). In Group B mean value increased from226.2 AU/ml at T0 to 273 AU/ml at T1 (p = not significant).Conclusions: These results suggest that the assessment of SCCA-IgM may behelpful in monitoring the outcome of loco-regional treatments in patients withHCC.

HEPATOLOGY

Diagnostic and prognostic role of SCCA-IgM serum levels inhepatocellular carcinoma (HCC)Caterina Pozzan,* Romilda Cardin,* Marika Piciocchi,* Nora Cazzagon,* Gemma Maddalo,*Veronica Vanin,* Anna Giacomin,* Patrizia Pontisso,† Umberto Cillo* and Fabio Farinati*

Departments of *Surgery, Oncology and Gastroenterology and †Clinical and Experimental Medicine, University of Padova, Padova, Italy

Key words

diagnosis, hepatocellular carcinoma,prognosis, SCCA-IgM, serological biomarkers.

Accepted for publication 18 February 2014.

Correspondence

Fabio Farinati, Department of Surgery,Oncology and Gastroenterology, PaduaUniversity, Policlinico Universitario, ViaGiustiniani 2, IT–35128 Padova, Italy. Email:[email protected]

AbstractBackground and Aim: The serpin squamous cell carcinoma antigen complexed with IgM(SCCA-IgM) has been reported as a promising serological marker for hepatocellularcarcinoma (HCC). We aimed to further evaluate SCCA-IgM diagnostic accuracy and todetermine its prognostic role.Methods: SCCA-IgM levels were determined in 327 sera obtained from 81 HCC patients,206 cirrhotics and 40 healthy blood donors (controls). Sensitivity, specificity, correlationwith clinical and tumor parameters and with survival were evaluated.Results: HCC patients had SCCA-IgM levels significantly higher than controls and cir-rhotics (P < 0.0001). Sensitivity, specificity, positive and negative predictive values forHCC were 89%, 50%, 41% and 92%, respectively. In comparison, sensitivity and speci-ficity for alphafetoprotein were 48% and 85%. SCCA-IgM levels were not significantlycorrelated with clinical or biological variables. With a cut-off of 130 AU/mL (receiveroperating characteristic curves), SCCA-IgM proved efficient in the prediction of prognosis,identifying the patients with long overall survival (efficiency validated in the homoge-nous subgroup of patients with intermediate-stage HCC undergoing transarterialchemoembolization) and predicting progression-free survival. A Cox multivariate analysisconfirmed SCCA-IgM predictive value, identifying tumor size and SCCA-IgM levels asindependent predictors of survival. A reduction in SCCA-IgM levels correlated withresponse to treatment.Conclusions: SCCA-IgM is a sensitive marker of HCC in patients with cirrhosis eventhough lacking in specificity. The determination of the levels of the marker in HCC patientsis highly efficient in predicting the patients’ prognosis, identifying those with long overalland progression-free survival and the responders and should be introduced in the clinicalpractice.

IntroductionConventional biomarkers, including alphafetoprotein (AFP) anddes-gamma-carboxyprothrombin (DCP), are of limited help inthe diagnosis of hepatocellular carcinoma (HCC) and only par-tially useful in the definition of the patients’ prognosis.1 AFP,however, is still considered by some authors2,3 as a clinicallymeaningful instrument for HCC surveillance and diagnosis. Thisis based on the results of two randomized surveillance studiescarried out in China, in which AFP determination led to a higherpercentage of patients diagnosed at an early stage4 and, associ-ated with ultrasound (US), reduced mortality.5 From the prog-nostic point of view, AFP has recently shown a relevantprognostic impact in specific settings, such as in patients on thewaiting list for liver transplantation6 or in predicting response inpatients undergoing loco-regional treatment.7 However, in a

study based on the results obtained in a series of 1158 patientswith HCC, we confirmed not only the low sensitivity of AFP inthe diagnosis of HCC but also its limited prognostic value, sincea multivariate analysis did not identify AFP as an independentpredictor of survival.1

The discovery of a novel class of tumor markers constituted bycirculating IgM antibodies forming immune complexes with spe-cific cancer biomarkers has provided interesting opportunities forHCC patient management.8–19 IgM immune complexes can bedetected in the serum of HCC patients as reported for squamouscell carcinoma antigen (SCCA),9–13,15 for AFP15–18 and for DCP,19

and the assessment of AFP-IgM, DCP-IgM and SCCA-IgMimmune complexes has allowed a higher diagnostic performancethan the determination of the free, not complexed, biomarker.According to the revised hypothesis of cancer immune surveil-lance,20 these immune-complexed biomarkers might be involved in

doi:10.1111/jgh.12576

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cancer immune-editing, reflecting the host immune-protectivemechanisms, aimed at suppressing tumor growth.

