Clinical Utility of Multigene Profiling Assays in Early-Stage Invasive Breast Cancer: An Ontario Health (Cancer Care Ontario) Clinical Practice Guideline

Objective: The purpose of this guideline is to determine the clinical utility of multigene profiling assays in individuals with early-stage invasive breast cancer. Methods: This guideline was developed by Ontario Health (Cancer Care Ontario)’s Program in Evidence-Based Care (PEBC) through a systematic review of relevant literature, patient- and caregiver-specific consultation and internal and external reviews. Recommendation 1: In patients with early-stage estrogen receptor (ER)-positive/human epidermal growth factor 2 (HER2)-negative breast cancer, clinicians should consider using multigene profiling assays (i.e., Oncotype DX, MammaPrint, Prosigna, EndoPredict, and the Breast Cancer Index) to help guide the use of systemic therapy. Recommendation 2: In patients with early-stage node-negative ER-positive/HER2-negative disease, clinicians may use a low-risk result from Oncotype DX, MammaPrint, Prosigna, EndoPredict/EPclin, or Breast Cancer Index assays to support a decision not to use adjuvant chemotherapy. Recommendation 3: In patients with node-negative ER-positive/HER2-negative disease, clinicians may use a high-risk result from Oncotype DX to support a decision to offer chemotherapy. A high Oncotype DX recurrence score is capable of predicting adjuvant chemotherapy benefit. Recommendation 4: In postmenopausal patients with ER-positive/HER2-negative tumours and one to three nodes involved (N1a disease), clinicians may withhold chemotherapy based on a low-risk Oncotype DX or MammaPrint score if the decision is supported by other clinical, pathological, or patient-related factors. Recommendation 5: The evidence to support the use of molecular profiling to select the duration of endocrine therapy is evolving. In patients with ER-positive disease, clinicians may consider using a Breast Cancer Index (H/I) high assay result to support a decision to extend adjuvant endocrine therapy if the decision is supported by other clinical, pathological, or patient-related factors

Tumor BRCA Testing in High Grade Serous Carcinoma: Mutation Rates and Optimal Tissue Requirements

Background: Approximately 25% of women diagnosed with tubo-ovarian high-grade serous carcinoma have germline deleterious mutations in BRCA1 or BRCA2, characteristic of hereditary breast and ovarian cancer syndrome, while somatic mutations have been detected in 3–7%. We set out to determine the BRCA mutation rates and optimal tissue requirements for tumor BRCA testing in patients diagnosed with tubo-ovarian high-grade serous carcinoma. Methods: Sequencing was performed using a multiplexed polymerase chain reaction-based approach on 291 tissue samples, with a minimum sequencing depth of 500X and an allele frequency of >5%. Results: There were 253 surgical samples (87%), 35 biopsies (12%) and 3 cytology cell blocks (1%). The initial failure rate was 9% (25/291), including 9 cases (3%) with insufficient tumor, and 16 (6%) with non-amplifiable DNA. Sequencing was successful in 78% (228/291) and deemed indeterminate due to failed exons or variants below the limit of detection in 13% (38/291). Repeat testing was successful in 67% (28/42) of retested samples, with an overall success rate of 86% (251/291). Clinically significant (pathogenic, likely pathogenic) variants were identified in 17% (48/276) of complete and indeterminate cases. Successful sequencing was dependent on sample type, tumor cellularity and size (p ≤ 0.001) but not on neoadjuvant chemotherapy or age of blocks (p > 0.05). Conclusions: Our study shows a 17% tumor BRCA mutation rate, with an overall success rate of 86%. Biopsy and cytology samples and post-chemotherapy specimens can be used for tumor BRCA testing, and optimal tumors measure ≥5 mm in size with at least 20% cellularity

Mll5 Is Required for Normal Spermatogenesis

Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects

The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

BACKGROUND: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. RESULTS: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. CONCLUSIONS: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Standardized Definitions for Efficacy End Points in Neoadjuvant Breast Cancer Clinical Trials: NeoSTEEP.

