Histology in the Decision-making Process -...

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Histology in the Decision-making Process

Histology in the Decision-making Process

Giorgio V. ScagliottiUniversity of Torino

Department of Clinical & Biological [email protected]

Giorgio V. ScagliottiUniversity of Torino

Department of Clinical & Biological [email protected]

Lung CancerFour Main Histological Subtypes

Squamous Cell Carcinoma Adenocarcinoma Large Cell Carcinoma

Small Cell CarcinomaTravis WD et al. WHO Classification of Lung Tumors 2004

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Lung CancerClinical Distinction for Therapy


Non-Small Cell Lung Cancer

Small Cell Lung Cancer

Lung CancerClinical Distinction for Therapy


Non-Small Cell Lung Cancer• Similarities in Biological Behaviour & Clinical

Outcomes• Same Chemotherapy Options

Small Cell Lung Cancer

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Relative Contribution of Prognostic Factors in NSCLC

Prog

nost

ic I

nfor

mat

ion

Stanley KE JNCI 1980; 65:25 I=Institution; H=Histology; TS=Tumor size WL=Weight loss; ED=Extent of disease; PS=Performance Status

“Efficacy Plateau” of CytotoxicChemotherapy in NSCLC

“Efficacy Plateau” of CytotoxicChemotherapy in NSCLC

Study Drugs # Pts%,

St. IV%,

ORR MST%,

1-YS

Kelly,2001SWOG 9503

Vnr/CisTax225/Cb

202208

8889

2825

88

3336

Schiller,2002ECOG 1594

Tax135/CisGem/CisTxt/Cis

Tax225/Cb

292288293290

89868686

21.321

17.315.3

8.18.17.48.3

31363135

Scagliotti,2002ILCP

Vnr/CisGem/Cis

Tax225/Cb

201205201

818182

303032

9.59.89.9

373743

Belani,2002TAX 326

Vnr/CisTxt/CisTxT/Cb

404408402

676767

253224

10.111.39.4

414638

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Scagliotti G et al. J. Thorac. Oncol. 2009;4:1568

Retrospective Analysis of a 3-Arm Randomized Trial

Pairwise Comparisons for Survival (P-values)

Squamous Adenocarcinoma Large cell Other

Squamous (N=187) - 0.0021 0.1607 0.9724

Adenocarcinoma (N=310) - 0.6953 0.0239

Large cell (N=45) - 0.2090

Other (N=65) -

• Squamous histology was associated with longer overall survival than adenocarcinoma

• To a lesser extent, “other” histology was associated with longer survival than adenocarcinoma

Scagliotti G et al. J. Thorac. Oncol. 2009;4:1568

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Retrospective Analysis by Histology in ECOG 1594

• Regardless of histology types, overall survival and progression-free survival were similar in chemo-naive patients treated with standard platin-based doublets involving paclitaxel, docetaxel or gemcitabine

Tien H, Dahlberg S, Schiller J, Johnson DJ., J. Thorac. Oncol. 2009; 4 (9 suppl.):S493

Prognostic & Predictive Role of Histologyin Advanced NSCLC

Prognostic & Predictive Role of Histologyin Advanced NSCLC

• A literature search of the last 25 years ofpublications in NSCLC including phase II-III clinical trials, meta-analyses and retrospectivereviews revealed :

• 11 reports found some degree of association betweenhistology and prognosis

• In 7 reports histology predicted outcomes in patientstreated with specific chemotherapeutic agents

• A prognostic/predictive role of histology wasreported in 12 studies with EGFR TKis

• A literature search of the last 25 years ofpublications in NSCLC including phase II-III clinical trials, meta-analyses and retrospectivereviews revealed :

• 11 reports found some degree of association betweenhistology and prognosis

• In 7 reports histology predicted outcomes in patientstreated with specific chemotherapeutic agents

• A prognostic/predictive role of histology wasreported in 12 studies with EGFR TKis

Hirsch F. , Novello S. et al. JTO 2009Hirsch F. , Novello S. et al. JTO 2009

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Critical Issues with Lung Cancer Histology

• Relevance of histopathological subtyping of lung cancer

• Subtyping problems in small samples(biopsies or FNA cytology)

