Granulocyte Colony-StimulatingFactor-Producing Small-CellCarcinoma of the Uterine Cervix:Report of a CaseAkihiko Watanabe, M.D.,

1* Toshiki Wachi, M.D.,1 Hiroko Omi, M.D.,

1

Hiroshi Nishii, M.D.,1 Kazuhiko Ochiai, M.D.,

1 Tadao Tanaka, M.D.,2 and

Yasuhiko Endo, M.D.3

A case of granulocyte colony-stimulating factor (G-CSF)-produc-ing small-cell carcinoma of the uterine cervix is described. In a70-yr-old woman, clusters of small cells with hyperchromaticnuclei at a high nuclear/cytoplasmic ratio were detected cytolog-ically in the cervix. These clusters were diagnosed as a small-cellneuroendocrine carcinoma by the concomitant use of Grimeliusstaining and immunohistochemical staining, in addition to elec-tron microscopic observation. This patient showed a significantincrease in peripheral leukocytes, despite the absence of infectioussigns. Immunohistochemical findings, together with a high bloodG-CSF level, suggested the production of G-CSF from the tumor.Consistent with the knowledge that both small-cell carcinoma andG-CSF-producing tumors have a poor prognoses, the patient hadno or partial response to therapies performed, and died from thecancer 11 mo after it was diagnosed. This case strongly indicatesthe need for early diagnosis of this type of tumor, based oncytological features. Diagn. Cytopathol. 2000;23:269–274.© 2000 Wiley-Liss, Inc.

Key Words: small-cell carcinoma; neuroendocrine carcinoma;uterine cervix; G-CSF-producing tumor; cytological diagnosis

Small-cell carcinoma arising in the uterus is rare. To diag-nose this type of tumor, it should be definitely determined tohave not only morphological characteristics of a small-cellcarcinoma under light microscopy, but also characteristicfeatures of a neuroendocrine carcinoma. However, it is notalways easy to make an accurate diagnosis because of somedifficulties in histopathological classification. Small-cell

neuroendocrine carcinoma often progresses very rapidlyand resists therapy. We herein describe the cytological andhistopathological findings of small-cell carcinoma of theuterine cervix, occurring in a patient with marked peripheralleukocytosis. An immunohistochemical study indicated theproduction of granulocyte colony-stimulating factor (G-CSF) from the tumor.

Case ReportThe patient was a 70-yr-old female, gravida 1 para 1; sheunderwent menopause at age 53. Her medical history wasunremarkable. A history of the present illness follows.

Physical FindingsThe patient visited our hospital with the chief complaint ofgenital bleeding. Her uterus was found to be swollen, andinspection with a speculum revealed an invasive tumorarising in the cervix.

Cytology of the Uterine CervixIn cytological examination with Papanicolaou staining,dense clusters of small cells with a high nuclear/cytoplasmic(N/C) ratio, or lacking cytoplasm, were observed in a scat-tered presence (Fig. 1). The nuclei were hyperchromatic andshowed a coarse granular chromatin pattern with inconspic-uous nucleoli. In the cell clusters, cells were densely pro-liferative and exhibited nuclear molding (Fig. 2). Thesefindings indicated either squamous-cell carcinoma of thesmall-cell nonkeratinizing type, poorly differentiated ade-nocarcinoma, or small-cell carcinoma.

Histopathological FindingsIn histopathological observation of a hematoxylin-eosin(H&E)-stained biopsy specimen of the uterine cervix, tumorcells widely infiltrated the entire area of the specimen. The

1Department of Obstetrics and Gynecology, Jikei University School ofMedicine Aoto Hospital, Tokyo, Japan

2Department of Obstetrics and Gynecology, Jikei University School ofMedicine, Tokyo, Japan

3Department of Pathology, Jikei University School of Medicine, Tokyo,Japan

*Correspondence to: Akihiko Watanabe, M.D., Department of Obstet-rics and Gynecology, Jikei University School of Medicine Aoto Hospital,6-41-2, Aoto, Katsushika-ku, Tokyo 125-8506, Japan.E-mail: wtnb-khk@dd.iij4u.or.jp

Received 19 March 1999; Accepted 20 March 2000

© 2000 WILEY-LISS, INC. Diagnostic Cytopathology, Vol 23, No 4 269

tumor formed solid lesions, but partially showed diffuseinvasion into the stroma. No glandular structures were ob-served (Fig. 3). Individual tumor cells were small and hadround or elliptical nuclei, with a high N/C ratio (Fig. 4). Notendency toward keratinization was observed. Grimeliusstaining demonstrated argyrophilic granules (Fig. 5).