PURPOSE: The Standardized Definitions for Efficacy End Points (STEEP) criteria, established in 2007 and updated in 2021 (STEEP 2.0), provide standardized definitions of adjuvant breast cancer (BC) end points. STEEP 2.0 identified a need to separately address end points for neoadjuvant clinical trials. The multidisciplinary NeoSTEEP working group of experts was convened to critically evaluate and align neoadjuvant BC trial end points. METHODS: The NeoSTEEP working group concentrated on neoadjuvant systemic therapy end points in clinical trials with efficacy outcomes-both pathologic and time-to-event survival end points-particularly for registrational intent. Special considerations for subtypes and therapeutic approaches, imaging, nodal staging at surgery, bilateral and multifocal diseases, correlative tissue collection, and US Food and Drug Administration regulatory considerations were contemplated. RESULTS: The working group recommends a preferred definition of pathologic complete response (pCR) as the absence of residual invasive cancer in the complete resected breast specimen and all sampled regional lymph nodes (ypT0/Tis ypN0 per AJCC staging). Residual cancer burden should be a secondary end point to facilitate future assessment of its utility. Alternative end points are needed for hormone receptor-positive disease. Time-to-event survival end point definitions should pay particular attention to the measurement starting point. Trials should include end points originating at random assignment (event-free survival and overall survival) to capture presurgery progression and deaths as events. Secondary end points adapted from STEEP 2.0, which are defined from starting at curative-intent surgery, may also be appropriate. Specification and standardization of biopsy protocols, imaging, and pathologic nodal evaluation are also crucial. CONCLUSION: End points in addition to pCR should be selected on the basis of clinical and biologic aspects of the tumor and the therapeutic agent investigated. Consistent prespecified definitions and interventions are paramount for clinically meaningful trial results and cross-trial comparison

The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13-14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13-14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies.The METABRIC project was funded by Cancer Research UK, the British Columbia Cancer Foundation and Canadian Breast Cancer Foundation BC/Yukon. This sequencing project was funded by CRUK grant C507/A16278 and Illumina UK performed all the sequencing. The authors also acknowledge the support of the University of Cambridge, Hutchinson Whampoa, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre, the Centre for Translational Genomics (CTAG) Vancouver and the BCCA Breast Cancer Outcomes Unit. We thank the Genomics, Histopathology, and Biorepository Core Facilities at the Cancer Research UK Cambridge Institute, and the Addenbrooke’s Human Research Tissue Bank (supported by the National Institute for Health Research Cambridge Biomedical Research Centre).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1147

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome

A tumor DNA complex aberration index is an independent predictor of survival in breast and ovarian cancer.

Complex focal chromosomal rearrangements in cancer genomes, also called "firestorms", can be scored from DNA copy number data. The complex arm-wise aberration index (CAAI) is a score that captures DNA copy number alterations that appear as focal complex events in tumors, and has potential prognostic value in breast cancer. This study aimed to validate this DNA-based prognostic index in breast cancer and test for the first time its potential prognostic value in ovarian cancer. Copy number alteration (CNA) data from 1950 breast carcinomas (METABRIC cohort) and 508 high-grade serous ovarian carcinomas (TCGA dataset) were analyzed. Cases were classified as CAAI positive if at least one complex focal event was scored. Complex alterations were frequently localized on chromosome 8p (n = 159), 17q (n = 176) and 11q (n = 251). CAAI events on 11q were most frequent in estrogen receptor positive (ER+) cases and on 17q in estrogen receptor negative (ER-) cases. We found only a modest correlation between CAAI and the overall rate of genomic instability (GII) and number of breakpoints (r = 0.27 and r = 0.42, p < 0.001). Breast cancer specific survival (BCSS), overall survival (OS) and ovarian cancer progression free survival (PFS) were used as clinical end points in Cox proportional hazard model survival analyses. CAAI positive breast cancers (43%) had higher mortality: hazard ratio (HR) of 1.94 (95%CI, 1.62-2.32) for BCSS, and of 1.49 (95%CI, 1.30-1.71) for OS. Representations of the 70-gene and the 21-gene predictors were compared with CAAI in multivariable models and CAAI was independently significant with a Cox adjusted HR of 1.56 (95%CI, 1.23-1.99) for ER+ and 1.55 (95%CI, 1.11-2.18) for ER- disease. None of the expression-based predictors were prognostic in the ER- subset. We found that a model including CAAI and the two expression-based prognostic signatures outperformed a model including the 21-gene and 70-gene signatures but excluding CAAI. Inclusion of CAAI in the clinical prognostication tool PREDICT significantly improved its performance. CAAI positive ovarian cancers (52%) also had worse prognosis: HRs of 1.3 (95%CI, 1.1-1.7) for PFS and 1.3 (95%CI, 1.1-1.6) for OS. This study validates CAAI as an independent predictor of survival in both ER+ and ER- breast cancer and reveals a significant prognostic value for CAAI in high-grade serous ovarian cancer

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BACKGROUND: Invasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells. METHODS: We examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR. RESULTS: Using GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC. CONCLUSION: IDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies