C. Blake Gilks

Immunohistochemical and Clinical Characterization of the Basal-Like Subtype of Invasive Breast Carcinoma

Purpose: Expression profiling studies classified breast carcinomas into estrogen receptor (ER)+/luminal, normal breast-like, HER2 overexpressing, and basal-like groups, with the latter two associated with poor outcomes. Currently, there exist clinical assays that identify ER+/luminal and HER2-overexpressing tumors, and we sought to develop a clinical assay for breast basal-like tumors. Experimental Design: To identify an immunohistochemical profile for breast basal-like tumors, we collected a series of known basal-like tumors and tested them for protein patterns that are characteristic of this subtype. Next, we examined the significance of these protein patterns using tissue microarrays and evaluated the prognostic significance of these findings. Results: Using a panel of 21 basal-like tumors, which was determined using gene expression profiles, we saw that this subtype was typically immunohistochemically negative for estrogen receptor and HER2 but positive for basal cytokeratins, HER1, and/or c-KIT. Using breast carcinoma tissue microarrays representing 930 patients with 17.4-year mean follow-up, basal cytokeratin expression was associated with low disease-specific survival. HER1 expression was observed in 54% of cases positive for basal cytokeratins (versus 11% of negative cases) and was associated with poor survival independent of nodal status and size. c-KIT expression was more common in basal-like tumors than in other breast cancers but did not influence prognosis. Conclusions: A panel of four antibodies (ER, HER1, HER2, and cytokeratin 5/6) can accurately identify basal-like tumors using standard available clinical tools and shows high specificity. These studies show that many basal-like tumors express HER1, which suggests candidate drugs for evaluation in these patients.

Ovarian Carcinoma Subtypes Are Different Diseases: Implications for Biomarker Studies

Background Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis. Methods and Findings In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%–2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%–2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%–0.8%). Conclusions The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma.

Mutation of FOXL2 in granulosa-cell tumors of the ovary

BACKGROUND Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T reg cells

Although immune mechanisms can suppress tumour growth, tumours establish potent, overlapping mechanisms that mediate immune evasion. Emerging evidence suggests a link between angiogenesis and the tolerance of tumours to immune mechanisms. Hypoxia, a condition that is known to drive angiogenesis in tumours, results in the release of damage-associated pattern molecules, which can trigger the rejection of tumours by the immune system. Thus, the counter-activation of tolerance mechanisms at the site of tumour hypoxia would be a crucial condition for maintaining the immunological escape of tumours. However, a direct link between tumour hypoxia and tolerance through the recruitment of regulatory cells has not been established. We proposed that tumour hypoxia induces the expression of chemotactic factors that promote tolerance. Here we show that tumour hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumour tolerance and angiogenesis. Thus, peripheral immune tolerance and angiogenesis programs are closely connected and cooperate to sustain tumour growth.

Mucinous Tumors of the Appendix Associated With Mucinous Tumors of the Ovary and Pseudomyxoma Peritonei: A Clinicopathological Analysis of 22 Cases Supporting an Origin in the Appendix

Twenty-two cases in which mucinous tumors of the appendix were associated with mucinous tumors of the ovary are reported. The patients ranged from 23 to 83 (average 49) years of age and usually presented with increasing abdominal girth. The appendiceal and ovarian tumors were synchronous in 21 cases. Laparotomy typically disclosed large cystic ovarian tumors that averaged 16 cm in diameter and were usually multilocular, an appendix that was usually dilated and covered with mucus, and abundant intra-abdominal mucus. The ovarian tumors were bilateral in seven cases. The ovarian and appendiceal tumors were typically similar histologically, with features similar to those of ovarian mucinous cystadenomas and cystadenomas of borderline malignancy. In most of the ovarian tumors, mucin dissected through the ovarian stroma (so-called pseudomyxoma ovarii). Eight of the 20 patients with follow-up information were well when last seen, but the duration of follow-up was 3 years or less in six of them. Two patients died of pseudomyxoma peritonei 4 and 5.5 years after presentation. One patient died of a myocardial infarct shortly after laparotomy for recurrent pseudomyxoma peritonei at 11 years. The remaining patients had definite or probable recurrent or residual disease but were alive at the time of the last follow-up information. The typical synchronous presentation of the ovarian and appendiceal tumors, their histologic similarity, the frequency of bilaterality of the ovarian tumors, the predominance of right-sided ovarian involvement, and the usual presence of mucin and atypical mucinous cells on the ovarian surfaces all point toward the probable secondary nature of the ovarian tumors.

Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities

BackgroundSubclassification of ovarian carcinomas can be used to guide treatment and determine prognosis. Germline and somatic mutations, loss of heterozygosity (LOH), and epigenetic events such as promoter hypermethylation can lead to decreased expression of BRCA1/2 in ovarian cancers. The mechanism of BRCA1/2 loss is a potential method of subclassifying high grade serous carcinomas.MethodsA consecutive series of 49 ovarian cancers was assessed for mutations status of BRCA1 and BRCA2, LOH at the BRCA1 and BRCA2 loci, methylation of the BRCA1 promoter, BRCA1, BRCA2, PTEN, and PIK3CA transcript levels, PIK3CA gene copy number, and BRCA1, p21, p53, and WT-1 immunohistochemistry.ResultsEighteen (37%) of the ovarian carcinomas had germline or somatic BRCA1 mutations, or epigenetic loss of BRCA1. All of these tumours were high-grade serous or undifferentiated type. None of the endometrioid (n = 5), clear cell (n = 4), or low grade serous (n = 2) carcinomas showed loss of BRCA1, whereas 47% of the 38 high-grade serous or undifferentiated carcinomas had loss of BRCA1. It was possible to distinguish high grade serous carcinomas with BRCA1 mutations from those with epigenetic BRCA1 loss: tumours with BRCA1 mutations typically had decreased PTEN mRNA levels while those with epigenetic loss of BRCA1 had copy number gain of PIK3CA. Overexpression of p53 with loss of p21 expression occurred significantly more frequently in high grade serous carcinomas with epigenetic loss of BRCA1, compared to high grade serous tumors without loss of BRCA1.ConclusionHigh grade serous carcinomas can be subclassified into three groups: BRCA1 loss (genetic), BRCA1 loss (epigenetic), and no BRCA1 loss. Tumors in these groups show distinct molecular alterations involving the PI3K/AKT and p53 pathways.

Systematic Analysis of Immune Infiltrates in High-Grade Serous Ovarian Cancer Reveals CD20, FoxP3 and TIA-1 as Positive Prognostic Factors

Background Tumor-infiltrating T cells are associated with survival in epithelial ovarian cancer (EOC), but their functional status is poorly understood, especially relative to the different risk categories and histological subtypes of EOC. Methodology/Principal Findings Tissue microarrays containing high-grade serous, endometrioid, mucinous and clear cell tumors were analyzed immunohistochemically for the presence of lymphocytes, dendritic cells, neutrophils, macrophages, MHC class I and II, and various markers of activation and inflammation. In high-grade serous tumors from optimally debulked patients, positive associations were seen between intraepithelial cells expressing CD3, CD4, CD8, CD45RO, CD25, TIA-1, Granzyme B, FoxP3, CD20, and CD68, as well as expression of MHC class I and II by tumor cells. Disease-specific survival was positively associated with the markers CD8, CD3, FoxP3, TIA-1, CD20, MHC class I and class II. In other histological subtypes, immune infiltrates were less prevalent, and the only markers associated with survival were MHC class II (positive association in endometrioid cases) and myeloperoxidase (negative association in clear cell cases). Conclusions/Significance Host immune responses to EOC vary widely according to histological subtype and the extent of residual disease. TIA-1, FoxP3 and CD20 emerge as new positive prognostic factors in high-grade serous EOC from optimally debulked patients.