Hematological StudyWhite blood cells (WBC) and granulocyte counts weremarkedly increased, besides an increase in neuron-specificenolase (NSE) (see Table I). However, no clinical evidenceof infection or inflammation was noted. A blood G-CSFlevel was measured, due to suspicion of production ofG-CSF from the tumor, because the increased peripheralleukocytes were mainly composed of normal mature neu-trophils, and the G-CSF level was found to be as high as 269pg/ml (normal,,30). Because the cytological, histopatho-

logical, and hematological findings indicated that the tumorwas a small-cell neuroendocrine carcinoma with the abilityto produce G-CSF, we further examined this tumor withimmunohistochemical staining and electron microscopy, tomake a definite diagnosis.

ImmunohistochemistryA biopsy specimen was again obtained from the uterinecervix, fixed with buffered 10% formalin solution, andembedded in paraffin, to prepare sections for immunohisto-chemical study. The sections were stained by a streptavidin-biotin (SAB) method, using the following antibodies: rabbitanti-chromogranin A polyclonal antibody (Nichirei Co., To-kyo, Japan) and rabbit anti-NSE polyclonal antibody(Nichirei Co.), which had been diluted at time of purchase;rabbit anti-synaptophysin monoclonal antibody (BiodesignInternational, Kennebunk, ME), diluted at 1:5 with phos-phate-buffered saline (PBS); and mouse anti-G-CSF poly-

Fig. 1. Cytological specimen of the uterine cervix. Clusters of small cellswith a high N/C ratio, or lacking cytoplasm, are observed scattered on anecrotic background (Papanicolaou stain,320).

Fig. 2. Nuclei are hyperchromatic and show a coarse granular chromatinpattern. Cells are densely proliferated with nuclear molding (Papanicolaoustain,3100).

Fig. 3. Historical specimen obtained by biopsy of the uterine cervix.Tumor cells extensively infiltrate over the entire area of the specimen(H&E stain,310).

Fig. 4. Individual tumor cells are small, and they have a high N/C ratio andround or elliptical nuclei with nuclear molding (H&E stain,340).

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clonal antibody (Oncogene Research Products, Cambridge,MA), diluted at 1:100 with PBS.

The specimen was weakly positive for chromogranin Aand synaptopysin, but it was not stained strongly enough toensure its positivity for these markers, while it was deeplystained with NSE and G-CSF (Figs. 6, 7).

Electron Microscopic ObservationAfter a portion of the biopsy specimen was fixed with 2.5%glutaraldehyde and postfixed with 1% osmic acid, and em-bedded in epon resin, ultrathin sections of 60-nm thicknesswere prepared and observed under electron microscopy.Dark round granules, which measured 200–300 nm in di-ameter and resembled neuroendocrine granules, were spo-radically observed in the cytoplasm of tumor cells (Fig. 8).

Imaging FindingsOn CT and MRI, the uterus appeared swollen and had atumor in the cervix, which grew into the parametrium in aninfiltrative manner and formed a pyometra (Fig. 9). Retro-peritoneal nodes and mediastinal nodes were found to beswollen (Fig. 10), suggesting lymph node metastasis, whileno lesions were detected in the lung fields. Thus thesemetastatic lymph nodes were thought to be derived from thecarcinoma of the uterine cervix, and not from a primarypulmonary tumor. From these findings, the patient wasclassified as stage IVb.

Fig. 5. Grimelius-positive staining pattern (3100).