• Role of immunohistochemical markers

• Tissue identification of prognostic and predictive factors (potentially useful forselecting therapy)






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CISCA - IPD Meta-analysisCISCA - IPD Meta-analysis

Ardizzoni A. et al. JNCI 2007;99:847

Histological Subtype of NSCLC and Chemotherapy Selection

Pemetrexed (non-squamous)UFT (adenocarcinoma)EGFR TKIs (?papillary & ‘BAC”)

Adenocarcinoma

Squamous cell carcinoma

IGFR inhibitorsBevacizumab contraindicated

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Carboplatin + Paclitaxel ± Bevacizumab in Advanced NSCLC (ECOG 4599): Survival by

Histology Subtype

Baseline characteristics

PC (n=444) PCB (n=434)

95% CITotal

N N Median

(months) NMedian

(months) HR

All patients 878 444 10.3 434 12.3 0.80 0.69–0.93

Histologic type

Adenocarcinoma 602 302 10.3 300 14.2 0.69 0.58–0.83

Large cell 48 30 8.7 18 10.0 1.15 0.60–2.24

Squamous 3 2 12.3 1 22.4 0.00 0.00

BAC 23 11 17.7 12 10.0 1.48 0.57–3.69

NSCLC, NOS 165 86 10.0 79 9.5 1.16 0.84–1.61

Other 34 11 12.6 23 8.4 0.92 0.43–1.98

Sandler A, et al. Proc IASLC Chicago 2008

BAC = brochioalveolar carcinoma; NOS = not otherwise specified; PC = paclitaxel + carboplatin; PCB = paclitaxel + carboplatin + bevacizumab

Study DesignStudy Design

Gemcitabine 1250 mg/m2 + Cisplatin 75 mg/m2 day 1;Gemcitabine 1250 mg/m2 day 8

Gemcitabine 1250 mg/m2 + Cisplatin 75 mg/m2 day 1;Gemcitabine 1250 mg/m2 day 8

§ Stage IIIB/IV NSCLC§ PS 0 - 1§ No prior chemo§ Randomization:

gender, PS, stage, histo vs cyto dx, brain mets

§ Stage IIIB/IV NSCLC§ PS 0 - 1§ No prior chemo§ Randomization:

gender, PS, stage, histo vs cyto dx, brain mets

RR

Pemetrexed 500 mg/m2 + Cisplatin 75 mg/m2 day 1Pemetrexed 500 mg/m2 + Cisplatin 75 mg/m2 day 1

Primary objective: Overall Survival

15% Non-inferiority margin (HR 1.17)

N = 1700 Patients , Power 80%

Primary objective: Overall Survival

15% Non-inferiority margin (HR 1.17)

N = 1700 Patients , Power 80%

B12, folate, and dexamethasone given in both arms

Scagliotti GV et al. JCO 2008; 26:3543

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Pre-Specific Subset AnalysesPre-Specific Subset Analyses

•• RandomizationRandomization FactorsFactors plus…plus…....

•• AgeAge GroupGroup

•• EthnicityEthnicity

•• Smoking StatusSmoking Status

•• HistologyHistology

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TS m

RNA

leve

ls

Thymidilate Synthase Expression in NormalLung Tissue & Lung Cancer

Thymidilate Synthase Expression in NormalLung Tissue & Lung Cancer

Significantly Higher in Squamous Cell Carcinoma of the Lung

Significantly Higher in Lung Cancer than in normal lung tissue

Snap Frozen Tissues FFPE Tissues

Ceppi P. et al. Cancer 2006Ceppi P. et al. Cancer 2006

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Cis/Pem vs. Cis/Pem : Subgroup AnalysesCis/Pem vs. Cis/Pem : Subgroup Analyses


Efficacy by Histology in Pemetrexed Studies

NSCLCHistologicGroup

Second-linePem vs. Docetaxel

First-linePem/Cis

vs. Gem/CisMaintenance

Pem vs. Placebo

Pem Doc Cis/Pem Cis/Gem Pem Placebo

Non-squamous n=205 n=194 n=618 n=634 n=325 n=156

Median OS, months 9.3 8.0 11.0 10.1 15.5 10.3

Adjusted HR (95% CI)

P value0.78 (0.61–1.00)

0.0480.84 (0.74–0.96)

0.0110.70 (0.56–0.88)

0.002

Squamous n=78 n=94 n=244 n=229 n=116 n=66

Median OS, months 6.2 7.4 9.4 10.8 9.9 10.8

Adjusted HR (95% CI)P value

1.56 (1.08–2.26)0.018

1.23 (1.00–1.51)0.050

1.07 (0.77–1.50)0.678

Non-squamous = adenocarcinoma, large cell carcinoma, and other/indeterminate NSCLC histology

Scagliotti GV, et al. Oncologist 2009

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Meta-Analysis of Postoperative Adjuvant CT with Tegafur-Uracil in NSCLC

Meta-Analysis of Postoperative Adjuvant CT with Tegafur-Uracil in NSCLC

Hamada C. et al. JCO 2005; 23:4999






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‘Biopsy’ Techniques in Lung Cancer Diagnosis

• Sputum cytology• Bronchial brushings and washings• Fluids• FNA cytology – primary or mets• Transbronchial biopsy• Bronchial biopsy• Core biopsy – primary or mets• Liver biopsy• Mediastinoscopy• Lymph node excision• VATS biopsy / resection• Thoracotomy & tumour excision

Increase in Cell number

and Tissue architecture

Kerr K. , 2008






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IHC is the most rapid (1 day) and less expensive method (2-8 euro x reaction) to display cell differentiation when morphology cannot distinguish clear-cut criteria

Immunohistochemistry of Lung Cancer

Poorly-differentiatedNSCLC ???

IHC

Poorly-differentiatedadenocarcinoma

TTF-1 +

Immunohistochemistry of Lung CancerImmunohistochemistry of Lung Cancer

• TTF-1

• Surfactant Apoproteins(A & B)

• Napsin A

• CK 7

• p63

• HMWCK (CK5-6, 34βE12)

• Desmocollin 3

ADENOCARCINOMA SQUAMOUS CELL CARCINOMA

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H&E TTF-1 p63

NSCLCon H&Eê

SQCat IHC

NSCLCon H&Eê

ADCat IHC

Lung Cancer Diagnosis (Bx and/or Cx)

• (1999/2004) Malignant Cancer NSCLC

• (2009) NSCLC SCCPoorly diff. NSCLC ADC

Architecture (pap/acinar/solid)Grade- IHC (CK5/6, 34bE12, p63, DSC-3, TTF-1)

- (Mucin stain)- Expert referral- Another sample

If clinically relevant

TTF-1, (mucin +ve), others –ve …..”favours ADC”TTF-1, (mucin –ve), others +ve …..”favours SQC”

CLINICAL DATAIMAGING DATA

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Inhibition of Folate Enzymes by Pemetrexed, Raltitrexed, and Methotrexate

Inhibition of Folate Enzymes by Pemetrexed, Raltitrexed, and Methotrexate

CompoundCompound TS (nM)TS (nM) DHFR DHFR (nM)(nM)

GARFT GARFT (nM)(nM)

† Shih et al. Cancer Res 57:1116-1123, 1997‡ Chabner et al. J Clin Invest, 76:907-912, 1985† Shih et al. Cancer Res 57:1116-1123, 1997‡ Chabner et al. J Clin Invest, 76:907-912, 1985

Pemetrexed †Pemetrexed † 109109 ±±9.09.0 7.07.0 ±±1.91.9 9,3009,300 ±±690690

Pemetrexed Glu5 †Pemetrexed Glu5 † 1.31.3 ±±0.30.3 7.27.2 ±±0.40.4 6565 ±±1616

Raltitrexed †Raltitrexed † 6.06.0 ±±0.90.9 4545 ±±33 424,000424,000

Raltitrexed Glu5 †Raltitrexed Glu5 † 1.41.4 ±±0.10.1 3030 ±±33 132,000132,000

Methotrexate ‡Methotrexate ‡ 13,00013,000 0.0040.004 80,00080,000

MTX Glu5 ‡MTX Glu5 ‡ 4747 0.0040.004 2,5002,500

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Classification of Human Lung Carcinomas by mRNA Expression Profiling Reveals Distinct

