Seth Rummel

Genomic instability in the breast microenvironment? A critical evaluation of the evidence

Tumor-associated stroma plays an active role in breast pathogenesis, with alterations in cell signaling, proliferation and angiogenesis contributing to successful tumorigenesis. Although epigenetic modifications to DNA, as well as changes in RNA and protein expression have been identified in tumor-associated stroma, there is considerable debate regarding the presence of chromosomal alterations, detected as loss of heterozygosity/allelic imbalance events in histologically normal stromal cells adjacent to the breast carcinomas. Previous studies have detected loss of heterozygosity/allelic imbalance at varying distances from the tumor margin, and patterns of chromosomal change have been linked to tumor grade and lymph node status. By contrast, recent reports challenge the presence of genomic instability in stromal cells of the breast tumor microenvironment, leading to the speculation that the loss of heterozygosity/allelic imbalance studies, based almost exclusively on microsatellite-based profiling of formalin-fixed, paraffin-embedded tissues, do not accurately measure the true genomic content of the tumor microenvironment but rather are a reflection of technological artifact. In this perspective, we present evidence surrounding the debate and critically evaluate arguments, both pros and cons, over the presence or absence of genomic instability tumor-associated stroma.

The role of the histoblood ABO group in cancer

Since the first link between blood type and cancer was described in 1953, numerous studies have sought to determine whether the histoblood ABO group is associated with tumorigenesis. In 2009, the first significant association between a SNP located within the ABO glycosyltransferase gene and increased risk of pancreatic cancer was reported. Here, we describe the history and possible functions of the histoblood ABO group and then provide evidence for a role of blood group antigens in the most common cancer types worldwide using both blood type and SNP data. We also explore whether confusion regarding the role of blood type in cancer risk may be attributable to heterogeneity within tumor types.

PSPHL and breast cancer in African American women: causative gene or population stratification?

BackgroundPhophoserine phosphatase-like (PSPHL) is expressed at significantly higher levels in breast tumors from African American women (AAW) compared to Caucasian women (CW). How overexpression of PSPHL contributes to outcome disparities is unclear, thus, molecular mechanisms driving expression differences between populations were evaluated.ResultsPCR was used to detect deletion of 30-Kb of chromosome 7p11 including the first three exons of PSPHL using genomic DNA from AAW (199 with invasive breast cancer, 360 controls) and CW (invasive breast cancer =589, 364 controls). Gene expression levels were evaluated by qRT-PCR using RNA isolated from tumor tissue and blood. Data were analyzed using chi-square analysis and Mann–Whitney U-tests; P < 0.05 was used to define significance. Gene expression levels correlated with deletion status: patients homozygous for the deletion had no detectable expression of PSPHL, while heterozygous had expression levels 2.1-fold lower than those homozygous for retention of PSPHL. Homozygous deletion of PSPHL was detected in 61% of CW compared to 6% of AAW with invasive breast cancer (P < 0.0001); genotype frequencies did not differ significantly between AAW with and without breast cancer (P = 0.211).ConclusionsThus, deletion of 7p11, which prevents expression of PSPHL, is significantly higher in CW compared to AAW, suggesting that this 30-kb deletion and subsequent disruption of PSPHL may be a derived trait in Caucasians. The similar frequency of the deletion allele in AAW with and without invasive breast cancer suggests that this difference represent population stratification, and does not contribute to cancer disparities.

Genomic (in)stability of the breast tumor microenvironment.

The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations reflects technological artifact rather than the true genomic content of the tumor microenvironment. Thus, breast stroma specimens from 112 women undergoing reductive mammoplasty (n = 7), prophylactic mastectomy (n = 6), or mastectomy for a breast disease (n = 99) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was conducted using a panel of 52 microsatellite markers in 484 stromal specimens from 98 women, of which 92% had no detectable AI events. When compared with previously generated AI data from 77 formalin-fixed, paraffin-embedded (FFPE) stroma specimens, 42% of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P < 0.001) than that found in frozen specimens (0.45%). This comparison of AI between FFPE and research-grade specimens suggests that past reports of AI in breast stroma reflect artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage. Furthermore, SNP data were generated from a subset of 86 stromal specimens using SNP arrays and copy number alterations were identified using Partek Genomics Suite. For 95% of the specimens, no detectable copy number alterations were found and the 11 changes that were detected were small and not shared between specimens. These data, therefore, support a model in which the tumor microenvironment is genetically stable. Mol Cancer Res; 10(12); 1526–31. ©2012 AACR.

Relationships between the ABO blood group SNP rs505922 and breast cancer phenotypes: a genotype-phenotype correlation study

BackgroundTo date, evaluation of the association of the ABO blood group and breast cancer has yielded mixed results. SNP rs505922, located within the first intron of the ABO gene, has been associated with the adenocarcinoma subtype of pancreatic cancer. To evaluate the association between genetic variation in the ABO blood group and risk of breast cancer, rs505922 was genotyped in 629 Caucasian women with invasive breast cancer, representing a variety of clinical and pathological tumor types.MethodsGenomic DNA was isolated from blood. TaqMan SNP assay C_2253769_10 was used to determine genotypes for each patient at rs505922. Statistical analysis was performed using chi-square analysis using a P-value <0.05 to define significance.ResultsGenotypes were generated for 100% of the 629 patients in this study. Allele and genotype frequencies did not vary significantly for age at diagnosis, tumor stage, size or grade, hormone, HER2 or lymph node status, intrinsic subtype, tumor type or patient outcome.ConclusionsAllele frequencies for rs505922 did not differ between women with breast cancer and published HapMap frequencies from women of European descent. Further stratification into different tumor phenotypes also failed to reveal an association between rs505922 and any clinical characteristics. Together, these data suggest that the minor allele of rs505922 and the resulting non-O blood types are not associated with increased risk or less favorable tumor characteristics or prognosis in breast cancer.

Tumour location within the breast: Does tumour site have prognostic ability?

Introduction Tumour location within the breast varies with the highest frequency in the upper outer quadrant (UOQ) and lowest frequency in the lower inner quadrant (LIQ). Whether tumour location is prognostic is unclear. To determine whether tumour location is prognostic, associations between tumour site and clinicopathological characteristics were evaluated. Materials and Methods All patients enrolled in the Clinical Breast Care Project whose tumour site—UOQ, upper inner quadrant (UIQ), central, LIQ, lower outer quadrant (LOQ)—was determined by a single, dedicated breast pathologist were included in this study. Patients with multicentric disease (n = 122) or tumours spanning multiple quadrants (n = 381) were excluded from further analysis. Clinicopathological characteristics were analysed using chi-square tests for univariate analysis with multivariate analysis performed using principal components analysis (PCA) and multiple logistic regression. Significance was defined as P < 0.05. Results Of the 980 patients with defined tumour location, 30 had bilateral disease. Tumour location in the UOQ (51.5%) was significantly higher than in the UIQ (15.6%), LOQ (14.2%), central (10.6%), or LIQ (8.1%). Tumours in the central quadrant were significantly more likely to have higher tumour stage (P = 0.003) and size (P < 0.001), metastatic lymph nodes (P < 0.001), and mortality (P = 0.011). After multivariate analysis, only tumour size and lymph node status remained significantly associated with survival. Conclusions Evaluation of tumour location as a prognostic factor revealed that although tumours in the central region are associated with less favourable outcome, these associations are not independent of location but rather driven by larger tumour size. Tumours in the central region are more difficult to detect mammographically, resulting in larger tumour size at diagnosis and thus less favourable prognosis. Together, these data demonstrate that tumour location is not an independent prognostic factor.

Sequence-based detection of mutations in cadherin 1 to determine the prevalence of germline mutations in patients with invasive lobular carcinoma of the breast

BackgroundLoss of cadherin 1 (CDH1) expression, which is normally involved in cell adhesion and maintenance of tissue architecture, is a hallmark of invasive lobular carcinoma (ILCA). Because hereditary cancers may require different risk reduction, counseling and treatment options than sporadic cancer, it is critical to determine the prevalence of germline CDH1 mutations in patients with ILCA.MethodsAll patients with ILCA (n = 100) previously enrolled in the Clinical Breast Care Project were identified. Genomic DNA was isolated from peripheral blood samples and DNA variants were detected for each exon of CDH1 using high-resolution melting technology followed by direct sequencing.ResultsWithin the 100 samples screened, four nonsynonymous variants were detected: A592T in one Hispanic patient, A617T in two patients, both African American, P825L in a Causasian patient whose grandmother had stomach cancer, and G879S in a Caucasian patient. Further evaluation of A617T in an additional 165 African American patients found that 11 patients, none with ILCA, carried this variant including one patient who was homozygous for the variant.ConclusionsCDH1 mutations are infrequent in patients with ILCA, and the variants that were detected have been classified as non-pathogenic. These data suggest that ILCA does not have a significant hereditary component and do not support CDH1 gene mutation testing in patients with ILCA.

Abstract P3-08-11: Contribution of germline mutations in cancer predisposition genes to tumor etiology in women diagnosed with invasive breast cancer before 40 years

Background: Although breast cancer in young women (YW) accounts for Methods: All patients diagnosed before 40 years were identified. Family history was classified as average (No first or second degree relatives with breast or ovarian cancer or 1 second degree relative with breast cancer diagnosed >50 years), moderate (1 first degree relative with breast cancer, 2 first or second degree relatives with breast cancer diagnosed >50 years or 1 first or second degree relative with ovarian cancer) or strong (>1 first or second degree relative with bilateral breast cancer, breast and ovarian cancer or male breast cancer, >2 first or second degree relatives with breast cancer before age 50, breast and ovarian cancer in different relatives, ovarian cancer at any age or >3 first or second degree relatives with breast cancer at any age). Genomic DNA was isolated from blood samples and targeted sequencing was performed using the TruSight Cancer panel (Illumina). Pathogenic mutations were identified using VariantStudio. Results: Seven percent (132/1950) of patients enrolled in the CBCP were diagnosed Discussion: Pathogenic mutations were found in 21% of young women with breast cancer with an additional 22% harboring potentially pathogenic mutations. BRCA1 mutations were associated with triple negative breast tumors in individuals with moderate to strong family history and BRCA2 mutations were associated with ER+ tumors in young women without strong family histories. These data demonstrate that although genetic predisposition may account for 21-43% of tumors, >50% of tumors in young women are not attributable to genetic causes, and identification of those non-genetic factors is critical to reduce the burden of breast cancer in this population. Citation Format: Ellsworth RE, Rummel SK, Shriver CD. Contribution of germline mutations in cancer predisposition genes to tumor etiology in women diagnosed with invasive breast cancer before 40 years [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-08-11.

Abstract 1329: PSPHL and breast cancer in African American women: Causativegene or population stratification.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The phophoserine phosphatase-like (PSPHL) gene, first identified as overexpressed in fibroblasts from patients with Fanconi anemia, is expressed at significantly higher levels in breast tumors as well as stromal tissues from African American women (AAW) compared to Caucasian women (CW). The function of PSPHL has not been determined, thus how overexpression of PSPHL impacts tumor etiology or contributes to outcome disparities in African American women is unclear. Here, we evaluated the molecular mechanisms driving expression differences between populations to determine whether expression differences are causative or represent population stratification. Methods: A PCR-based approach was used to detect the retention or deletion of a 30-kb segment of 7p11 that includes the first three exons of PSPHL. Genotypes were determined using genomic DNA from AAW with invasive breast cancer (n=220), AAW without breast cancer (n = 352) and CW with invasive breast cancer (n=508). Gene expression levels were evaluated by qRT-PCR using RNA isolated from breast tumors from 52 CW and 43 AAW. Genotype frequencies were analyzed using chi-square analysis and gene expression levels by a Mann-Whitney U-test; P<0.05 was used to define significance. Results: Gene expression levels were correlated with the presence or absence of the 30-kb deletion of 7p11: patients homozygous for the deletion had no detectable expression of PSPHL, while patients heterozygous for the deletion had expression levels 2.5-fold lower than those homozygous for retention of PSPHL. When the frequency of the deletion was evaluated by ethnicity, 62% of CW were homozygous for the deletion of PSPHL compared to 5% of AAW with invasive breast cancer (P<0.00005). In contrast, genotype frequencies did not differ significantly between AAW with invasive breast cancer compared to AAW controls (P=0.116). Conclusions: The deletion of 30-kb of chromosome 7p11 prevents expression of PSPHL. The prevalence of the deletion allele is 77% in CW compared to 23% in AAW, suggesting that this 30-kb deletion and subsequent disruption of PSPHL may be a derived trait in Caucasians. Although the deletion frequency and associated expression differ significantly between AAW and CW with invasive breast cancer, the similar frequency of the deletion allele in AAW with and without invasive breast cancer suggests that these genetic differences represent population stratification, and do not contribute to outcome differences between AAW and CW with breast cancer. Citation Format: Seth Rummel, Cayla Penatzer, Craig D. Shriver, Rachel E. Ellsworth. PSPHL and breast cancer in African American women: Causativegene or population stratification. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1329. doi:10.1158/1538-7445.AM2013-1329

Abstract 2624: Evaluation of the association of the ABO blood group SNP rs505922 with breast cancer phenotypes

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: To date, evaluation of the association of the ABO blood group and breast cancer has yielded mixed results. SNP [rs505922][1], located within the first intron of the ABO gene, has been associated with the adenocarcinoma subtype of pancreatic cancer. To determine whether the ABO blood group is associated with risk of breast cancer, [rs505922][1] was genotyped in 629 Caucasian women with invasive breast cancer, representing a variety of clinical and pathological tumor types. Methods: Genomic DNA was isolated from blood clots. TaqMan SNP assay C\_2253769\_10 was used to determine the genotypes for rs505922. Statistical analysis was performed using chi-square analysis using a P-value <0.05 to define significance. Results: Genotypes were generated for 100% of the 629 patients in this study. Allele and genotype frequencies did not vary significantly for age at diagnosis, tumor stage, size or grade, hormone, HER2 or lymph node status, intrinsic subtype, tumor type or patient outcome. Conclusions: Allele frequencies for [rs505922][1] did not differ between women with breast cancer and published HapMap frequencies. Further stratification into different tumor phenotypes also failed to reveal an association between [rs505922][1] and any tumor characteristics. Together, these data suggest that the minor allele of [rs505922][1] and the resulting non-O blood types, are not associated with increased risk or less favorable tumor characteristics or prognosis in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2624. doi:1538-7445.AM2012-2624 [1]: /lookup/external-ref?link_type=GEN&access_num=rs505922&atom=%2Fcanres%2F72%2F8_Supplement%2F2624.atom