SCCA-IgM was recently shown to be a predictive biomarker ofHCC risk in patients with cirrhosis, because it was found that anincrease in time of serum SCCA-IgM levels is associated with ahigher incidence of HCC.12 SCCA-IgM also showed a prognosticrole in patients with chronic HCV-related hepatitis in whom themarker predicted histologic progression13 and response to inter-feron treatment.14 In this study, we evaluated the efficiency of thedetermination of serum SCCA-IgM levels in identifying patientswith HCC and defining their prognosis, in search of a biomarker tobe used to modulate treatment aggressiveness, in parallel with theBarcelona Clinic Liver Cancer (BCLC)/American Association ofthe Study of Liver Diseases (AASLD) treatment and prognosticalgorithm.21

MethodsThe study was retrospectively performed on sera prospectivelycollected from 327 patients providing informed consent to blooddrawing, consecutively recruited between 2005 and 2009. Theseries included 81 HCC patients, 206 cirrhosis patients withoutHCC, and 40 blood donors as a healthy control group. Sera fromcirrhotics were obtained at our outpatient clinic from patients withchronic liver disease fulfilling the following inclusion criteria:

• International normalized ratio (INR) higher than 1.20;• White blood cell (WBC) lower than 4.40 × 109 /L;• Platelets (PLTs) below 150 × 109 /L; (at least two out of three

criteria);• US examination showing findings compatible with cirrhosis:

coarse liver structure, irregular profiles, dilated portal tract,reduced flow velocity, enlarged spleen diameter (at least threeout of five criteria).

The absence of HCC was confirmed by routine US examination (atthe time of entry in the study and every six months), with aone-year negative follow-up.

Sera from HCC patients were obtained from patients admittedin our unit for treatment purposes. The diagnosis of HCC wasbased on the 2005 AALSD guidelines,22 although, for a localpolicy, almost always (96%) finally biopsy-confirmed, with themain lesion or the most accessible and “safe for biopsy” lesionbeing selected for histological sampling. The clinical features ofpatients with HCC and with cirrhosis are shown in Tables 1 and 2,respectively.

As shown by a preliminary Kolmogorov–Smirnov test, the dis-tribution of the data was not normal (P < 0.0001) and non-parametric tests were therefore used throughout the study. Thediagnostic accuracy of SCCA-IgM was evaluated by the receiveroperating characteristic (ROC) method and the area under thecurve (AUC) calculated. With respect to the prognostic accuracy,the HCC study population was arbitrarily subgrouped into long-term and short-term survivors (> or < than 36 months). This choicewas supported by the fact that in patients in BCLC/AASLD inter-mediate stage, that represented the majority of our sample, theexpected survival is calculated at 36 months, and ranges between20 to 40%. The clinical features of the two subgroups of patientsare shown in Table 3.

The prognostic cut-off was again established with the ROCmethod at the value that maximized sensitivity and specificity.Overall survival (OS) curves were calculated by Kaplan–Meiermethod and compared by the log–rank test. The prognostic accu-racy of SCCA-IgM levels was then evaluated, restricting the analy-sis only to patients with “intermediate stage” HCC whounderwent transcatheter arterial chemoembolization (TACE),carried out using the standard selective/superselective methodwith lipiodol (5–10 mL) and epirubicin (10–40 mg). To furtherevaluate the prognostic value of SCCA-IgM, the correlation

Table 1 Clinical, biological and tumoral features of patients with hepa-tocellular carcinoma (HCC)

n %

Gender Male 63 78Female 18 22

Mean age (years) 66 ± 11Status at the end of follow-up Death 56 69

Alive 25 31Etiology HBV 9 11

HCV 41 51Alcohol 18 22Mixed 7 9Others 6 7

Histology No 3 4Yes 78 96

Child–Pugh A 54 67B 26 32C 1 1

AFP(ng/mL) < 20 42 52≥ 20 e < 200 24 30≥ 200 15 18

Number of nodules ≤ 3 58 72> 3 and ≤ 5 16 20> 5 7 8

Maximal size (cm) ≤ 3 53 65> 3 and ≤ 5 17 21> 5 11 14

Portal thrombosis No 76 94Yes 5 6

Metastasis No 80 99Yes 1 1

Edmonson’s grading 1 42 542 34 443–4 2 2

First treatment TACE 49 60Surgery 2 3Percutaneous ablation 19 23Supportive cares 11 14

CLIP score 0 15 181 41 512 20 253–4 5 6

BCLC staging very early/early 21 26intermediate/advanced 60 74

HBV, hepatitis B virus; HCV, hepatitis C virus; Mixed, HBV+HCV,HBV+alcohol, HCV+alcohol; Others, primary biliary cirrhosis, hemochro-matosis, cryptogenic.

SCCA-IgM in hepatocellular carcinoma C Pozzan et al.

1638 Journal of Gastroenterology and Hepatology 29 (2014) 1637–1644


between SCCA-IgM levels and the progression-free survival(PFS) was calculated considering only patients in whom treatment(TACE) was effective (i.e. those with at least partial response, nostable disease, or disease progression at the one-month CT scan-ning, according to modified Response Evaluation Criteria In SolidTumors [RECIST] criteria23). OS and PFS curves were calculatedby Kaplan–Meier method and compared by the log–rank test.Finally, in a subgroup of 22 homogenous patients more recentlytreated by drug eluting beads-TACE (DEB-TACE), response totreatment was correlated with trends in time of SCCA-IgM levels,in samples obtained the day of treatment and the day of control CTscanning, performed as a routine four weeks later (chi-squaredanalysis).

The correlation between SCCA-IgM levels and the followingvariables was evaluated: number of nodules, tumor size (diameterof the largest lesion on CT scanning/nuclear magnetic resonance(NMR), grading according to Edmonson,24 Child–Pugh status (A,B, C25), AFP values (< 20, 20–200, > 200 U/mL), BCLC staging(very early/early vs intermediate/advanced), presence or absenceof thrombosis and metastasis. The impact of the same variablesand of SCCA-IgM levels (> /< than the prognostic cut-off) onsurvival was first evaluated with univariate analysis; only the vari-ables that were statistically significant or borderline in the univari-ate analysis (P < 0.1) were included in the Cox regression analysisto finally establish the independent predictors of survival. A Pvalue < 0.05 was held as significant.