Table I. Laboratory Data Before Therapya

Hematological studyWBC 17,100/mlGranulocytes 83%Lymphocytes 11%Monocytes 5%Eosinocytes 1%Basocytes 0%Red blood cells 3303 104/mlHb 9.3 g/dlHt 28.3%Thrombocyte 44.13 104/mlBlood chemistryGOT 20 IU/lGPT 8 IU/lLDH 603 IU/lALP 312 IU/lBUN 16.7 mg/dlCr 0.5 mg/dlSerologic testCRP 0.8 mg/dlTumor markersCEA 79.1 ng/mlCA125 35 U/mlSCC 1.1 ng/ml (normal,,1.5)CA19-9 ,7 U/mlNSE 39.0 ng/ml (normal,,10.0)OthersACTH 18 pg/mlCortisol 21.0mg/dlG-CSF 268 pg/ml (normal,,30)aHb, hemoglobin; Ht, hematocrit; GOT, glutamic oxaloacetic transami-nase; GPT, glutamic pyruvic transaminase; LDH, lactate dehydrogenase;ALP, alkaline phosphatase; BUN, blood urea nitrogen; Cr, creatinine;CRP; C-reactive protein; SCC, squamous-cell carcinoma antigen.

Fig. 6. NSE-positive immunohistochemical staining pattern. Streptomycinbiotin complex method without counterstain (340).

Fig. 7. G-CSF-positive immunohistochemical staining pattern. Streptomy-cin biotin complex method without counterstain (340).

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Therapeutic CourseThe patient first received two courses of chemotherapy withcyclophosphamide, pirarubicin, and carboplatin (CAJ ther-apy). Although the mediastinal nodes diminished in size, theprimary lesion remained unchanged, and renal function wasimpaired. Thus the CAJ therapy was replaced by radiother-apy, which subsequently diminished the primary lesion.However, liver metastasis was detected during the oraltreatment with 5-fluorouracil following radiotherapy, andthe patient died due to multiple organ failure associated withtumor progression 11 mo after the diagnosis was made. HerWBC count increased to 124,400/ml, as measured on the dayshe died. In addition, her WBC and granulocyte counts de-creased or increased according to the diminution of lesionsby therapy or the appearance of recurrent lesions (Fig. 11).

DiscussionSquamous-cell carcinoma of the small-cell nonkeratinizingtype is representative of cancers that arise in the uterine

cervix and consist of small cells. A cancer composed ofsmall cells and that clinically shows rapid progression,leading to a poor prognosis, is traditionally classified as ofthe undifferentiated type. These cancers have a morphologysimilar to that of pulmonary small-cell carcinoma and areclassified as small-cell carcinoma.1–5 Small-cell carcinomaof the uterine cervix is a very rare tumor, accounting forabout 1% of all cervical carcinomas.4,6

On cytodiagnosis of small-cell carcinoma, small roundcells with a high N/C ratio, or lacking cytoplasm, are foundto form dense clusters that are observed to be scattered on anecrotic background. Although it may be difficult to distin-guish small-cell carcinoma of the cervix from squamous-cell carcinoma of the small-cell nonkeratinizing type, theformer tumor shows weaker cell linkage than the latter.Nuclei are hyperchromatic and often show a coarse granularchromatin pattern with inconspicuous nucleoli. Dense cellproliferation is observed in individual cell clusters, withnuclear molding as a characteristic feature. In histopatho-logical observation under light microscopy, small-cell car-cinoma of the cervix is composed of small cells with littlecytoplasm, like those observed at cytology, and it formspoorly defined trabeculae and clusters with marked stromalinfiltration. The cell border is not clearly observed. Otherthan these findings, it is important to demonstrate Grime-lius-positive histopathological findings3 and positive immu-nohistochemical staining with neuroendocrine markers,such as chromogranin A, synaptophysin, and NSE.7 Inaddition, roundish neuroendocrine granules, encircled witha limiting membrane, should also be demonstrated underelectron microscopy.8 Our case, however, was only weaklystained with chromogranin A and synaptopysin. Thisseemed to result from improper fixation conditions. Thuswe considered that our case was a neuroendocrine tumor,based on the NSE-positive result and electron microscopicfindings.

From the viewpoint of the functional types of tumors, ithas been reported that small-cell carcinoma of the uterus

Fig. 10. Horizontal section of the chest on CT: mediastinal nodes areswollen (arrows), suggesting lymph node metastasis.

