Migrating the SNP array-based homologous recombination deficiency measures to next generation sequencing data of breast cancer

The first genomic scar-based homologous recombination deficiency (HRD) measures were produced using SNP arrays. As array-based technology has been largely replaced by next generation sequencing approaches, it has become important to develop algorithms that derive the same type of genomic scar scores from next generation sequencing (whole exome “WXS”, whole genome “WGS”) data. In order to perform this analysis, we introduce here the scarHRD R package and show that using this method the SNP array-based and next generation sequencing-based derivation of HRD scores show good correlation (Pearson correlation between 0.73 and 0.87 depending on the actual HRD measure) and that the NGS-based HRD scores distinguish similarly well between BRCA mutant and BRCA wild-type cases in a cohort of triple-negative breast cancer patients of the TCGA data set

Biological Stoichiometry in Human Cancer

A growing tumor in the body can be considered a complex ecological and evolutionary system. A new eco-evolutionary hypothesis (the "Growth Rate Hypothesis", GRH) proposes that tumors have elevated phosphorus (P) demands due to increased allocation to P-rich nucleic acids, especially ribosomal RNA, to meet the protein synthesis demands of accelerated proliferation.We determined the elemental (C, N, P) and nucleic acid contents of paired malignant and normal tissues from colon, lung, liver, or kidney for 121 patients. Consistent with the GRH, lung and colon tumors were significantly higher (by approximately two-fold) in P content (fraction of dry weight) and RNA content and lower in nitrogen (N):P ratio than paired normal tissue, and P in RNA contributed a significantly larger fraction of total biomass P in malignant relative to normal tissues. Furthermore, patient-specific differences for %P between malignant and normal tissues were positively correlated with such differences for %RNA, both for the overall data and within three of the four organ sites. However, significant differences in %P and %RNA between malignant and normal tissues were not seen in liver and kidney and, overall, RNA contributed only approximately 11% of total tissue P content.Data for lung and colon tumors provide support for the GRH in human cancer. The two-fold amplification of P content in colon and lung tumors may set the stage for potential P-limitation of their proliferation, as such differences often do for rapidly growing biota in ecosystems. However, data for kidney and liver do not support the GRH. To account for these conflicting observations, we suggest that local environments in some organs select for neoplastic cells bearing mutations increasing cell division rate ("r-selected," as in colon and lung) while conditions elsewhere may select for reduced mortality rate ("K-selected," as in liver and kidney)

Benefits, risks, and safety of external beam radiation therapy for breast cancer

Lindsay C Brown,1 Robert W Mutter,1 Michele Y Halyard2 1Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 2Department of Radiation Oncology, Mayo Clinic, Scottsdale, AZ, USA Abstract: Breast cancer is a common and complex disease often necessitating multimodality care. Breast cancer may be treated with surgical resection, radiotherapy (RT), and systemic therapy, including chemotherapy, hormonal therapy, and targeted therapies, or a combination thereof. In the past 50 years, RT has played an increasingly significant role in the treatment of breast cancer, resulting in improvements in locoregional control and survival for women undergoing mastectomy who are at high risk of recurrence, and allowing for breast conservation in certain settings. Although radiation provides significant benefit to many women with breast cancer, it is also associated with risks of toxicity, including cardiac and pulmonary toxicity, lymphedema, and secondary malignancy. RT techniques have advanced and continue to evolve dramatically, offering increased precision and reproducibility of treatment delivery and flexibility of treatment schedule. This increased sophistication of RT offers promise of improved outcomes by maintaining or improving efficacy, reducing toxicity, and increasing patient access and convenience. A review of the role of radiation therapy in breast cancer, its associated toxicities and efforts in toxicity reduction is presented. Keywords: breast malignancy, radiotherapy, toxicity, outcome

Abstract P3-04-07: Functional BRCA-pathway defects in sporadic breast cancers rarely show a significant reduction in BRCA-pathway proteins