SCCA-IgM assay. The serum levels of SCCA-IgM immunecomplexes were assessed by commercial ELISA Kit (Hepa-IC,Xeptagen, Xeptagen SpA, Marghera, Venice, Italy) according tothe manufacturer’s instructions. The amount of SCCA-IgMimmune complexes was expressed in arbitrary units (AU)/mL byinterpolation of samples absorbance on the calibration curvesplotted with SCCA-IgM calibrators. Briefly, plates precoated withpolyclonal anti-human SCCA antibody were incubated with100 μL of serum diluted 1:8 in the dilution buffer. SCCA-IgMimmune complexes were revealed by the addition of secondary

antibody and ABTS chromogen solution. All reagents were pro-vided in the kit and results were processed with XEREPRO soft-ware (Xeptagen).

Results

Diagnostic accuracy. Controls had median SCCA-IgMlevels (40 AU/mL, C.I. 31–65) significantly lower than both cir-rhotics and HCC patients (P < 0.0001). The difference betweenmedian SCCA-IgM levels in the two last groups of patients (cir-rhosis 90.5 AU/mL [C.I.80–111.2], HCC 130 [C.I. 121–170]), washighly statistically significant (P = 0.0001). Considering the cut-off for SCCA-IgM obtained by the ROC curves (89 AU/mL, AUC66%), the levels of the immune complex were increased in 89%(72/81) of HCC patients and in 50% (104/206) of cirrhotics.

Sensitivity and specificity of SCCA-IgM determination forHCC were therefore 89% and 50% respectively, with a positivepredictive value (PPV) and a negative predictive value (NPV) of41% and 92%. On the other hand, sensitivity and specificity forHCC of AFP, considering the established normality cut-off of20 U/mL, were 48% and 85% respectively, with a PPV and a NPVof 71% and 68%; only 15/81 HCC patients (18%) presented AFPlevels higher than the generally accepted diagnostic cut-off(200 U/mL).

Combining the two markers together (i.e., considering as diag-nostic either SCCA-IgM > 89 AU/mL or AFP > 20 U/mL, orboth) the sensitivity reached 94%, but at the cost of a furtherreduction in specificity (both markers negative), which dropped to45% (Table 4), with a PPV of 40% and NPV of 95%. Using adiagnostic AFP cut-off of 200 U/mL (Table 5), the gain in speci-ficity was not substantial (from 45% to 50%).

Survival prediction. The three-year survival of all the HCCpatients, irrespective of the treatment they underwent, was 36%(median survival time 29 months, confidence interval [C.I.] 24–34).The AUC of SCCA-IgM in the prediction of survival was notparticularly high (AUC = 0.63), the cut-off being fixed at 130 AU/mL. With the above cut-off, SCCA-IgM presented a 67% sensitivityand a 49% specificity in the prediction of long-term survival.

The survival curves of the two subgroups (SCCA-IgM levelslower or higher than the cut-off) again irrespectively of the treat-ment, calculated by the Kaplan–Meier/log–rank tests, divergedstarting at 24 months from diagnosis, with a clear-cut and signifi-cant difference (P = 0.018). The 48-month survival was 40% inpatients with SCCA-IgM < 130 AU/mL versus 7% in those withSCCA-IgM > 130 AU/mL. Median survival was 48 months (CI29–66) for patients with SCCA-IgM < 130 AU/mL and 26 months(CI 22–30) for those with SCCA-IgM > 130 AU/mL. Similarly,the hazard plot, describing the risk of death in the two groups,showed a forceps opening at 24 months (Fig. 1).

The Kaplan–Meier curves obtained considering only thepatients with intermediate stage HCC who underwent TACEshowed an analogous trend: median survival was 30 months (CI16–44) for patients with SCCA-IgM < 130 AU/mL and 20 months(CI 13–27) for those with SCCA-IgM > 130 AU/mL, the differ-ence being statistically significant (log–rank P = 0.039).

The PFS curves (Fig. 2) calculated by the Kaplan–Meier/log–rank tests in the 18 intermediate stage patients (out of 49, 37%)

Table 2 Clinical and biological features of patients with cirrhosis

n %

Gender Male 140 68Female 66 32

Mean age (years) 57 ± 11Etiology HBV 39 19

HCV 124 60Alcohol 16 8Mixed 25 12Cryptogenic 2 1

Child–Pugh A 146 71B 58 28C 2 1

AFP(ng/mL) < 20 175 85≥ 20 e < 200 31 15≥ 200 0 0

C Pozzan et al. SCCA-IgM in hepatocellular carcinoma



who showed a radiologic complete response to TACE treatment,confirmed the prognostic value of SCCA-IgM: median PFS was 14months (C.I. 11–17) for patients with SCCA-IgM < 130 AU/mLand 6 (C.I. 5–7) for those with SCCA-IgM > 130 AU/mL, a dif-ference that was highly statistically significant (log–rankP = 0.003).

In the subgroup of 22 patients treated by DEB-TACE, a drop inSCCA-IgM levels significantly correlated with a complete orpartial tumor response, as assessed by the CT scanning per-formed four weeks following treatment (P = 0.0039)(Table 6).Patients with stable disease or tumor progression had always stableor increased levels of the marker at four weeks, while patientswith complete response always showed a reduction. Patients

with partial response had an intermediate behavior. In the sameseries, AFP did not predict complete response (data notshown).

