metaplastic cancers of the breast (mcbs) are a distinct subgroup of breast cancers. these are a...

Metaplastic cancers of the breast (MCBs) are a distinct subgroup of breast cancers. These are a heterogeneous group of tumours with mixed epithelial and sarcomatoid components, or mixed adenocarcinoma and squamous cell carcinoma components. (1) Many studies have reported aggressive clinical behavior of these tumours associated with poor survival. (1-4) Recently, few studies (5, 6) have proposed that MCBs belong to basal-like subtype of invasive ductal cancers (IDC). High-throughput gene expression profiling followed by hierarchical clustering led to identification of breast cancer subtypes. (7) Such approach can reliably determine whether MCBs belong to basal-like breast cancers (BBCs). However, rarity of these tumors has been a handicap for conventional gene expression profiling. (8) If MCBs belong to basal-like subtype, these should cluster together on multi-parameter hierarchical clustering as in microarray. Number of parameters to be used for such clustering should be more than 100; (9) smaller number of markers (5, 6) can yield spurious results. We tested this hypothesis by performing quantitative gene expression analysis of 502 cancer-related genes on archived FFPE tumors using a novel cDNA-mediated annealing, selection, extension, and ligation (DASL) assay to identify clustering pattern and differentially expressed genes between MCBs and BBCs identified by their triple negative receptor status.

Gene expression profiling:Whole genomeFocused (DASL on FFPE), standard panel or customizedDASL Whole genome

Genotyping: Standard panel or customized, up to 1536 SNPs

miRNA expression profiling

Methylation profiling

Ancillary services:Nucleic Acid quantitation using NanoDropRNA QC using BioanalyzerAdditional details available at: http://www.cancer.iu.edu/research/facilities/translational_genomics/

Translational Genomics CoreMS-B037,635 Barnhill Drive, Indianapolis, IN 46202Phone: 317-274-5583/5565Fax: 317-274-5565 Email: transgen@iupui.edu

Dr. Sunil Badve (Core Director)CPL 4050, 350 W 11th st., Indianapolis, IN 46202Phone: 317-491-6417Fax: 317-491-6417Email: sbadve@iupui.edu

• All nucleic acid extractions have been standardized for each type of material.• All nucleic acid material from samples to be used on assay is checked for quality using NanoDrop &/or Bioanalyzer &/or pre-assay qPCR.• Appropriate duplicates incorporated in each assay for making inter-chip comparisons and checking assay performance.• Excellent R2 values obtained routinely for technical duplicates; above 0.99 for intact RNA and above 0.95 for degraded RNA.

We selected 8 MCBs with squamous differentiation (Figure 1A), and 25 IDCs, 11 of which were triple negative tumors (TN) (Figure 1B); clinical characteristics of these patients are described in Table 1. RNA (200 ng) was extracted using HighPure RNA Paraffin Kit (Roche Applied Bioscience, Indianapolis, IN, USA). RNA was pre-qualified using iScript (Bio-Rad Laboratories Inc, Hercules, CA, USA) to reverse transcribe and SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) to perform qPCR. DASL assay was performed using the Sentrix Universal Array (Illumina Corp., San Diego, CA, USA) of 502 known cancer genes. Statistical analyses and clustering were performed using BeadStudio v3.0 (Illumina Corp.).

BACKGROUND

METHODS

LIST OF SERVICES

QUALITY CONTROL AND ASSURANCES

CONTACT INFORMATION

Mangesh A. Thorat 1, Tanuja M Shet 2, Akira Morimiya 1, Roshni F Chinoy 2, Rajendra A. Badwe 3, Sunil Badve 1

1Dept. of Pathology & Translational Genomics Core, IU School of Medicine, 2Dept. of Pathology, Tata Memorial Hospital, Mumbai, India, 3Dept. of Surgical Oncology, Tata Memorial Hospital, Mumbai, India

COMPARISON OF THE GENE EXPRESSION PROFILES OF METAPLASTICBREAST CANCER AND BASAL-LIKE BREAST CANCER

Unsupervised hierarchical clustering of 33 cases showed two main branches, MCB and IDC (Figure 2). IDC group broadly comprised two subgroups; 9-case-subgroup clustering farthest from MCB branch contained 7 TN (78%), and 16-case-subgroup between these branches contained 4 TN (25%). Analysis of TN (n=11) vs. MCBs (n=8) revealed significant down-regulation of 10 genes (after correction for false-discovery) in MCBs (Table 2). Number of differentially expressed genes without correction for false-discovery was 53.

RESULTS

MCBs and TN have distinct gene expression profiles and do not cluster together as one group. Contrary to recent hypothesis, MCBs are a distinct group of breast cancers and do not belong to BBCs.

CONCLUSION

Figure 1: A) Metaplastic breast cancer with squamous differentiation. (200X) B) Basal (triple negative) breast cancer. (400X)

ParameterGroups Metaplastic (%) Triple Negative

(%)

All IDCs

(%)

Age<50 3 (37.50) 4 (36.36) 11 (44.00)

>50 5 (62.50) 7 (64.64) 14 (56.00)

Tumor

Size

<1 cm 0 (0) 0 (0) 2 (8.00)

1-2 cm 0 (0) 0 (0) 4 (16.00)

>2 cm 8 (100) 11 (100) 19 (76.00)

Grade

Low 0 (0) 0 (0) 1 (4.00)

Mod. 2 (25.00) 1 (9.09) 7 (28.00)

Poor 3 (37.50) 10 (90.91) 17 (68.00)

No info. 3 (37.50) 0 (0) 0 (0)

ER+ 2 (25.00) 0 (0) 9 (36.00)

- 6 (75.00) 11 (100) 16 (64.00)

PR+ 1 (12.50) 0 (0) 3 (12.00)

- 7 (87.50) 11 (100) 22 (88.00)

HER2+ 0 (0) 0 (0) 10 (40.00)

- 8 (100) 11 (100) 15 (60.00)

Gene p-value

TSG101 0.0046

CCNA2 0.0048

XPA 0.0082

VEGFB 0.0119

RAP2A 0.0166

VIL2 0.0297

LYN 0.0363

SOD1 0.0393

NTRK2 0.0486

MMP7 0.0493

Table 1: Patient Characteristics

Table 2: Genes under-expressed in squamous MCBs

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Figure 2: Unsupervised Hierarchical clustering