CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women's Circle of Health Study and the Women's Circle of Health Follow-Up Study.

BACKGROUND: Tumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes. METHODS: We investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women's Circle of Health Study and Women's Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43-12.33). RESULTS: Women with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16-0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12-0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71-1.10). CONCLUSIONS: In contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer. Trial registration Not applicable.

Racial differences in CD8+ T cell infiltration in breast tumors from Black and White women.

BACKGROUND: African American/Black women with breast cancer have poorer survival than White women, and this disparity persists even after adjusting for non-biological factors. Differences in tumor immune biology have been reported between Black and White women, and the tumor immune milieu could potentially drive racial differences in breast cancer etiology and outcome. METHODS: We examined the association of CD8+ cytotoxic T cells with clinical-pathological variables in the Women's Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We evaluated 688 invasive breast tumor samples (550 Black, 138 White) using immunohistochemical staining of tissue microarray slides. CD8+ T cells were scored for each patient tumor sample with digital image analysis. RESULTS: Black women had a significantly higher percentage of high-grade, estrogen receptor (ER)-negative, and triple-negative tumors than White women and significantly higher CD8+ T cell density (median 87.6/mm2 vs. 53.1/mm2; p < 0.001). Within the overall population and in the population of Black women only, CD8+ T cell density was significantly higher in younger patients and patients with high-grade and ER/PR-negative tumors. No significant associations were observed between CD8+ T cell density and overall survival or breast cancer-specific survival in the overall population, or when Black patients were analyzed as a separate group. However, when stratified by subtype, Black women with triple-negative breast cancer and high CD8+ T cell density showed a trend towards better overall survival in comparison with patients with low CD8+ T cell density (HR = 0.51; 95% CI 0.25-1.04). CONCLUSIONS: Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups.

Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast.

BACKGROUND: A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs). This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. METHODS: We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs). Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH) analysis to identify genes showing altered expression in LOH regions. RESULTS: Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. CONCLUSION: We have demonstrated the utility of the application of integrated analysis using high resolution CGH and whole genome transcript analysis for detecting driver genes in IDC. The high resolution platform allowed a refined demarcation of CNAs and gene expression profiling provided a mechanism to detect genes directly impacted by the CNA. This is the first report of LOH integrated with gene expression in IDC using a high resolution platform.

Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

Loss of immunoreactivity in tissue sections has been shown to occur when slide sections are stored at room temperature for prolonged periods of time. We conducted a systematic investigation to determine the extent of staining loss in various storage conditions to determine an optimal storage method. We investigated 6 antibodies that are commonly used for breast cancer subtyping in research studies with immunohistochemistry (ER, PR, HER2, CK5/6, EGFR, and Ki67) in formalin-fixed paraffin-embedded breast tissue microarrays consisting of 148 patients. Tissue microarrays were sectioned at various time points: fresh, 1 week, 1 month, 6 months, and 12 months before staining. Slides sectioned at each time point were stored in 5 storage conditions: desiccator, paraffin dipped, 4°C, -20°C, and -80°C. Immunohistochemistry scores were assessed over time with McNemar Test and Bowker Test of Symmetry. Desiccator storage was the only storage condition that did not show any loss in immunoreactivity for any antibody or time point in our study. Paraffin coated slides were the most difficult storage method operationally and also showed the most loss in immunoreactivity. Storing sections in a desiccator was the most effective method for minimizing immunoreactivity loss. Cold storage at 4°C is an intermediate option that is not as protective as a desiccator, but offers the advantage of being accessible to virtually all research labs.

Identification of genes involved in squamous cell carcinoma of the lung using synchronized data from DNA copy number and transcript expression profiling analysis.

Lung cancer is the leading cause of cancer deaths in the world and squamous cell carcinoma (SqCC) is the second most common in this group. Genomic DNA copy number alterations are fundamental genetic events in the development and progression of SqCC as well as other epithelial-derived cancers. The ability to identify tumor suppressor genes (TSGs) and oncogenes that are affected during tumor initiation and progression could facilitate the identification of novel molecular targets for therapeutic intervention and provide diagnostic biomarkers. Despite the association of many genetic alterations in lung cancer the molecular mechanisms of tumor progression remain ambiguous since often too many candidates are revealed using conventional genetic microarray analysis. To overcome this limitation, we have identified genes in SqCC which show concordant gene expression changes defined using microarray analysis with DNA copy number alterations defined by BAC-array comparative genomic hybridization (aCGH) in the same tumors. An in-house overlay algorithm was used to synchronize the data resulting from the two analyses. Although the expression levels of many genes were altered when compared to normal controls, those which correlated with copy number changes were far fewer, providing a manageable number for biological studies. We identified over 2000 genes which displayed both gene expression alterations and mapped to BACs which demonstrated a corresponding loss or gain. A further stringent statistical analysis identified minimal regions of overlap for losses or gains which displayed a coincident decrease or increase in the expression of genes mapping to those regions. Consistent gains involved 3q23-q29, 5p15.1-q11.1 and chromosomes 18 and 20, while consistent losses involved 3p26.3-p12.3, 9p24.3-q34.3, and chromosomes 17 and 19. The concordance finding between these two approaches suggests that DNA copy number alterations can directly influence gene expression patterns that impact on tumorigenesis in SqCC of the lung.