Univariate and multivariate analysis. Only diagnosis(Cirrhosis/HCC) was significantly correlated with SCCA-IgMlevels (Rho = 0.167, 0.010–0031 95% CI, P = 0.037, Spearman’srank correlation analysis). In HCC, none of the clinical variableswas significantly correlated with SCCA-IgM levels, includingChild–Pugh status or AFP levels (P = 0.975), in the univariateanalysis.

At univariate analysis tumor size, number of nodules, Child–Pugh status, BCLC staging and SCCA-IgM were significantly

Table 3. Clinical, Biological and Tumoral Features of Patients With Hepatocellular Carcinoma Subgrouped on the Basis of Scca-IgM PrognosticCut-Off (>/< 130 AU/ml)

SCCA IgM< 130 AU/ml (40)

SCCA IgM> 130 AU/ml (41)

n % n %

Child–Pugh A 36 90 18 44 Chi2 = 24.224781p < 0.0001B 4 10 22 54

C 0 0 1 1AFP(ng/ml) < 20 28 70 14 34 Chi2 = 11.256037

p = 0.0036≥ 20 e < 200 6 15 18 44≥ 200 6 15 9 22

Number of nodules ≤ 3 35 87 23 56 p = n.s.> 3 and ≤ 5 5 13 11 26> 5 0 0 7 18

Maximal size (cm) ≤ 3 30 75 23 56 p = n.s.> 3 and ≤ 5 7 18 10 24> 5 3 7 8 20

Portal thrombosis No 37 93 39 95 p = n.s.Yes 3 7 2 5

Metastasis No 39 99 41 100 p = n.s.Yes 1 1 0 0

Edmonson’s grading 1 22 55 21 52 p = n.s.2 18 45 18 443–4 0 0 2 4

BCLC staging Very early/early 12 30 9 22 p = n.s.Intermediate/advanced 28 70 32 78

n.s., not significant.

Table 4 Combination of SCCA-IgM and AFP testing. The diagnosticcut-off of SCCA-IgM and AFP were 89 AU/mL and 20 U/mL respectively

Serological diagnosis Clinical diagnosis

SCCA-IgM AFP HCC (81) Cirrhosis (206)

n % n %

+ + 35 43 22 10+ − 37 46 82 40+ +/− 72 89 104 50− + 4 5 9 4Overall (+/+, +/−, −/+) 76 94 113 55− − 5 6 93 45

Table 5 Combination of SCCA-IgM and AFP testing. The diag-nostic cut-off of SCCA-IgM and AFP were 89 AU/mL and 200 U/mLrespectively

Serological diagnosis Clinical diagnosis

SCCA-IgM AFP HCC (81) CIRRHOSIS (206)

n % n %

+ + 14 17 0 0+ − 58 72 104 50+ +/− 72 89 104 50− + 1 1 0 0Overall (+/+, +/−, −/+) 73 90 104 5− − 8 10 102 50




associated with survival (Table 7); in particular, AFP was not evenclose to be significantly associated with survival (P = 0.45). Whenthe patients were subgrouped according to their SCCA-IgM levels(< or > the cut-off), patients with lower levels of the marker weresignificantly more frequently Child–Pugh A and with lower levelsof AFP (see Table 3).

The Cox multivariate analysis showed that, amongst the fiveparameters, tumor size (P < 0.001, [HR] 2.47, C.I. 1.31–3.61) andSCCA-IgM levels (P = 0.004, Hazard ratio [HR] 2.34, C.I. 1.28–3.64), selected in this order in the “forward conditional” model,were identified as independent predictors of survival.

0 20 40 600

1

2

3

Haz

ard

HAZARD PLOT

Time (months)

SURVIVAL PLOT (PL ESTIMATES)

0 20 40 600,00

0,25

0,50

0,75

1,00

Su

rviv

or

Time (months)

Figure 1 Survival and hazard plot for the two subgroups of patients: in black, those with SCCA-IgM > 130 AU/mL; in grey, those with SCCA-IgM< 130 AU/mL. The curves of survival and hazard diverge starting at 24 months. , < 130 AU/mL; , > 130 AU/mL.

Figure 2 Progression-free survival (PFS) curves for the two subgroupsof intermediate-stage patients treated by TACE: in black, those withSCCA-IgM > 130 AU/mL; in grey, those with SCCA-IgM < 130 AU/mL.Patients with SCCA-IgM levels below the cut-off have a significantlylonger PFS. , < 130 AU/mL; , > 130 AU/mL.

Table 6 Correlation between trend in time of SCCA-IgM levels andresponse to DEB-TACE

Disease Stable/progression

Partialresponse

Completeresponse

SCCA-IgM levels =/>† 6 (100%) 6 (55%) 0 (0%)SCCA-IgM levels <‡ 0 (0%) 5 (45%) 5 (100%)

P = 0.0039, chi square 11.090.†=/> no variation or increase.‡< reduction.

Table 7 Significant predictors of survival: the univariate analysis

Variables Hazard ratio C.I. P value

Tumor size 3.61 1.77–4.81 0.001Number of nodules 2.00 0.99–2.48 0.093Child–Pugh status 2.02 1.06–3.75 0.031BCLC Staging 1.41 0.94–2.10 0.088SCCA-IgM 2.12 1.08–3.85 0.017




Discussion

The search of reliable and efficient biomarkers for a diagnostic andprognostic evaluation of HCC is still an open issue. This search isclearly justified by the fact that the discovery of a biomarkerefficient enough, both in terms of sensitivity and specificity, toavoid performing the often long list of examinations required for adiagnosis of HCC (US, CT scanning, NMR, liver biopsy) woulddramatically cut costs and risks involved in the diagnostic algo-rithm. AFP is still universally used, although several studies haverepeatedly demonstrated that both the sensitivity and the specific-ity of the marker are not high enough to justify its routine use,26

despite some recent attempt to rehabilitate the marker.2,3 Betterperformances are provided by AFP in defining the prognosis ofHCC patients, and for instance the marker is used as a prognostictool in one of the more widely adopted staging systems for HCC,the Cancer of the Liver Italian Program (CLIP) score,27,28 as well asin the post-treatment evaluation, because it correlates with radio-logic response, disease-free and overall survival.29–31 However, ona large scale, even the prognostic definition in the individualpatient lacks adequate efficiency,1 as we also recently confirmed.32

As reported, immunohistochemical studies recently demonstratedan overexpression of SCCA variants (SCCA-1, SCCA-2 e SCCA-PD) in HCC tissues.33 SCCA is a member of the serine protease(serpin) inhibitors, and “in vivo” and “in vitro” studies demonstratethat it has a role in cancer development and progression because itinterferes with apoptosis, prompts tumor growth34 and promotesepithelial-to-mesenchymal transition, thus facilitating tumorinvasion.35 SCCA expression is an early event in hepato-carcinogenesis with a progressive increase of expression in cirrho-sis, dysplastic nodes and HCC,9,36,37 this increase heralding, incirrhotics, HCC development.12

Our study confirms, at least in part, the results obtained with theuse of SCCA-IgM in the diagnosis of HCC. With the indicatedcut-off, the marker showed a high (89%) sensitivity, with however,a relatively low specificity (50%), as previously reported also inother studies,38 and the increased SCCA-IgM levels in cirrhotics isjustified by the data obtained at immunohistochemistry.

Even though we do not support a diagnostic approach based onthe combination of multiple diagnostic markers, which inevitablyleads to associate an increased sensitivity to a lower specificity, itmust be reported that the association of SCCA-IgM and AFPdetermination (> 20 U/mL) raised the sensitivity to 94% (5%higher than with SCCA-IgM alone) with a specificity of 45% (5%lower than with SCCA-IgM alone) and a high NPV (95%). With adiagnostic AFP cut-off (> 200 U/mL), the sensitivity was basicallythe same than with SCCA-IgM alone.

In contrast with what was observed with respect to AFP,1

SCCA-IgM serum levels do not correlate with the patients’ tumorburden, in terms of number and size of the nodules, nor theyappear to depend on tumor grading, as already reported37 or withthe Child–Pugh status or the BCLC/AASLD stage. It seems there-fore that the determinants for the expression of the marker are notlinked to the extent of the neoplastic involvement or with the liverfunction. They appear to rather reflect the intrinsic biologicalactivity of the tumor and/or the extent of the patients’ immuneresponse.

If several studies already reported the diagnostic role of SCCA-IgM, much less information is available on its potential prognostic

role. We subgrouped the series of HCC patients on the basis of thecut-off identified by using the ROC curves (SCCA-IgM >/<130AU/mL) with as the end-point, a short (<36 months) or long (>36months) survival. As said, the 36 months survival limits was arbi-trarily chosen, but this time point is routinely utilized in the BCLC/AASLD guidelines to define the survival of HCC patients in theintermediate stage, that were, in this study, the majority of thesample.

The difference in survival between patients with SCCA-IgMlevels higher or lower than the cut-off was statistically significant(P = 0.018), with a median survival in patients with SCCA-IgM < 130 AU/mL almost twice as high than in those with SCCA-IgM above the cut-off (48 vs 26 months). It is interesting to notethat the survival curves of the two groups of patients remainsubstantially similar up to 24 months, to then diverge very clearlyafter that time point, to suggest that SCCA-IgM levels reflect abiologic feature potentially related to more aggressive livertumors. Analogous results were obtained if considering onlypatients in intermediate stage undergoing TACE, a homogenoussubgroup of HCC patients, thus confirming, with an internal vali-dation carried out in patients in a specific stage and undergoing aspecific treatment, the results obtained in the overall group. Addi-tionally, SCCA-IgM levels determination showed a very clearcorrelation with PFS in patients with complete response, with amedian time to progression of 14 months in patients with lowversus six months in those with high SCCA-IgM levels. Again, thispoints to either a different biologic behavior of the tumor, with ahigher aggressiveness mediated by the effect of the serpin ontumor growth and invasiveness,34,35 or to an adaptive immuneresponse in patients with different levels of expression of thebiomarker. The antigenic changes involved in malignant transfor-mation in cancer cells are indeed recognized by the immunesystem and trigger an immune response that is aimed at controllingtumor growth. The significant impact of SCCA-IgM determinationin defining the patients’ prognosis was confirmed, in this study,also by the data showing that, in the Cox analysis, SCCA-IgMlevels were identified as independent predictors of OS, with arelative weight just slightly lower than that of tumor size. Finally,even though these data are to be interpreted with caution, given thesmall sample size, SCCA-IgM determination also demonstrated toefficiently and significantly predict response to treatment since adrop in the marker at four weeks from DEB-TACE was onlyobserved in patient with complete or partial response while, con-versely, no patient with stable disease or progression showed areduction in SCCA-IgM levels, a predictive capacity not shown byAFP. A recent paper has described a prognostic impact of SCCAtissue expression in 61 patients with HCC in whom the expressionwas evaluated in either bioptic or surgical samples. The authors’findings indicated that the absence of SCCA expression was cor-related with a higher proliferation rate and was a significant inde-pendent negative predictor of survival.39 These findings are incontrast with our own results, but it must be kept in mind that nocorrelation between serum SCCA-IgM levels and SCCA tissueexpression has been documented in a study that specificallyaddressed the point37 and also that serum SCCA-IgM determina-tion reflects not only the tumor biology but, as already discussed,also the patients’ immune response.

In summary, SCCA-IgM sensitivity is, in this study, quiteencouraging, but the specificity is not high enough to justify the




use of the marker in HCC diagnosis, at least as a single marker.We, however, do not support the use of a “multiple-markers”approach that, in our mind, may ameliorate sensitivity, but with acost of an even lower specificity and with an increase in direct(tests) and indirect (imaging procedure-related) costs. Overall, therole of the marker is to be confirmed in larger, prospectivelycollected, series of patients. On the other hand, the performance ofSCCA-IgM in defining the prognosis of HCC patients is quiteclear in terms of OS, PFS and treatment response. Obviously,additional studies, which should be aimed at confirming our dataand analyzing the levels of the marker in relation also to differenttreatment methodologies on a larger patient sample, are againrequired.

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HEPA-IC Literature

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52. Biasiolo A, Tono N, Zaninotto M, Merkel C, Fassina G, Plebani M, Gatta A, Pontisso P. Specificity of squamous cell carcinoma antigen (SCCA)-IgM detection in patients with HCV infection and rheumatoid factor seropositivity. J Med Virol. 85:1005-8. 2013.

51. Morisco F, Di Costanzo G G, Guarino M, Tortora R, Loperto I, Auriemma F, Ferraiuoli C, Tuccillo C, Beneduce L, Caporaso N . SCCA-IgM: a biomarker to monitor the outcome of therapy with sorafenib in advanced HCC. Dig Liver Dis. 45:S217-18. 2013.

50. Morisco F, Di Costanzo G G, Guarino M, Tortora R, Loperto I, Clery E, Auriemma F, Tuccillo C, Beneduce L, Caporaso N. Circulating SCCA-IgM complex is able to monitor the success of loco-regional therapy in HCC patients. Dig Liver Dis. 45:S218. 2013.

49. Morisco F, Di Costanzo G G, Guarino M, Tortora R, Loperto I, Tuccillo C, Ferraiuoli C, Beneduce L, Auriemma F, Caporaso N. Su1001 Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCC Therapy. Gastroenterology. 144:S-991. 2013.

48. Biasiolo A, Tono N, Ruvoletto M, Quarta S, Turato C, Villano G, Beneduce L, Fassina G, Merkel C, Gatta A, Pontisso P. IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease. PLoS ONE. 7:e40658. 2012.

47. Buccione D, Fatti G, Gallotta A, Loggi E, Di Donato R, Testa L, Saitta C, Santi V, Di Micoli A, Erroi V, Fazio V, Picciotto A, Biasiolo A, Degos F, Pontisso P,

Raimondo G, Trevisani F. SCCA-IgM as predictor of hepatocellular carcinoma in patients with liver cirrhosis. OJGas. 2:56-61. 2012.

46. Fransvea E, Trerotoli P, Sacco R, Bernabucci V, Milella M, Napoli N, Mazzocca A, Renna E, Quaranta M, Angarano G, Villa E, Antonaci S, Giannelli G. SCCA-IC serum levels are predictive of clinical response in HCV chronic hepatitis to antiviral therapy: a multicentric prospective study. J Viral Hepat. 19:704-710. 2012.

45. Gallotta A, Orzes E, Fassina G. Biomarkers Quantification with Antibody Arrays in Cancer Early Detection. Clin Lab Med. 32:33-45. 2012.

44. Morisco F, Guarino M, Tortora R, Donnarumma L, Loperto I, Camera S, Mariniello A, Tuccillo C, Beneduce L, Di Costanzo GG, Caporaso N . Serum Levels of SCCA-IgM are related to the efficacy of HCC treatments. Dig Liver Dis. 44:S135. 2012.

43. Pozzan C, Cardin R, Pichiocchi M, Cazzagon N, Sergio A, Vanin V, Giacomin A, Pontisso P, Cillo U, Farinati F. Prognostic role of SCCA-IgM serum levels in hepatocellular carcinoma (HCC) . Dig Liver Dis. 44:S141-S142. 2012.

42. Zorzetto V, Maddalo G, Rugge M, Parente P, Zaninotto G, Ruol A, Biasiolo A, Zanatta L, Farinati F. SCCA-IgM determination in barrett's esophagus and esophageal cancer might regulate endoscopic follow-up schedule. Dig Liver Dis. 44:S151. 2012.

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40. Biasiolo A, Tono N, Briarava M, Beneduce L, Fassina G, Farinati F, Giacomin A, Matteucci C, Sorrentino R, Nitti D, Gatta A , Pontisso P. Improved diagnostic accuracy for HCC using a combination of IgM-linked biomarkers. Dig Liver Dis. 43:S91. 2011.

39. Buccione D, Fatti G, Gallotta A, Loggi E, Di Donato R, Testa L, Saitta C, Santi V, Di Micoli A, Fazio V, Picciotto A, Biasiolo A, Pontisso P, Raimondo G, Trevisani F. Serum SCCA-IgM as a predictor of hepatocellular carcinoma (HCC) in patients with liver cirrhosis. Dig Liver Dis. 43:S95. 2011.

