Sonika Dahiya

Gene expression profiling of the tumor microenvironment during breast cancer progression.

IntroductionThe importance of the tumor microenvironment in breast cancer has been increasingly recognized. Critical molecular changes in the tumor stroma accompanying cancer progression, however, remain largely unknown. We conducted a comparative analysis of global gene expression changes in the stromal and epithelial compartments during breast cancer progression from normal to preinvasive to invasive ductal carcinoma.MethodsWe combined laser capture microdissection and gene expression microarrays to analyze 14 patient-matched normal epithelium, normal stroma, tumor epithelium and tumor-associated stroma specimens. Differential gene expression and gene ontology analyses were performed.ResultsTumor-associated stroma undergoes extensive gene expression changes during cancer progression, to a similar extent as that seen in the malignant epithelium. Highly upregulated genes in the tumor-associated stroma include constituents of the extracellular matrix and matrix metalloproteases, and cell-cycle-related genes. Decreased expression of cytoplasmic ribosomal proteins and increased expression of mitochondrial ribosomal proteins were observed in both the tumor epithelium and the stroma. The transition from preinvasive to invasive growth was accompanied by increased expression of several matrix metalloproteases (MMP2, MMP11 and MMP14). Furthermore, as observed in malignant epithelium, a gene expression signature of histological tumor grade also exists in the stroma, with high-grade tumors associated with increased expression of genes involved in immune response.ConclusionsOur results suggest that the tumor microenvironment participates in tumorigenesis even before tumor cells invade into stroma, and that it may play important roles in the transition from preinvasive to invasive growth. The immune cells in the tumor stroma may be exploited by the malignant epithelial cells in high-grade tumors for aggressive invasive growth.

A five-gene molecular grade index and HOXB13:IL17BR are complementary prognostic factors in early stage breast cancer.

Purpose: Histologic tumor grade is a well-established prognostic factor for breast cancer, and tumor grade–associated genes are the common denominator of many prognostic gene signatures. The objectives of this study are as follows: (a) to develop a simple gene expression index for tumor grade (molecular grade index or MGI), and (b) to determine whether MGI and our previously described HOXB13:IL17BR index together provide improved prognostic information. Experimental Design: From our previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, we selected five cell cycle–related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n = 323), we developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR. Results: MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each others prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI. Conclusions: We developed and validated a five-gene reverse transcription PCR assay for MGI suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The combination of MGI and HOXB13:IL17BR outperforms either alone and identifies a subgroup (∼30%) of early stage estrogen receptor–positive breast cancer patients with very poor outcome despite endocrine therapy.

Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Background Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover. Methodology/Principal Findings Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated. Conclusions/Significance This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

Analysis of the MammaPrint Breast Cancer Assay in a Predominantly Postmenopausal Cohort

Purpose: Most node-negative breast cancer patients are older and postmenopausal and are increasingly being offered adjuvant chemotherapy despite their low overall risk of distant relapse. A molecular diagnostic test with high negative predictive value (NPV) for distant metastasis in this subgroup would spare many older breast cancer patients adjuvant treatment. Experimental Design: We determined the NPV and positive predictive value of the MammaPrint assay in breast cancer patients who were consecutively diagnosed and treated at the Massachusetts General Hospital between 1985 and 1997. Primary tumors from 100 patients with node-negative, invasive breast cancer (median age, 62.5 years; median follow-up, 11.3 years) were subjected to MammaPrint analysis and classified as being at either low or high risk for distant metastasis. Results: The MammaPrint 70-gene signature displayed excellent NPV as in previous studies, correctly identifying 100% of women at low risk for distant metastases at 5 years. However, this assay had a lower positive predictive value (12% at 5 years) than previously observed. Conclusions: The MammaPrint assay was originally designed to identify younger breast cancer patients at low risk for distant metastasis, who might consequently be spared systemic treatment. We show here that the same signature has a very high NPV for distant recurrence after adjuvant treatment in older breast cancer patients.