Fig. 8. Electron microscopic appearances: dark round granules with adiameter of 200–300 nm are seen in the cytoplasm of tumor cells. They arethought to be neuroendocrine granules (34,400).

Fig. 9. Horizontal section of the pelvis on CT: a tumor invading theparametrium is detected in the cervix (single arrow) with a pyomtra(double arrows).

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produces adrenocorticotropic hormone (ACTH)9 or antidi-uretic hormone.10 However, this type of tumor has not beenreported to produce any cytokine, such as G-CSF, withmanifestations of clinical symptoms. In our patient, in-creased peripheral leukocytes, particularly mature neutro-phils, aroused our suspicion that her tumor was producingG-CSF. This patient complained only of genital bleeding,without symptoms of infection or inflammation, but herblood G-CSF level greatly increased. Asano et al. firstreported the production of G-CSF from tumor tissue.11

G-CSF production from tumor tissue can be verified bydetecting G-CSF in extracts from tumor tissue, or by mea-suring the activity of G-CSF in nude mice implanted withthe tumor cells following tumor-cell subcultures. Since spe-cific antibodies to G-CSF were established in 1990, anincreasing number of cases have been immunohistochemi-cally determined to produce G-CSF.12,13 However, suchimmunohistochemical staining cannot always detect cases

that are actually positive, probably because cytokines arerapidly released, even if they have been produced.14 An-other problem inherent to immunostaining is that tumorcells may have G-CSF-receptors15; namely, G-CSF pro-duced in some site, other than the tumor, may be detected.In our case, the tumor cells were thought to definitelyproduce G-CSF because the patient’s WBC and granulocytecounts decreased as the tumor diminished in size by therapy,along with decreases in tumor markers.

Although G-CSF production by a tumor was first detectedin the lung, this type of tumor has been observed in manyother organs. Various histological types, including squa-mous-cell carcinoma and adenocarcinoma, have been iden-tified as G-CSF-producing tumors, but G-CSF can also beproduced by poorly differentiated or undifferentiated cancerat a relatively high frequency.16 G-CSF-producing tumorsare considered to have poor prognoses. The cancer found inour patient was already advanced at the time of diagnosis

Fig. 11. WBC counts, percentage of granulocytes, and NSE levels show synchronous changes with diminution of lesions by therapy or appearance ofrecurrent lesions.

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and showed unsatisfactory response to therapies, followedby rapid progression. Such a poor prognosis was thought tobe explained by the histological and functional types of thetumor, a small-cell neuroendocrine carcinoma with the abil-ity to produce G-CSF. Although the effect of G-CSF pro-duced from the tumor on the growth of the tumor itself or onthe immunological system remains obscure, it has beenreported that G-CSF may serve as an autocrine growthfactor.17–19

The small-cell carcinoma detected in our patient showedno particular cellular features in relation to the production ofG-CSF. Although it is difficult to make a diagnosis ofsmall-cell carcinoma with cytological observation alone, webelieve that small-cell carcinoma should always be consid-ered in the differential diagnosis if small undifferentiatedmalignant cells with a high N/C ratio are cytologicallydetected in the uterus.

AcknowledgmentsWe thank Ms. Isoko Arasaki for her skillful assistance inpreparing cytological, histopathological, and immunohisto-chemical specimens.

References1. Pazdur R, Bonomi P, Slayton R, Gould VE, Miller A, Jao W, Dolan T,

Wilbanks G. Neuroendocrine carcinoma of the cervix: implications forstaging and therapy. Gynecol Oncol 1981;12:120–128.

2. Groben P, Reddick R, Askin F. The pathologic spectrum of small cellcarcinoma of the cervix. Int J Gynecol Pathol 1985;4:42–57.

3. Barrett RJ, Davos I, Leuchter RS, Lagasse LD. Neroendocrine featuresin poorly differentiated and undifferentiated carcinomas of the cervix.Cancer 1987;60:2325–2330.

4. van Nagell JR, Powell DE, Gallion HH, Elliott DG, Donaldson ES,Carpenter AE, Higgins RV, Kryscio R, Pavlik EJ. Small cell carci-noma of the uterine cervix. Cancer 1988;62:1586–1593.