38. Plebani M, Wu AH. Circulating macrocomplexes: old wine in new bottles?. Clin Chem Lab Med. 49:759-60. 2011.

37. Pozzan C, Cardin R, Cazzagon N, Picciocchi M, vanin V, Giacomin A, Sergio P, Pontisso P, Farinati F.

Diagnostic and prognostic role of SCCA-IgM in hepatocellular carcinoma (HCC). J Hepatol. 54: S101. 2011.

36. Zorzetto V, Rugge M, Parente P, Zaninotto G, Castoro M, Ruol A, Maddalo G, Zanatta L, Farinti F. SCCA-IgM and CEA-IgM in barrett's esophagus and esophageal cancers. Dig Liver Dis. 43:S156. 2011.

35. Giannini EG, Basso M, Bazzica M, Contini P, Marenco S, Savarino V, Picciotto A. Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen-associated (SCCA) variants' serum levels in anti-HCV positive cirrhotic patients. J Viral Hepat. 17:563-568. 2010.

34. Savitskaya Y, Rico G, Linares L, Gonzalez R, Tellez R, Estrada E, Marin N, Martinez E, Alfaro A, Ibarra C. Circulating Natural IgM Antibodies Against Angiogenin in the Peripheral Blood Sera of Patients with Osteosarcoma as Candidate Biomarkers and Reporters of Tumorigenesis. Biomark Cancer. 2:65-78. 2010.

33. Stefaniuk P, Janusz Cianciara J, Wiercinska-Drapalo A. Present and future possibilities for early diagnosis of hepatocellular carcinoma. World J Gastroenterol. 16:418-24. 2010.

32. Stefaniuk P, Krygier R, Dusza M, Jablonowska M, Mikula T, Dabrowska M, Cianciara J, Wiercinska-Drapalo A. Do we have more predictive HCC biomarkers? Usefulness of new generation of HCC biomarkers in immune complexes with immunoglobulins of the IgM class. Exp Clin Hep. 6:AB18-18. 2010.

31. Teofanescu I, Gologan E, Stefanescu G, Balan G. Surveillance of cirrhosis for hepatocellular carcinoma clinical validation of new serological biomarkers for improved hepatocellular carcinoma diagnosis. Rev Med Chir Soc Med Nat Iasi. 114:39-46. 2010.

30. Yim S, Chung Y. An Overview of Biomarkers and Molecular Signatures in HCC. Cancers (Basel). 2:809-23. 2010.

29. Zuin J, Veggiani G, Pengo P, Gallotta A, Biasiolo A, Tono N, Gatta A, Pontisso P, Toth R, Cerin D, Frecer V, Meo S, Gion M, Fassina G, Beneduce L. Experimental validation of specificity of SCCA-IgM assay in patients with cirrhosis. Clin Chem Lab Med. 48:217-23. 2010.

28. Biasiolo A, Tono N, Quarta S, Beneduce L, Gatta A, Fassina G, Pontisso P. SCCA1-IgM and SCCA2-IgM compared with total SCCA-IgM for diagnosis of hepatocellular carcinoma. Int J Biol Markers. 24:205. 2009.

27. Carrara S, Bhalla V, Stagni C, Benini C, Ferretti A, Valle F, Gallotta A, Riccio B, Samorì B. Label-free cancer markers detection by capacitance biochip.

Sens Actuators B Chem. 136:163-172. 2009.

26. Gallotta A, Pengo P, Beneduce L, Fassina G. Combinatorial approaches in cancer early detection. Drug Design and Discovery for Developing Countries (Eds. Megnassan E, Owono Owono L, Miertus S) ICS-UNIDO. 206-18. 2009.

25. Gallotta A, Zuin J, Veggiani G, Pengo P, Biasiolo A, Tono N, Gatta A, Pontisso P, Fassina G, Beneduce L. Combining SCCA-IgM and AFP-IgM levels increases accuracy of hepatocellular carcinoma detection. Dig Liver Dis. 41:A15. 2009.

24. Gallotta A, Zuin J, Veggiani G, Pengo P, Leon A, Gion M, Biasiolo A, Tono N, Gatta A, Pontisso P, Fassina G, Beneduce L. Combination of biomarkers-IgM by logistic regression improves diagnostic accuracy in hepatocellular carcinoma. Int J Biol Markers. 24:208. 2009.

23. Kumar M, Sarin SK. Biomarkers of diseases in medicine. Current Trends in Science. 403-417. 2009.

22. Turato C, Ruvoletto MG, Biasiolo A, Quarta S, Tono N, Bernardinello E, Beneduce L, Fassina G, Cavalletto L, Chemello L, Merkel C, Gatta A, Pontisso P. Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease. Dig Liver Dis. 41:212-6. 2009.

21. Vidalino L, Doria A, Quarta S, Zen M, Gatta A, Pontisso P. SERPINB3 apoptosis and autoimmunity. Autoimmun Rev. 9:108-12. 2009.

20. Zuin J, Veggiani G, Pengo P, Gallotta A, Biasiolo A, Tono N, Franzosi L, Pontisso P, Fassina G, Beneduce L. SCCA-IgM usefulness to monitor patients with cirrhosis is not affected by interfering IgM's. Dig Liver Dis. 41:A11. 2009.