Response of Some Head and Neck Cancers to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors May Be Linked to Mutation of ERBB2 rather than EGFR

Purpose: Small-molecule tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) have shown modest yet reproducible response rates in patients with squamous cell carcinoma of the head and neck (SCCHN). Somatic mutations in EGFR have recently been shown to be predictive of a clinical response in patients with non–small cell lung cancer (NSCLC) treated with these inhibitors. The objective of this study was to determine if such mutations, or recently reported mutations in ERBB2, also underlie EGFR-TKI responsiveness in SCCHN patients. Experimental Design: We sequenced the kinase domain of EGFR and exon 20 of ERBB2 in tumor specimens from eight responsive patients. In addition, mutational analysis was done on tumor specimens from nine gefitinib nonresponders and 65 unselected cases of SCCHN. Results: None of eight TKI-responsive specimens had mutations within the kinase domain of EGFR. EGFR amplification was also not associated with drug responsiveness. However, a single responsive case had a somatic missense mutation within exon 20 of ERBB2. Conclusion: Our data indicate that unlike NSCLC, EGFR kinase mutations are rare in unselected cases of SCCHN within the United States and are not linked to gefitinib or erlotinib responses in SCCHN. Alternative mechanisms, including ERBB2 mutations, may underlie responsiveness in this tumor type.

HOXB13 promotes ovarian cancer progression

Deregulated expression of HOXB13 in a subset of estrogen receptor-positive breast cancer patients treated with tamoxifen monotherapy is associated with an aggressive clinical course and poor outcome. Because the ovary is another hormone-responsive organ, we investigated whether HOXB13 plays a role in ovarian cancer progression. We show that HOXB13 is expressed in multiple human ovarian cancer cell lines and tumors and that knockdown of endogenous HOXB13 by RNA interference in human ovarian cancer cell lines is associated with reduced cell proliferation. Ectopic expression of HOXB13 is capable of transforming p53−/− mouse embryonic fibroblasts and promotes cell proliferation and anchorage-independent growth in mouse ovarian cancer cell lines that contain genetic alterations in p53, myc, and ras. In this genetically defined cell line model of ovarian cancer, we demonstrate that HOXB13 collaborates with activated ras to markedly promote tumor growth in vivo and that HOXB13 confers resistance to tamoxifen-mediated apoptosis. Taken together, our results support a pro-proliferative and pro-survival role for HOXB13 in ovarian cancer.

The prognostic biomarkers HOXB13, IL17BR, and CHDH are regulated by estrogen in breast cancer.

Purpose: We previously identified three genes, HOXB13, IL17BR, and CHDH, that strongly predict clinical outcome in estrogen receptor (ER)–positive breast cancer patients receiving tamoxifen monotherapy. The biological mechanisms linking these genes to estrogen signaling and tamoxifen response in breast cancer remain to be determined. Experimental Design: In a consecutive series of 148 ER-positive and ER-negative breast cancers, HOXB13, IL17BR, and CHDH gene expression was measured by quantitative real-time PCR and correlated with ER, PR, and HER2 expression. The role of estrogen and ER in the regulation of these three genes was assessed in several ER-positive and ER-negative breast cancer cell lines. Results: In primary breast tumors, HOXB13 expression correlated negatively, and IL17BR and CHDH expression correlated positively, with ER status, and all three genes exhibited an ER-dependent correlation pattern with HER2 status that differs from PR and PS2, two canonical estrogen-regulated genes. Results using breast cancer cell lines show that these genes are regulated by estradiol in an ER-dependent manner, and that this regulation is abrogated by tamoxifen. Conclusions:HOXB13, IL17BR, and CHDH are estrogen-regulated genes, but their pattern of correlation with known positive (ER, PR) and negative (HER2) predictors of tamoxifen response differs from canonical ER signature genes. These results provide a biological rationale for the prognostic utility of these three genes in early-stage ER-positive breast cancer and for their potential to predict anti-estrogen resistance.

