Lise Sveen

Prevalence of BRCA1 and BRCA2 mutations among clinic-based African American families with breast cancer

Abstract. To define the prevalence and relative contributions of BRCA1 and BRCA2 mutations among African American families with breast cancer, we analyzed 28 DNA samples from patients identified through two oncology clinics. The entire coding regions of BRCA1 and BRCA2 were screened by protein truncation test, heteroduplex analysis, or single-stranded conformation polymorphism followed by DNA sequencing of variant bands. Deleterious protein-truncating BRCA1 and BRCA2 mutations were identified in five patients or 18% of the entire cohort. Only 8% (1 of 13) of women with a family history of breast cancer, but no ovarian cancer, had mutations. The mutation rates were higher for women from families with a history of breast cancer and at least one ovarian cancer (three of six, 50%). One woman with a family history of undocumented cancers was also found to carry a deleterious mutation in BRCA2. The spectrum of mutations was unique in that one novel BRCA1 mutation (1625del5) and three novel BRCA2 mutations (1536del4, 6696delTC, and 7795delCT) were identified. No recurrent mutations were identified in this cohort, although one BRCA2 (2816insA) mutation had been previously reported. In addition, two BRCA1 and four BRCA2 missense mutations of unknown significance were identified, one of which was novel. Taken together with our previous report on recurrent mutations seen in unrelated families, we conclude that African Americans have a unique mutation spectrum in BRCA1 and BRCA2 genes, but recurrent mutations are likely to be more widely dispersed and therefore not readily identifiable in this population.

MYC is amplified in BRCA1-associated breast cancers.

Purpose: Germ-line mutations in the BRCA1 tumor suppressor gene predispose to early onset breast cancers with a distinct phenotype characterized by high tumor grade, aneuploidy, high proliferation rate, and estrogen receptor-negativity. The molecular mechanisms and cooperative oncogenes contributing to multistep tumor progression in cells lacking BRCA1 are not well defined. To examine whether C-MYC (MYC), a transforming oncogene associated with genetic instability, contributes to multistep tumor progression in BRCA1-associated breast cancer, we have analyzed tumors from women with hereditary BRCA1-mutated and sporadic breast cancers. Experimental Design: We performed fluorescence in situ hybridization using a MYC:CEP8 assay on formalin-fixed paraffin-embedded tumor tissues from 40 women with known deleterious germ-line BRCA1 mutations and 62 sporadic cases, including 20 cases with hypermethylation of the BRCA1 gene promoter. Results: We observed a MYC:CEP8 amplification ratio ≥2 in 21 of 40 (53%) BRCA1-mutated tumors compared with 14 of 62 (23%) sporadic tumors (P = 0.003). Of the 14 sporadic cases with MYC amplification, 8 (57%) were BRCA1-methylated. In total, MYC amplification was found in a significantly higher proportion of tumors with BRCA1 dysfunction (29 of 60, 48% versus 6 of 42, 14%; P = 0.0003). In a multivariable regression model controlling for age, tumor size, and estrogen receptor status, BRCA1-mutated tumors demonstrated significantly greater mean MYC:CEP8 ratio than sporadic tumors (P = 0.02). Conclusions: Our data indicate that MYC oncogene amplification contributes to tumor progression in BRCA1-associated breast cancers. Thus, we conclude that the aggressive histopathological features of BRCA1-associated tumors are in part due to dysregulated MYC activity.

Complete allelic analysis of BRCA1 and BRCA2 variants in young Nigerian breast cancer patients

Breast cancer is a leading cause of cancer deaths among women, and is expected to claim the lives of nearly 40 000 individuals in the USA each year (American Cancer Society Breast Cancer Facts and Figures 2003–2004 ). Only 5–10% of breast cancers are associated with mutations in the susceptibility genes BRCA1 and BRCA2 . However, in cases associated with strong family history, mutation rates are higher, ranging from 16% to 26% for BRCA1 1–3 and from 7% to 13% for BRCA2 .2,3 However, many breast cancer patients with strong family histories have no obvious mutations in BRCA1 /2. While there is an active search for other breast cancer susceptibility genes, it is possible that the true contributions of BRCA1 and BRCA2 to early onset breast cancer have been underestimated. Indeed, one study has shown that only 63% of breast cancer families linked to BRCA1 are associated with detectable mutations in BRCA1 .4 Several reasons for this discrepancy are possible. For example, mutations in BRCA1 promoter sequences might be undetectable by current detection techniques. Additionally, inherited genomic rearrangements that inactivate BRCA1 and BRCA2 but cannot be detected by conventional polymerase chain reaction (PCR) based assays have been reported.5,6,7,8,9,10 Finally, it is possible that some genetic variants previously dismissed as “unclassified variants” or “polymorphisms” may have hitherto underappreciated effects on protein synthesis or function. Most studies of BRCA1 and BRCA2 associated breast cancers have focused on white populations, yet several observations suggest that there might be a genetic component to breast cancer susceptibility in families of African ancestry.11 Breast cancer is less common in African populations than in other populations but, when it does occur, it is characterised by an early age of onset and a higher mortality.12–14 Additionally, …