Adenocarcinoma Subclasses

Classification of Human Lung Carcinomas by mRNA Expression Profiling Reveals Distinct

Adenocarcinoma Subclasses

SCLC SCLC –– Very High TS ; Squamous Very High TS ; Squamous –– Intermediate TS , Adenocarcinoma Intermediate TS , Adenocarcinoma –– Low TSLow TS

BhattacharjeeBhattacharjee A et al. PNAS 2001; 98:13790A et al. PNAS 2001; 98:13790

TS TS mRNAmRNA ExpressionExpression in Lung Cancer (n=146)in Lung Cancer (n=146)

ADC SCC non-NE LCC LCNEC SCLC0

1

2

3

4

5

TS

rela

tive

mR

NA

leve

ls

Scagliotti GV et al. Poster Discussion ASCO 2009 - Abstract # 7521

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Negative Positive

DSC3 protein expression

0

10

20

30

40

50

60

70

80

90

100T

S pr

otei

n ex

pres

sion

leve

lsP=0.018

Correlations Between DSC3 Staining and TS Expression Levels in Large Cell Carcinoma of the Lung

Phase III Study ED-SCLC Pemetrexed/Carboplatin vs

Etoposide/ Carboplatin

Socinski MA. JCO 2009;27:4787

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TS p

ositi

veTS

neg

ativ

eCytological sample Histological sample

TS Immunoreactivity in Cytological NSCLC-NOS

Gene Expression According to Histology

Squamous CellCarcinoma

Adenocarcinoma P value

Median (range) Median (range)

ERCC1 1.41 (0.45-7.34) 0.72 (0.23-2.45) 0.0001

MZF1 0.62 (0.06-6.72) 0.25 (0.03-1.49) 0.0001

Twist 10.37 (0.30-76.01) 2.50 (0.14-19.16) 0.0001

RRM1 2 (0.6-6.9) 1.2 (0.4-2.9) 0.0001

TRX 2.13 (0.40-11.88) 0.91 (0.31-7.94) 0.0001

Tpd1 1.7 (0.6-7.3) 1.3 (0.1-2.6) 0.02

NFAT 0.4 (0.1-2.3) 0.5 (0.1-1.8) 0.65

BRCA1 4.26 (0.55-18.48) 1.50 (0.09-8.08) 0.0001

BubR1 16.3 (1.4-90) 7 (0.8-25) 0.0001

Rosell R. et al. PLoS ONE 2:e1129

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Are Other Genomic Markers Differentially Expressed in NSCLC?

Author Squamous Adenocarcinoma P ValueOlauseen, NEJM 2006

ERRC1 Positive * 70% 45%0.001

ERCC1 Negative * 21% 40%

Zheng, NEJM 2007Median ERCC1** 56.8

(1.9-178.7)68.0

(6.6-153.1)

Median RRM1 ** 62.3(13.2-96.2)

40.5(8.3-95.1)

* H Score (semiquantitative IHC) ; ** AQUA Scores

Five Gene Signature and Outcome in NSCLC

High-risk Low-risk P value

Original Cohort (n=101)Adenocarcinoma 61% 36% 0.03

Squamous Cell Ca. 32% 47%Others 7% 17%

Validation Cohort (n=60)Adenocarcinoma 32% 50% 0.19

Squamous Cell Ca. 59% 42%Others 9% 8%

Chen HY et al. NEJM 2007; 356:11

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hsa-miR-205 Expression Distinguishes Squamous From Non-Squamous NSCLChsa-miR-205 Expression Distinguishes

Squamous From Non-Squamous NSCLC• MicroRNA expression levels in 122

adenocarcinoma and squamous cell carcinoma

• qRT-PCR platform in an independent dataset of 20+27 NSCLC FFPE samples

• Assay validated in a blinded cohort of 79 NSCLC FFPE samples

• Hsa-miR-2005 highly specific marker for SCC of the lung

• Cut-off score of 2.5, sensitivity 96%, specificity 90%

Lebanovy D et al. JCO2009; 27:2030-37

Conclusion• Histologic subtyping should be taken into account in making

treatment decisions

• WHO classification should remain the common language for exchanging information and treatment decision.