19. Zuin J, Veggiani G, Pengo P, Gallotta A, Biasiolo A, Tono N, Gatta A, Pontisso P, Toth R, Cerin D, Frecer V, Meo S, Gion M, Fassina G, Beneduce L. Evaluation of analytical specificity of SCCA-IgM assay for monitoring patients with cirrhosis. Int J Biol Markers. 24:209. 2009.

18. Beneduce L, Pesce G, Gallotta A, Zampieri F, Biasolo A, Tono N, Pontisso P, Fassina G. Hepatocellular carcinoma induces specific IgM-biomarkers immune complexes. Dig Liver Dis. 40:A32. 2008.

17. Biasiolo A, Chemello L, Quarta S, Cavalletto L, Bortolotti F, Caberlotto C, Beneduce L, Bernardinello E, Tono N, Fassina G, Gatta A, Pontisso P. Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis. J Viral Hepat. 15:246-9. 2008.

16. Biasiolo A, Tono N, Quarta S, Beneduce L, Gatta A, Fassina G, Pontisso P. Squamous cell carcinoma antigen (SCCA)-IgM complex: SCCA1-IgM and SCCA-

IgM compared to total SCCA-IgM for diagnosis of hepatocellular carcinoma. Gut. 57:A147. 2008.

15. Cagol M, Ruol A, Biasiolo A, Zanchettin G, Tono N, Castoro C, Alfieri R, Pontisso P, Ancona E. Serum squamous cell carcinoma antigen-IgM (SCCA-IgM) immunocomplex: a new biomarker for cancer of the esophagus. Gut. 57:A113. 2008.

14. Giannelli G, Antonaci S. Hepatocellular carcinoma: tumoral or peritumoral disease?. Recenti Prog Med. 98:23-8. 2007.

13. Giannelli G, Fransvea E, Trerotoli P, Beaugrand M, Marinosci F, Lupo L, Nkontchou G, Dentico P, Antonaci S. Clinical validation of combined serological biomarkers for improved hepatocellular carcinoma diagnosis in 961 patients. Clin Chim Acta. 383:147-52. 2007.

12. Giannelli G, Iannone F, Fransvea E, Chialà A, Lapadula G, Antonaci S. Squamous cellular carcinoma immunocomplexed is increased in scleroderma patients with lung fibrosis. Clin Exp Rheumatol. 25:794-5. 2007.

11. Turato C, Ruvoletto M, Biasiolo A, Quarta S, Tono N, Cavalletto L, Chemello L, Merkel C, Gatta A, Pontisso P. HFE, TGF-BETA1 and squamous cell carcinoma antigen-1 polymorphisms and liver disease stage in chronic HCV infection. Dig Liver Dis. 39:A27. 2007.

10. Beneduce L, Gallotta A, Marino M, Fassina G. Improvement of sensitivity for liver cancer detection by simultaneous evaluation of SCCA-IgM, AFP-IgM complexes and free AFP. J Hepatol. 44:S97. 2006.

9. Giannelli G, Antonaci S. New frontiers in biomarkers for hepatocellular carcinoma. Dig Liver Dis. 38:854-9. 2006.

8. Pontisso P, Quarta S, Caberlotto C, Beneduce L, Marino M, Bernardinello E, Boscato N, Fassina G, Cavalletto L, Gatta A, Chemello L. Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma. Int J Cancer. 119:735-40. 2006.

7. Quarta S, Caberlotto C, Beneduce L, Marino M, Fassina G, Tono N, Cavalletto L, Chemello L, Gatta A, Pontisso P. Monitoring SCCA-IgM complex predicts HCC development in cirrhotic patients. J Hepatol. 44:S107. 2006.

6. Beneduce L, Castaldi F, Marino M, Pontisso P, Fassina G. Comparison of free and IgM-complexed squamous cell carcinoma antigen in hepatocellular carcinoma. J Hepatol. 42:89. 2005.

5. Beneduce L, Castaldi F, Marino M, Quarta S, Ruvoletto M, Benvegnù L, Calabrese F, Gatta A, Pontisso P, Fassina G. Squamous cell carcinoma antigen-immunoglobulin M complexes as novel

biomarkers for hepatocellular carcinoma. Cancer. 103:2558-65. 2005.

4. Quarta S, Caberlotto C, Beneduce L, Castaldi F, Marino M, Fassina G, Tono N, Cavalletto L, Chemello L, Gatta A, Pontisso P. Serum SCCA-IgM complexes increase over time and HCC development in cirrhotic patients. Dig Liver Dis. 37:A38-A39. 2005.

3. Beneduce L, Castaldi F, Marino M, Quarta S, Ruvoletto M, Pontisso P, Fassina G. Circulating squamous cell carcinoma antigen-IgM complexes as novel biomarkers for hepatocellular carcinoma. Dig Liver Dis. 36:A2-A3. 2004.

2. Beneduce L, Castaldi F, Marino M, Quarta S, Ruvoletto M, Pontisso P, Fassina G. Serological detection of squamous cell carcinoma antigen-IgM complexes in hepatocellular carcinoma. J Hepatol. 40:77. 2004.

1. Pontisso P, Fassina G, Quarta S, Ruvoletto M, Tono N, Castaldi F, Nigro A, Marino M, Beneduce L, Gatta A. Nanotecnologie nella diagnostica dell'epatocarcinoma. Atti del 14° Conv. Int. Attualità e Prospettive in Epatologia. 144-8. 2004.

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HEPA-IC · 2Xeptagen, Pozzuoli (Na), Italy 3Istituto Oncologico Veneto (IOV), Padova, Italy About...

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