Abstract 462: Cytotoxic CD8+ T cells and immunosuppressive T regulatory cells are associated with aggressive breast cancer subtypes

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: An increase in tumor infiltrating lymphocytes (TILs) indicates an immune response to tumor antigens. CD8+ TILs play a vital role in cancer progression, and may have tumor suppressive effects. FoxP3+ T regulatory cells (Tregs), however, suppress a variety of immune cells - including CD4+ TILs, CD8+ TILs, dendritic cells, B cells, and macrophages - and thus may inhibit the beneficial effect these cells have on tumor suppression. Specifically, CD8+ TILs - which are a critical component of tumor-specific adaptive immunity - are a major target of Tregs. This study was aimed at understanding how these TILs correlate with breast cancer molecular subtypes in diverse population of breast cancer patients. Methods: Tissue Microarrays (TMAs) of breast cancer cases from 1992-2010 were obtained from the University of Chicago breast cancer biospecimen bank under IRB approved protocols. Molecular subtype was assigned based on immunohistochemical (IHC) staining into the following groups: luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), HER2-like (ER/PR-, HER2+) and basal-like (ER/PR/HER2-, EGFR+ and/or CK5/6+). IHC for CD8 and FoxP3 was performed on TMAs using DAKO, cat # M7103 antibody, and Abcam antibody cat #ab20034. The amount of CD8+ and FoxP3+ cells presents within the neoplastic tissue were counted manually in two high-powered fields (x400) by two independent pathologists, and recalculated for 1 mm2. The ratio of FoxP3+/CD8+ was calculated and correlated with molecular subtype. Results: Of the 292 cores evaluated, 268 were evaluable representing 134 individual cases. There were 28 basal-like (20.9%), 14 HER2 (10.4%), 78 luminal A (58.3%), and 14 luminal B (10.4%). Basal-like tumors showed the highest infiltration of both Tregs and CD8+ TILs when compared to other breast cancer subtypes, and the ratio of FoxP3+: CD8+ TILs in basal-like tumors were 1.7, 2.5, and 4.5-fold higher than in HER2, Luminal A, and Luminal B tumors respectively (p<0.05 for all correlations). Summary: FoxP3+ and CD8+ TIL infiltration density were significantly different among molecular subtypes of breast cancer. Future studies will examine the genomic determinants and prognostic significance of TILs in aggressive breast cancer subtypes, especially basal-like breast cancers. Citation Format: Galina F. Khramtsova, Rita Nanda, Ekaterina A. Khramtsova, Lise Sveen, Sope Olugbile, Olufunmilayo I. Olopade. Cytotoxic CD8+ T cells and immunosuppressive T regulatory cells are associated with aggressive breast cancer subtypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 462. doi:10.1158/1538-7445.AM2015-462