Universal Reference RNA is not a representative normal sample for oligonucleotide microarray studies.

Translational research has been defined as the scientific study using human material that will ultimately generate patient specific data. A major caveat in human directed study is the availability of high quality and quantities of patient derived homogeneous cells for analysis. Whereas there exist sources for which tumor tissue and blood samples can be made available, the same cannot be said for normal tissue. The absence of normal control tissue has led to the creation of pooled cell lines and tissues for purchase known as "reference RNA". Although initially created for purposes of standardization, the difficulty associated with acquiring normal tissue has led some investigators to use sources of universal pooled RNA for comparative analysis with clinical tissue specimens. In order to study the effects of using Universal Reference RNA on expression profiling experiments we have evaluated the performance of universal RNA compared to RNA obtained from a purified population of colon epithelial cells in defining a set of altered transcripts in colon cancer.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples.

BACKGROUND: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. METHODS: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. RESULTS: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. CONCLUSION: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient.

OBJECTIVE: The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. MATERIALS AND METHODS: Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. RESULTS: UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. CONCLUSION: We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

A Pilot Study of Stereotactic Body Radiation Therapy Combined with Cytoreductive Nephrectomy for Metastatic Renal Cell Carcinoma.

Purpose: While stereotactic body radiotherapy (SBRT) can reduce tumor volumes in patients with metastatic renal cell carcinoma (mRCC), little is known regarding the immunomodulatory effects of high-dose radiation in the tumor microenvironment. The main objectives of this pilot study were to assess the safety and feasibility of nephrectomy following SBRT treatment of patients with mRCC and analyze the immunological impact of high-dose radiation.Experimental Design: Human RCC cell lines were irradiated and evaluated for immunomodulation. In a single-arm feasibility study, patients with mRCC were treated with 15 Gray SBRT at the primary lesion in a single fraction followed 4 weeks later by cytoreductive nephrectomy. RCC specimens were analyzed for tumor-associated antigen (TAA) expression and T-cell infiltration. The trial has reached accrual (ClinicalTrials.gov identifier: NCT01892930).Results: RCC cells treated in vitro with radiation had increased TAA expression compared with untreated tumor cells. Fourteen patients received SBRT followed by surgery, and treatment was well-tolerated. SBRT-treated tumors had increased expression of the immunomodulatory molecule calreticulin and TAA (CA9, 5T4, NY-ESO-1, and MUC-1). Ki67+ -proliferating CD8+ T cells and FOXP3+ cells were increased in SBRT-treated patient specimens in tumors and at the tumor-stromal interface compared with archived patient specimens.Conclusions: It is feasible to perform nephrectomy following SBRT with acceptable toxicity. Following SBRT, patient RCC tumors have increased expression of calreticulin, TAA, as well as a higher percentage of proliferating T cells compared with archived RCC tumors. Collectively, these studies provide evidence of immunomodulation following SBRT in mRCC. Clin Cancer Res; 23(17); 5055-65. ©2017 AACR.

Characterization of cell-type specific profiles in tissues and isolated cells from squamous cell carcinomas of the lung.

Lung cancer accounts for 28% of all cancer deaths, a higher percentage than any other human cancer. Squamous Cell Carcinoma (SqCC) is the most common lung neoplasm and is a tumor that is extensively associated with tobacco use. Despite the association of many genetic alterations with lung cancer, the precise molecular mechanisms of tumorigenesis, for the most part, remain ambiguous. Although many studies of lung cancer have used global transcript profiling approaches designed to uncover genes or pathways that are important in lung tumorigenesis, no strong candidates have emerged. A lack of concurrence amongst these various studies can be attributed, in a large part, to the cellular heterogeneity within lung tissue. We have attempted to reduce this complication by designing a profiling strategy that will minimize the confounding involvement of tissue heterogeneity in gene expression of lung tumors. Specifically, we have profiled transcript expression levels in both isolated cells and tissues from SqCC and normal samples. Our strategy consists of combining and subtracting the input of these various cell types which has produced a unique transcript profile of the squamous carcinoma cell. We then analyzed the data using Pathways Assist analysis software to determine which processes may be involved in SqCC tumorigenesis. The MAP/ERK pathway involved in growth and differentiation was the pathway that was most frequently identified across all comparisons. In addition, biological interaction networks of the SqCC profile identified IL-8 as playing a potentially important role SqCC development.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer.