Abstract Background: Array comparative genomic hybridization (aCGH) studies have revealed that a subgroup of sporadic breast cancers have a similar genetic landscape as BRCA-mutation carriers, characterized by widespread large-size deletions across the genome. We have previously shown that a functional assay of homologous recombination, using radiation-induced RAD51 foci, correlates closely with the aCGH findings. The prevalence of functional defects in the BRCA-pathway is estimated to be ∼25%, and present in all subtypes of breast cancer regardless of estrogen receptor or HER2 status. Here we sought to investigate the candidate genes and proteins that may drive this BRCA-like phenotype in sporadic breast cancers. Methods: Fresh human breast cancer samples were obtained to query the BRCA-pathway, under an IRB-approved study. As previously, the ability to assemble RAD51 foci was the defining functional assay. A 4-day growth assay assessed whether BRCA-pathway defective tumors were sensitive to crosslinking agents. Nanostring-based gene expression analysis was performed to interrogate the transcript level of key players in the BRCA-pathway. Massively parallel whole exome sequencing analysis is in progress, since tumor and normal germline DNA was obtained for all these samples. Results: Previously we found the incidence of BRCA-pathway defective tumors was 18/71 (25.3%). The current analysis relates to samples obtained in the last 3 years, including 42 patients in the initial series and 18 additional samples, for which DNA, RNA and immunohistochemistry analyses were successfully performed. Known mutation carriers were excluded from the study. RAD51 focus induction revealed a bimodal distribution: no induction (0.75-1.25) or an induction by &amp;gt; 75%. 17 tumors were repair-defective and 43 were proficient, consistent with our previous analysis. 16 of the 17 repair-defective tumors had the lowest IC50 to mitomycin C, further supporting the functional phenotype classification. Since many of the BRCA-pathway proteins are cell cycle regulated, we determined that cell proliferation markers (e.g. Ki67, PCNA) were not different between the repair defective and proficient tumors. Nanostring gene expression analysis revealed no difference in the expression levels of BRCA1, BRCA2 and RAD51. Since BRCA1 protein recruitment was defective in the majority of repair-defective tumors, we also analysed RNF8, RNF168, RAP80, ABRAXAS, MERIT40 or BRCC36 and found no abnormality. The methylation status of the BRCA1 promoter is being tested for all cases, however our results suggest that reduced mRNA and protein levels of BRCA1, BRCA2 or RAD51 are not a major cause of BRCA-pathway defects. Conclusions: The cause of BRCA-pathway defects in sporadic breast cancer cannot be explained by a reduction in mRNA or protein for members of the BRCA-pathway in the vast majority of cases. Methylation of BRCA1 to produce a reduction in mRNA sufficient to cause a repair defective phenotype is likely to be relatively uncommon. Whole exome analysis may reveal a common explanation for this biologically and clinically relevant phenotype. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-07.</jats:p

PD10-02: Sporadic Breast Cancers Show Defects in the BRCA1-BRCA2 Pathway of Homologous Recombination in All Biomarker-Defined Sub-Types of Breast Cancer.

Abstract Background: Mutation carriers of BRCA1 and BRCA2 are well known to develop early onset breast cancer, with loss of the second allele occurring in the development of the tumor. However, by array comparative genomic hybridization (aCGH) studies, some sporadic breast cancers have a similar “genetic landscape” as BRCA-mutation carriers, showing large losses and gains across the genome. We have now identified that DNA repair defects involving the BRCA1-BRCA2 pathway can occur in the absence of mutations in either gene and in the absence of a deficiency in either protein. Methods: Fresh human breast cancer samples were irradiated, ex-vivo, to look for the ability to assemble RAD51 protein macrocomplexes or foci. Primary breast cancer specimens were obtained from consented patients with non-metastatic, invasive carcinomas following lumpectomy or mastectomy, without neoadjuvant cytotoxic or hormonal therapy. A single cell suspension was prepared from the tumor, with one half irradiated to 10Gy and the other half mock-treated. After 4h, cells were mounted, fixed on slides, and stained with anti-Rad51, anti-BRCA1, and anti-γH2AX antibodies. At least 200 nuclei were examined and scored using confocal microscopy. A failure to induce RAD51 nuclear foci by 2-fold after ionizing radiation was designated as defective in homologous recombination (HR). Results: For the 71 patient samples analyzed, we have 14 triple-negative tumors, of which 6 are HR-defective (42.8%); for Her2-amplified tumors, we have 6/19 (31.6%) that are HR-defective and for ER+ tumors 6/38 (15.8%). The overall incidence of HR-defective tumors is 18/71 (25.3%), which is substantially higher than we would have expected. Known mutation carriers were not included in the study, since these samples are BRCA-HR-defective in all cases we have tested. For the more recently acquired samples, we have undertaken additional tests to characterize the tumors: short-term growth assays in response to mitomycinC to validate that HR-defective tumors are indeed sensitive to cross-linking agents; and, a pilot analysis to study aCGH patterns in HR-defective tumors. The latter studies have compared 6 repair-deficient and 6 repair-proficient tumors using unsupervised cluster analysis of large block deletions or large block copy number increase, which clearly reveal that large block alterations are linked to repair-deficient tumors. Conclusions: There is a significant incidence of BRCA-HR defective sporadic breast cancers, as determined by RAD51 function in response to ionizing radiation plus genetic landscape alterations using aCGH. The pool of breast cancers that are susceptible to repair targeting strategies is larger than expected and is not readily defined by conventional diagnostic biomarker classification. These findings may account for the failure of the recent phase III study of the addition of iniparib to carboplatin and gemcitabine in triple-negative cancer as only a minority of the tumors will be susceptible to this targeting strategy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-02.</jats:p