Abstract B07: Molecular characterization of colorectal cancer in West Africans

Introduction: To reduce global disparities in cancer outcomes, there is a need for more concerted efforts to develop the capacity of health care providers to appropriately diagnose and treat common cancers. Colorectal cancer (CRC) incidence in indigenous Africans is significantly lower than in Caucasians but there is a paucity of data on the genetic determinants and molecular biology of colorectal cancer in Nigerians. Lynch syndrome (LS), defined by germ-line mutation in one of the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), is the most common heritable syndrome predisposing to CRC. Loss of MMR proteins can be readily detected by immunohistochemistry (IHC) in both Lynch syndrome and sporadic CRC, and further testing for mutated BRAF protein is helpful in distinguishing Lynch syndrome from sporadic CRC. Materials and Methods: With IRB approval and under a collaborative arrangement with the University of Chicago, seven tissue microarray (TMA) blocks were constructed from CRC samples obtained from the University College Hospital in Ibadan, Federal Teaching Hospital in Gombe, Olabisi Onabanjo University in Sagamu and Ahmadu Bello University in Zaria. TMAs included duplicate 1.0-mm cores of CRC tissue and adjacent normal. For the four MMR proteins, IHC was performed on these TMAs with the following antibodies: MLH1 (Thermo Scientific Pierce Biotech), MSH2 (Life Technologies), MSH6 (Novex), PMS2 (Pierce), anti-BRAF V600E (Roche). All IHC-stained slides (MLH1, MSH2, MSH6, PMS2) were evaluated for expression levels of those proteins in the tumor tissue relative to normal tissue (control) using the following scoring criteria: normal expression was defined as nuclear staining within tumor cells, negative protein expression was defined as complete absence of nuclear staining within tumor cells, and cases with immunoreactivity in 0-10% of tumor cells were scored as equivocal. The slides stained with anti-BRAF V600E (VE1) were scored on a scale of 0 to 3. Strong cytoplasmic staining was scored as 3, medium as 2, 1 as weak, and 0 when the staining was absent. Results: Of the total evaluable CRC cases, 152 of 293 (52%) were males aged between 16 and 90 years, mean age of 47.33 +/-17.57, while there were 141 female cases, with a mean age of 49.71+/-14.75. More than a quarter of the cases were under 40 years of age. By our IHC screening algorithm, 140 (48%) of the patients had intact MMR proteins, while 153 (52%) patients had at least one MMR protein absent. The most common proteins lost were MLH1/ PMS2 (141 cases, 48.13% of total) cases, followed by the loss of MSH2/MSH6 (12 cases, 4.10% of total). Testing of the group in which MLH1/ PMS2 proteins were absent (141 patients) showed mutated BRAF in only 31 cases (22% of this group). The cases which lack MLH1/ PMS2 and are negative for the BRAF mutation (110 cases), as well as those which are MSH2/MSH6 negative (12 cases), form a group of 122 cases (41.64% of total) which would need additional tests for hypermethylation and referral for genetic counseling. Conclusion: In this dataset of relatively young onset CRC cases, a large percentage of cases are of the HNPCC subtype. Use of IHC as a universal screening test for all CRC cases, as well as follow-up referral for genetic counseling, could be an innovative approach to CRC cancer control in the population. Citation Format: Aliyu Lawan, Galina Khramtsova, David Irabor, Mustapha Ajani, Lise Sveen, Yusuf M. Abdullah, Henry O. Ebili, Umar Saad, John O. Ogunbiyi, Olufunmilayo I. Olopade, Abideen O. Oluwasola. Molecular characterization of colorectal cancer in West Africans. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B07.

Abstract 2950: Biomarkers of immune infiltration for multiplex immunofluorescence in breast cancer

Introduction: Only a subset of patients have a clinical response to cancer immunotherapy. The molecular mechanisms that mediate the immunological response or tolerance to treatment are just beginning to be understood. Several tumor signaling pathways are involved in those mechanisms: Wnt/β-catenin, STAT3, NFkB, PI3K/PTEN/AKT, and TP53. To characterize the tumor, its immune infiltrate, and its microenvironment, we examined expression of several biomarkers and determined the relationships among them in various breast cancer subtypes. Methods: 148 breast cancer (BCa) cases from the University of Chicago Breast Cancer SPORE tissue bank under IRB approved protocols were classified into four biological subtypes based on immunohistochemical (IHC) staining: luminal A, luminal B, Her2 and basal-like. IHC staining for PD-L1, CD8, FoxP3, CD68, CD163, and β-catenin was performed on tissue microarrays. CD8+ and FoxP3+ cells were counted manually and recalculated for 1 mm2. Macrophage phenotype was determined using single and double staining with CD68 for total population and CD68/CD163 for M2. CD68+ and CD163+ cells were counted manually, confirmed by Image Analysis, and recalculated for 1 mm2. PD-L1 and β-catenin (membrane-associated, cytosolic and nuclear) were scored as negative, weak positive, moderate, and strong. Results: The study cohort subtypes were 20.9% basal-like, 10.4% Her2, 58.3% luminal A, and 10.4% luminal B. The ratio of M2 to M1 cells increased with disease progression (p Conclusions and Future Directions: Our data suggests that IHC using a panel of antibodies may be a robust and suitable method for evaluating level of immune infiltration. Future studies will evaluate multiplex immunofluorescence of multiple biomarkers in breast tissue. Citation Format: April Swoboda, Galina Khramtsova, Lise Sveen, Andrey Khramtsov, Rita Nanda, Olufunmilayo Olopade. Biomarkers of immune infiltration for multiplex immunofluorescence in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2950. doi:10.1158/1538-7445.AM2017-2950

Abstract 3278: The effect of cold ischemia time on protein expression in breast cancer tissues

Background: HER2 testing is a critical tool for determining the molecular subtypes of breast tumors. However there are various factors which may influence the accuracy of HER2 immunohistochemistry (IHC) results. Those factors have been addressed by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) and outlined in the updated 2013 guideline. The ASCO/CAP guidelines advise limiting the tissues cold ischemia time as much as possible specifically to no more than one hour. A tissue9s cold ischemia time is defined as the time from the tissues removal from the patient to the initiation of fixation. Our institution works with several collaborators for whom postoperative tissue banking is performed for the majority of breast resection specimens. In most cases, the procedure of tissue excision takes 30-60 minutes. However, in some cases cold ischemia time is increased more than 60 minutes. Presently, there is limited information available on the effect of cold ischemia on HER2 IHC staining. In this study, we compared HER2 IHC results for breast tumor specimens to determine the effect of prolonged cold ischemia time on protein expression. HER2 status was confirmed by the in-situ hybridization method (FISH). Design: In this study we evaluated HER2 status using two different FDA-approved FISH and an FDA-approved IHC assay in a large cohort of invasive breast carcinomas with differing cold ischemia time in pathology laboratories at the University of Chicago (U of C) and in Ibadan, Nigeria (IMRAT). Cold ischemia time was used to stratify the samples into two groups 1-3 h (n=27 U of C cases) and >3h (n=79 MRAT cases). Both FISH and IHC were evaluated using ASCO/CAP scoring criteria. Tissue microarrays (TMAs) including duplicate 1.0-mm cores of invasive breast carcinoma were constructed utilizing formalin-fixed, paraffin-embedded tissue of 106 breast cancer patients, who had surgery at the U of C and IMRAT. HER2 status determination was performed on the TMAs utilizing IHC with the HercepTest (DAKO, USA), and FDA-approved FISH assay (PathVysions, Abbott Molecular, USA). Results: Only 3.7% of the cases examined in the 1-3 h cold ischemia time group had a discrepancy in the HER2 IHC and FISH result. However, within the >3 h cold ischemia time cohort a total of 7.6 % were identified with a minor discrepancy between the HER2 IHC and FISH results. Conclusions: In this study, we compared the effect of cold ischemia time on standard HER2 testing methodologies including FISH and IHC. Comparison of FISH and IHC showed that IHC did not identify several FISH HER2-amplified cases at >3h cold ischemia times. These findings are important in consideration of specimen preparation and could have an effect on specimen handling and fixation guidelines. Citation Format: Galina F. Khramtsova, Andrey I. Khramtsov, Lise Sveen, Abayomi Odetunde, Oyinlolu Olorunsogo Adeyanju, Olufunmilayo I. Olopade, Oluwasola A. Olayiwola. The effect of cold ischemia time on protein expression in breast cancer tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2014-3278

Abstract 5066: Molecular profiles of breast cancer in Barbados

Background: Breast cancer is a complex disease with varying incidence and mortality rates across populations. This study is the first investigation of molecular subtypes of breast tumors in a Barbados, West Indies population which is predominantly African in origin. Methods: To identify molecular subtypes of breast cancer we collected 260 formalin-fixed and paraffin-embedded (FFPE) tissue blocks from the Pathology Laboratory, Queen Elizabeth Hospital, St. Michael, Barbados. Pathologic features, including diagnosis, grade, tumor size, and axillary lymph node metastasis, were abstracted from pathology reports. Histology diagnosis, grading of invasive breast cancer and carcinoma in situ were performed using protocols of the College of American Pathologists and World Health Organization. Tissue microarrays were constructed from these 260 confirmed breast cancer samples and adjacent normal breast tissue at the University of Chicago. Immunohistochemical (IHC) assays were performed with commercial antibodies. To identify breast cancer subtypes, the tissues were evaluated for the expression of ER, PR, HER2, CK5/6 and EGFR. Vimentin staining served as a control for tissue fixation quality in archival tumors. Allred scores for ER and PR were calculated. HER2 was evaluated by IHC according to ASCO/CAP guidelines. EGFR was evaluated according to PharmDX recommendations. Vimentin and CK 5/6 were evaluated according to Dabbs, 2006. Breast cancer subtypes were defined as luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+, and/or EGFR+), HER2+/ER- (HER2+, ER-, PR-), and unclassified (negative for all five markers). Results: The mean age of the breast cancer patients was 57 years old (SD=15 years). There were 63.7% patients with ER positive tumors, 50.6% with PR positive tumors, and 21.2% with HER2 positive tumors. The distribution of breast cancer subtypes was luminal A (52.5%), luminal B (12.7%), HER2+/ER- (8.5%), basal-like (24.6%), and unclassified (1.7%). The majority of patients had high grade tumors (52.3% grade II and 39.1% grade III). The luminal A and B tumors contained ductal and lobular types and several special types of breast carcinomas such as tubular, mucinous, cribriform, signet ring cells and acinic cell types. The basal-like tumors were more likely to be medullary carcinomas. The basal-like (70.2%) and HER2+/ER- (65.0%) tumors were more likely to be high-grade tumors than the luminal A (20.2%) and luminal B (40.0%). Conclusions: We found that the proportion of estrogen receptor positive tumors in Barbados was similar to that observed in African Americans, lower than that of Caucasians and Asians but higher than Nigerian. There was a relatively high proportion of basal-like breast cancer in this cohort of African descent. Further epidemiology studies are warranted to identify reasons for this similarity, including possibly the increasing Western influence on the Barbadian lifestyle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2011-5066