JOURNAL CLUB (07-04-15)Dr Sakshi Gupta

Guide – Col Sonia Badwal

Original ArticleVolume 26, Issue 11 (November 2013)

Introduction Gene expression profile studies(Perou

et al) 5 Intrinsic molecular subtypes of

Invasive Carcinoma were confirmed.

The ‘basal like’subtype Relevance To Patient Management, 15% Of All Invasive Breast Cancers High Number Of Metastatic Breast

Cancer Cases And Breast Cancer-related Deaths Early age of onset BRCA-related Herediteray Ca High Incd – African ethnicity

Poor prognosis Resistant to mol.targeted therapies Limited Rx option Basal – like = dx of exclusion- ER – Negative- PR – Negative- Her2 – Negativeo Rx = Cytotoxic CT

Problems in Dx Micro array based gene expression

profiling – Gold standard- not practicel for routine dx - high cost - complex procedure Result – very few published reports on

basal like cancer

Most studies are - based on FFPE based IHC – universally aval, Inexpensive tech. Surrogate definitions – - triple negative phonotype (TNP) negative ER,PR and Her2 but positive - CK5/6 and/or EGFR (Nielsen et al)

Accuracy of these IHC vs Gene expression based subtype assignment – moderate

76-79% - Sn 72-100 - Sp

Past studies on basal like Only some were validated or

independent Very few – compared with Gene

expression gold std None – compared large number of

biomarkers in a single validation series.

Current STUDY recent gene expression profile data & Published literature found 72 proposed basal – like IHC

biomarker in literature. Using same tissue microarray – in

which subtype assigned by PAM50 GEPassay

MATERIALS AND METHODS1. Tissue Microarray Construction and

PAM50 Molecular subtype assignment2. Immunohistochemical Staining and

Scoring

Tissue Microarray Construction & PAM50 Molecular subtype assignment

TMA - archival tumor blocks of 137 high-grade patients,

Washington University and Barnes-Jewish Hospital in St Louis from 1997 to 2003

Of 137 cases, 127 were successfully assigned an intrinsic molecular subtype.

Exclude : duplicate cases and normal-like Subtype.

Remaining 122 samples 42 basal-like and 80 non-basal-like breast

cancers (58 luminals and 22 HER2-enriched).

IHC Staining and Scoring 72 biomarkers -gene expression profile data. 61 of 72 – Suitable Abs acquired. 15/61 IHC - nonspecific staining on control

tissues or TMA after multiple attempts with varying antibody dilutions.

remaining 46 were stained. Stained slides were scanned using a BLISS

system (Bacus Laboratories/OlympusAmerica, Lombard, IL, USA), and

a pathologist scored each biomarker

Statistical Analysis PASW Statistics 18 for Windows (SPSS, 2009,

Chicago, IL, USA, www.spss.com) was used to perform contingency table analyses.

Pearson’s Chi-square analysis (or the Fisher’s exact test) was used to compare biomarker expression

95% CI for Sn and Sp for each biomarker were generated in R version 2.11.1

P<0.05 defined statistical significance.

ResultsTissue Microarray Staining

46 biomarkers – satisfactory staining In 42 Basal & 80- non Basal like (defined by

PAM50 GEP)

IHC Interp. & UnivariateAnalysis of Basal-Like Biomarkers

25/46 biomarkers – significant assoc with basal-like

MOST SENSITIVE - Ki 67 and PPH3 - Sn 92%

Both (ki67 & PPH3) marker of mitotic fig

Study showed : PPH3 staining is - strong - discrete and - easy to interpret

Lack of ER or PR expression – also 92% Sn for detection of basal-like breast ca.

HIGHEST SPECIFICITY – (100%) - Cytokeratin 14 (CK14) - Insulin like growth factor

mRNAbinding protein-3. (IMP3) - Nerve growth factor receptor

(NGFR) But – poor Sn-22%-27%

NEGATIVE INPP4B staining

Best combination of Sn (61.1%) and Sp (98.6%) with the overall highest individual OR (108.4) NEGATIVE INPP4B - (inositol

polyphosphate-4-phosphatase,type II) = absence may be the best single

diagnostic IHC biomarker for basal-like. Very minimal background staining was

noted.