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

Bortezomib mitigates adverse prognosis conferred by Bcl-2 overexpression in patients with relapsed/refractory multiple myeloma.

Overexpression of the Bcl-2 family of genes results in increased transcription of anti-apoptotic proteins. In vitro data suggest that this may enhance acquired chemoresistance and correlate with extramedullary invasion. This has led to pursuing the Bcl-2 family of proteins as therapeutic targets in several malignant disorders, including multiple myeloma (MM). The impact of novel therapeutic agents such as bortezomib on these molecular markers is not known. We investigated the association between the expression of anti-apoptotic members of the Bcl-2 family and the efficacy of bortezomib in patients with relapsed/refractory MM. Gene expression data generated prospectively from large clinical trials were utilized. Hypothesis testing using a multisample test for equivalence was performed. The association between Bcl-2 expression levels and clinical response was negated in bortezomib-treated patients (p = 0.014), while not so in dexamethasone-treated patients (p = 0.92). Similar results were noted for variant 2 of the Mcl-1 gene (p = 0.003). Results for Bcl-xl did not meet the level of significance. Thus, the importance of the Bcl-2 family of proteins as prognostic markers in MM should be reassessed in the novel therapeutic agent era. Our data suggest that bortezomib may overcome the prognostic effect conferred by overexpression of some of the anti-apoptotic Bcl-2 family of genes in patients with relapsed/refractory MM.

Copy number and gene expression alterations in radiation-induced papillary thyroid carcinoma from chernobyl pediatric patients.

BACKGROUND: Following exposure to radiation during the Chernobyl fallout tragedy, papillary thyroid carcinoma (PTC) increased significantly in individuals who were children at the time of the accident. We have used two high-throughput, whole genome platforms to analyze radiation-induced PTCs from pediatric patients from the Chernobyl region. METHODS: We performed comparative genomic hybridization using Affymetrix 50K Mapping arrays and gene expression profiling on 10 pediatric post-Chernobyl PTCs obtained from patients living in the region. We performed an overlay analysis of these two data sets. RESULTS: Many regions of copy number alterations (CNAs) were detected including novel regions that had never been associated with PTCs. Increases in copy numbers were consistently found on chromosomes 1p, 5p, 9q, 12q, 13q, 16p, 21q, and 22q. Deletions were observed less frequently and were mapped to 1q, 6q, 9q, 10q, 13q, 14q, 21q, and 22q. Gene expression analysis revealed that most of the altered genes were also perturbed in sporadic adult PTC; however, 141 gene expression changes were found to be unique to the post-Chernobyl tumors. The genes with the highest increases in expression that were novel to the pediatric post-Chernobyl tumors were TESC, PDZRN4, TRAa/TRDa, GABBR2, and CA12. The genes showing the largest expression decreases included PAPSS2, PDLIM3, BEXI, ANK2, SORBS2, and PPARGCIA. An overlay analysis of the gene expression and CNA profiles was then performed. This analysis identified genes showing both CNAs and concurrent gene expression alterations. Many of these are commonly seen in sporadic PTC such as SERPINA, COL8A, and PDX, while others were unique to the radiation-induced profiles including CAMK2N1, AK1, DHRS3, and PDE9A. CONCLUSIONS: This type of analysis allows an assessment of gene expression changes that are associated with a physical mechanism. These genes and chromosomal regions are potential markers for radiation-induced PTC.

TIMP1 and SERPIN-A overexpression and TFF3 and CRABP1 underexpression as biomarkers for papillary thyroid carcinoma.

BACKGROUND: No molecular pathways or specific genes are consistently associated with sporadic cases of papillary thyroid carcinoma (PTC), despite that it is the most common thyroid malignancy. Nodular goiter is an enlargement of the thyroid that is a compensatory response to a perturbation in normal thyroid homeostasis. It has been disputed in the literature that patients presenting with goiter have a higher incidence of PTC. The identification of molecular events that are common to both goiter and PTC could explain the overlap of these two disorders. METHODS: We used high-density oligonuleotide arrays to perform molecular profiling of PTC and nodular goiter with paired normal samples. RESULTS: Specifically, increased expression of SERPIN-A (proteinase inhibitor-alpha antitrypsin) and TIMP 1 (tissue inhibitor of metalloproteinase 1) identified these as candidate molecular biomarkers for PTC. Decreases in the CRABP1 (cellular retinoic acid binding protein 1) and TFF3 (trefoil factor 3) expression levels identified these as candidate molecular biomarkers as well. The same analysis was performed to identify genes showing specific alterations in goiter tissues. CONCLUSIONS: This is the first report to our knowledge that compares the gene expression profiles of PTC and goiter. Our results suggest that PTC and goiter share very limited overlap in transcript expression.