5. Sheets EE, Berman ML, Hrountas CK, Liao SY, Disaia PJ. Surgicallytreated, early-stage neuroendocrine small-cell cervical carcinoma. Ob-stet Gynecol 1988;71:10–14.

6. Abeler VM, Holm R, Nesland JM, Kjorstad KE. Small cell carcinomaof the cervix. Cancer 1994;73:672–677.

7. Kamiya M, Uei Y, Higo Y, Watanabe Y, Yamagishi K, Shimosato Y.Immunocytochemical diagnosis of small cell undifferentiated carci-noma of the cervix. Acta Cytol 1993;37:131–134.

8. Gersell DJ, Mazoujian G, Mutch DG, Rudloff MA. Small-cell undif-ferentiated carcinoma of the cervix. Am J Surg Pathol 1988;12:684–698.

9. Iemura K, Sonoda T, Hayakawa A, Abe S, Nemoto N, Adachi I, TsudaH, Teshima S. Small cell carcinoma of the uterine cervix showingCushing’s syndrome caused by ectopic adrenocorticotropin hormoneproduction. Jpn J Clin Oncol 1991;21:293–298.

10. Kothe MJC, Prins JM, de Wit R, Velden KVD, Schellekens PA. Smallcell carcinoma of the cervix with inappropriate antidiuretic hormonesecretion. Case report. Br J Obstet Gynaecol 1990;97:647–648.

11. Asano S, Urabe A, Okabe T, Sato N, Kondo Y, Ueyama Y, Chiba S,Ohsawa N, Kosaka K. Demonstration of granulopoietic factor(s) in theplasma of nude mice transplanted with a human lung cancer and in thetumor tissue. Blood 1977;49:845–852.

12. Shimamura K, Fujimoto J, Hata J, Akatsuka A, Ueyama Y, WatanabeT, Tamaoki N. Establishment of specific monoclonal antibodiesagainst recombinant human granulocyte colony-stimulating factor andtheir application for immunoperoxidase staining of paraffin-embeddedsections. J Histochem Cytochem 1990;38:283–286.

13. Nagatomo A, Ooike H, Harada A, Sasaki T, Hanada T, Matuoka Y,Irimajiri S, Kishimoto H. Direct identification of lung cancer cellsproducing granulocyte colony stimulating factor with monoclonal anti-G-CSF antibodies. Intern Med 1993;32:468–471.

14. Akatsuka A, Shimamura K, Katoh Y, Takekoshi S, Nakamura M,Nomura H, Hasegawa M, Ueyama Y, Tamaoki N. Electron micro-scopic identification of the intracellular secretion pathway of humanG-CSF in a human tumor cell line: a comparative study with a Chinesehamster ovary cell line (IA1-7) transfected with human G-CSF cDNA.Exp Hematol 1991;19:768–772.

15. Avalos BR, Gasson JC, Hedvat C, Quan SG, Baldwin GC, WeisbartRH, Williams RE, Golde DW, DiPersio JF. Human granulocyte col-ony-stimulating factor: biologic activities and receptor characteriza-tion on hematopoietic cells and small cell lung cancer cell lines. Blood1990;75:851–857.

16. Obara T, Ito Y, Kodama T, Fujimoto Y, Mizoguchi H, Oshimi K,Takahashi M, Hirayama A. A case of gastric carcinoma associatedwith excessive granulocytosis. Cancer 1985;56:782–788.

17. Inoue M, Minami M, Fujii Y, Matsuda H, Shirakura R, Kido T.Granulocyte colony-stimulating factor and interleukin-6-producinglung cancer cell line, LCAM. J Surg Oncol 1997;64:347–350.

18. Tachibana M, Miyakawa A, Uchida A, Murai M, Eguchi K, NakamuraK, Kubo A, Hata J-I. Granulocyte colony-stimulating factor receptorexpression on human transitional cell carcinoma of the bladder. Br JCancer 1997;75:1489–1496.

19. Tachibana M, Murai M. G-CSF production in human bladder cancerand its ability to promote autocrine growth: a review. Cytokines MolTher 1998;4:113–120.

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