• There are cheap ways to separate squamous from non squamous histology also in limited cytological samples.

• The use of pharmacogenomic markers holds the promise to improve results of cytotoxic chemotherapy

• Mandatory need of histology (pharmacogenomic) - driven prospective trials in any stage of NSCLC

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Biomarkers, Prognostication and Prediction

BRCA1 mRNAPCR

TS proteinIHC

RRM1 proteinIHCERCC1 protein

IHC

cMET amplificationFISH

EGFR proteinIHC

EGFR amplificationFISH

EGFR genemutations

TS mRNAPCR

RRM1 mRNAPCR

KRAS genemutation

ERCC1 mRNAPCR

P53 proteinIHC

Selected geneExpressionsignatures

What’sNext?

SerpinB3 proteinIHC

SerpinB3 mRNAPCR

p27kip1 proteinIHC

Cis/Pem vs. Cis/Pem : Overall SurvivalCis/Pem vs. Cis/Pem : Overall Survival


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Non-squamous group Squamous group

Pemetrexed (n=205)

Docetaxel (n=194)

Pemetrexed (n=78)

Docetaxel (n=94)

% ECOG PS 2 12.5 10.1 8.3 17.4% TSPC <3 months 51.0 51.0 48.7 41.9

% Stage IV 81.5 78.9 57.7 66.0

% Male 60.5 69.1 89.7 88.3Median OS, months 9.3 8.0 6.2 7.4

Adjusted OS HR (95% CI) 0.778 (0.607, 0.997) 1.563 (1.079, 2.264)

Median PFS, months 3.1 3.0 2.3 2.7

Adjusted PFS HR (95% CI) 0.823 (0.664, 1.020) 1.403 (1.006, 1.957)

Second -Line Study of Pemetrexed vs. Docetaxel : Efficacy by Histology

Second -Line Study of Pemetrexed vs. Docetaxel : Efficacy by Histology

Treatment by Histology Interaction: Survival Adjusted for Cofactors (p=0.001)

Peterson P. et al. 12th World Conference on Lung Cancer 2007

Median PFS, mos CR+PR+SD*, % Prelim Median

OS, mos

Pem Plac p-value Pem Plac p-value Pem Plac p-value

Nonsquamous (n=482)

4.37 1.84 <0.00001 54.3 26.6 <0.001 14.4 9.4 0.005

Adeno (n=329)

4.60 2.66 <0.00001 58.2 29.6 <0.001 16.4 11.7 0.091

Large cell (n=20)

4.53 1.45 0.104 30.0 25.0 0.999 9.1 5.5 0.154

Other (n=133)

4.11 1.58 0.0001 47.5 18.9 0.004 11.3 7.0 0.005

Squamous (n=181)

2.43 2.50 0.896 33.3 34.5 0.999 9.6 11.9 0.231

Double-blind, Placebo-controlled Phase III Trial of Maintenance Pemetrexed Maintenance Pemetrexed : Efficacy by Efficacy by HistologicHistologic GroupsGroups

* Clinical response (CR+PR+SD) was significantly improved with pemetrexed vs placebo in the intent-to-treat population (49% vs 29%, p <0.001).

Ciuleanu T. et al. Proc. ASCO 2008

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Carbo/Etoposide vs. Carbo/Pemetrexed in ED-SCLC: Interim PFS Analysis

Carbo/Etoposide vs. Carbo/Pemetrexed in ED-SCLC: Interim PFS Analysis

Median (95% CI)Pem-Cb: 3.68 (3.38, 4.2)Eto-Cb: 5.32 (5.03, 5.85)

Log rank p<.0001PFS HR = 1.79 (90% CI: 1.49, 2.15)

Patients at riskPem-Cb: 364 199 92 21 6 1 0Eto -Cb: 369 220 140 52 13 1 0

Prob

abili

ty W

ithou

t Eve

nt

Socinski M. et al. Proc. ASCO 2008

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