Differences in Tumor Type in Low-stage Versus High-stage Ovarian Carcinomas

Although there are recognized differences in the type of ovarian carcinomas between those tumors diagnosed at low versus high stage, there is a lack of data on stage distribution of ovarian carcinomas diagnosed according to the current histopathologic criteria from large population-based cohorts. We reviewed full slide sets of 1009 cases of 2555 patients diagnosed with ovarian carcinoma that were referred to the British Columbia Cancer Agency over a 16-year period (1984 to 2000). On the basis of the reviewed cases we extrapolated the distribution of tumor type in low-stage (I/II) and high-stage (III/IV) tumors. We then compared the frequencies with those seen in a large hospital practice. The overall frequency of tumor types was as follows: high-grade serous—68.1%, clear-cell—12.2%, endometrioid—11.3%, mucinous—3.4%, low-grade serous—3.4%, rare types—1.6%. High-grade serous carcinomas accounted for 35.5% of stage I/II tumors and 87.7% of stage III/IV tumors. In contrast, clear-cell (26.2% vs. 4.5%), endometrioid (26.6% vs. 2.5%), and mucinous (7.5% vs. 1.2%) carcinomas were relatively more common among the low-stage versus high-stage tumors. This distribution was found to be very similar in 410 consecutive cases from the Washington Hospital Center. The distribution of ovarian carcinoma types differs significantly in patients with low-stage versus high-stage ovarian carcinoma when contemporary diagnostic criteria are used, with consistent results seen in 2 independent case series. These findings reflect important biological differences in the behavior of the major tumor types, with important clinical implications.

Software tools for high-throughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays.

The creation of tissue microarrays (TMAs) allows for the rapid immunohistochemical analysis of thousands of tissue samples, with numerous different antibodies per sample. This technical development has created a need for tools to aid in the analysis and archival storage of the large amounts of data generated. We have developed a comprehensive system for high-throughput analysis and storage of TMA immunostaining data, using a combination of commercially available systems and novel software applications developed in our laboratory specifically for this purpose. Staining results are recorded directly into an Excel worksheet and are reformatted by a novel program (TMA-Deconvoluter) into a format suitable for hierarchical clustering analysis or other statistical analysis. Hierarchical clustering analysis is a powerful means of assessing relatedness within groups of tumors, based on their immunostaining with a panel of antibodies. Other analyses, such as generation of survival curves, construction of Cox regression models, or assessment of intra- or interobserver variation, can also be done readily on the reformatted data. Finally, the immunoprofile of a specific case can be rapidly retrieved from the archives and reviewed through the use of Stainfinder, a novel web-based program that creates a direct link between the clustered data and a digital image database. An on-line demonstration of this system is available at http://genome-www.stanford.edu/TMA/explore.shtml.

Gene Expression Profiling Reveals Reproducible Human Lung Adenocarcinoma Subtypes in Multiple Independent Patient Cohorts

PURPOSE Published reports suggest that DNA microarrays identify clinically meaningful subtypes of lung adenocarcinomas not recognizable by other routine tests. This report is an investigation of the reproducibility of the reported tumor subtypes. METHODS Three independent cohorts of patients with lung cancer were evaluated using a variety of DNA microarray assays. Using the integrative correlations method, a subset of genes was selected, the reliability of which was acceptable across the different DNA microarray platforms. Tumor subtypes were selected using consensus clustering and genes distinguishing subtypes were identified using the weighted difference statistic. Gene lists were compared across cohorts using centroids and gene set enrichment analysis. RESULTS Cohorts of 31, 72, and 128 adenocarcinomas were generated for a total of 231 microarrays, each with 2,553 reliable genes. Three adenocarcinoma subtypes were identified in each cohort. These were named bronchioid, squamoid, and magnoid according to their respective correlations with gene expression patterns from histologically defined bronchioalveolar carcinoma, squamous cell carcinoma, and large-cell carcinoma. Tumor subtypes were distinguishable by many hundreds of genes, and lists generated in one cohort were predictive of tumor subtypes in the two other cohorts. Tumor subtypes correlated with clinically relevant covariates, including stage-specific survival and metastatic pattern. Most notably, bronchioid tumors were correlated with improved survival in early-stage disease, whereas squamoid tumors were associated with better survival in advanced disease. CONCLUSION DNA microarray analysis of lung adenocarcinomas identified reproducible tumor subtypes which differ significantly in clinically important behaviors such as stage-specific survival.