Microproteomic analysis of 10,000 laser captured microdissected breast tumor cells using short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis and porous layer open tubular liquid chromatography tandem mass spectrometry

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

Spindle cell oncocytoma of the adenohypophysis: report of two cases

Spindle cell oncocytoma of the adenohypophysis is a recently described, spindle and granular cell, S-100 protein-positive, GFAP-negative, neuroendocrine marker-negative, mitochondria-rich neoplasm of uncertain histogenesis that can clinically and radiologically mimic pituitary adenoma. We present two cases of this entity to extend understanding of this unusual tumor. The first case was a 26-year-old man who presented with a 6-month history of progressive headache, blurred vision in the right eye, nausea, vomiting and impotence. Magnetic resonance imaging (MRI) revealed a rounded, 1.5to 2-cm homogeneously enhancing mass in the pituitary region, which involved the right cavernous sinus, impinged on the temporal lobe and expanded the sella. At transsphenoidal exploration, the mass was firm and difficult to dissect. Following this procedure, visual acuity in the right eye worsened and he underwent a right pterional craniotomy with subtotal resection of the tumor, with residual tumor remaining around the carotid artery. The patient received proton beam radiotherapy to a total dose of 54 Gy over a period of 2 months, during which his vision improved significantly. He has been followed with serial MRI, visual and endocrine examinations for 7 years and remains stable, without tumor regrowth. The second case was a 55-year-old woman who presented with headache and progressive visual deterioration over 6 months, worse in the left than right eye, as well as a 10-day history of vomiting. MRI showed a 6.5·3.3·4 cm sellar and parasellar mass. A transsphenoidal gross total excision was performed. The tumor was highly vascular with destruction of the sellar floor. Six months following surgery, her visual acuity is improved and she has no evidence of recurrence. The two tumors were histologically similar. Case 1 was predominantly composed of spindle cells in a fascicular arrangement (Fig. 1A) with foci of oncocytic appearance (Fig. 1B). Case 2, although also largely spindled in appearance, had more polygonal cells that formed ill-defined lobules (Fig. 1C, D). The cells in both cases had abundant, granular eosinophilic cytoplasm and nuclei that were only mildly pleomorphic. Neither mitotic activity nor necrosis was noted. On immunohistochemical evaluation, the tumor cells were positive for both S-100 protein (Fig. 1E) and epithelial membrane antigen, but did not stain for GFAP, pituitary hormones (prolactin, growth hormone, TSH, ACTH, FSH, LH), chromogranin or synaptophysin . The MIB1 index was approximately 1% in case 1 and 8% in case 2. Electron microscopy was performed on case 2, and showed the tumor cells to be rich in mitochondria with scattered rare secretory granules, but without complex membrane interdigitations or desmosomes (Fig. 1F). Only seven spindle cell oncocytomas of the adenohypophysis have been reported to date [5, 10]. These seven patients were older adults (ranging from 53 to 76 years), five presenting with pan-hypopituitarism and four complaining of visual defects. All five of the lesions initially reported [10] were considered benign since they lacked invasion and showed low proliferative activity. The mean follow-up period for this first series was 3 years, with the longest follow-up 68 months; none of the five tumors had recurred or metastasized. Kloub et al. [5], however, reported two cases of spindle cell oncocytoma of the adenohypophysis that recurred, raising a question about the predicted behavior of these lesions. Although the morphological features of the two primary tumors were not predictive of aggressive behavior, the recurrent tumors had high Ki-67 labeling indices (18–20%) in addition to one recurrent case with mitotic activity and necrosis. These higher proliferation S. Dahiya AE E. T. Hedley-Whyte AE D. N. Louis (&) Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA E-mail: dlouis@partners.org

Frequent met oncogene amplification in a Brca1/Trp53 mouse model of mammary tumorigenesis.

In a screen for gene copy number alterations in mouse mammary tumors initiated by loss of the Brca1 and Trp53 genes, we observed that the majority (11 of 15; 73%) had high-level amplification of wild-type Met, encoding a growth factor receptor implicated in tumor progression. Met amplification was localized to unstable double minute chromosomes and was uniquely found in mouse breast tumors driven by loss of Brca1 and Trp53. Whereas analogous MET amplification was not found in human breast cancers, the identification of a dominant somatic genetic lesion in the Brca1/Trp53 mouse model suggests that recurrent secondary hits may also exist in BRCA1-initiated human breast cancer.