Other single biomarkers Nestin (using a 1% cutpoint), negative ER staining (using a 1%

cutpoint), CK5 and c-kit.Best positively expressed IHC = Nestin and

CK5

Discussion1. Gene expression profiling-based

technologies2. Surrogate immunohistochemical

definitions.3. Validation of IHC against a gold standard

is a necessary step4. to identify the most useful

biomarkers that can best define the intrinsic molecular subtypes of breast cancer

DiscussionPAM50 assay, (gene expression assay applicable to

FFPE blocks)1. now greatly facilitates such endeavors.2. identifies breast cancer intrinsic

subtypes (luminal A, luminal B, HER2-enriched or basal-like) with prognostic and predictive implications

Discussion Loss of INPP4B expression IHC - most strongly associated with basal-like

breast cancer among the 46 biomarkers tested - (OR=108.4, 61.1% sensitivity and 98.6%

specificity) Loss of INPP4B expression IHC -- Could be misleading-- can be caused by technical problems at any of

the several steps--antigen fading in micro array

Discussion- NESTIN—a positive biomarker - possessed the second highest odd ratio

(OR=28.7, 54.1% sensitivity and 95.8% specificity)

consistency of nestin staining and interpretation across different studies may reflect

good antigen stability in clinical samples, typical of structural proteins

Discussion- NESTIN- Results obtained in this study were

consistent with other studies- good antigen stability in clinical

samples, typical of structural proteins

Study Basal like Non basalParry et al 15 /22

(68%) 3 /117 (2%)

Liu et al 9 /21(57%) 12 /129 (9%)

This study 20 /36 (55%)

3 of 72 (4%)

Ki67 or PPH3 Sn (-92%) Neither Ki67 nor PPH3 are Specific for

basal subtype Also seen in Luminal B and HER2-

enriched breast cancers.CK14, IMP3 and NGFR highest specificity (100%) for basal-like

breast cancer but significant expense of sensitivity, 22% to

27%.

high Sp and low Sn while highly Sn biomarkers had low Sp, MULTI-MARKER IMMUNOPANEL rather

than a single biomarker might be more useful

for phenotypic heterogeneity and increase overall sensitivity for

detection

2 – marker panel1. INPP4B negativity and/or nestin

positivity : 83% Sn and 96% Sp2. INPP4B and CK5 : 83% Sn and 91% Sp

Negative points large confidence intervals for OR values Limited sample size : so cautious interpretation

needed Pepe et al : independent contribution of a

biomarker to classification accuracy can be negligible despite a strong association with disease status.

However, all lowest confidence interval values remain above the null value (OR>1),

supporting a true association between tested biomarkers and basal-like breast cancer

Negative points automated immunostaining and

antigen retrieval techniques, commercialization of antibodies –

whether really capitalizing the gene expression profiling.

CONCLUSION the results of this comprehensive IHC survey may be able

to contribute to the development of a clinically

practical multi-marker immunopanel that best defines basal-like breast cancer in an inexpensive and widely accessible way.

Followed by rigorous evaluation classification accuracy and validation on large independent data sets, application of such an assay in retrospective analyses and prospective clinical trials will help to accurately identify basal-like breast cancer cases,

ultimately facilitating development of therapies for breast cancer patients with this Particularly aggressive form of the disease.

References1. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast

tumours. Nature 2000;406: 747–752.2. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical

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4. Li H, Cherukuri P, Li N, et al. Nestin is expressed in the basal/myoepithelial layer of the mammary gland and is a selective marker of basal epithelial breast tumors. Cancer Res 2007;67:501–510.

5. Parry S, Savage K, Marchio C, et al. Nestin is expressed in basal-like and triple negative breast cancers. J Clin Pathol 2008;61:1045–1050.

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