RESUMEN
PURPOSE: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. METHODS: HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. RESULTS: HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). CONCLUSION: The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/genética , Antígeno B7-H1/genética , Genes BRCA2 , Mutación , Recombinación Homóloga/genéticaRESUMEN
BACKGROUND: Subclonal evolution during primary breast cancer treatment is largely unexplored. We aimed to assess the dynamic changes in subclonal composition of treatment-naïve breast cancers during neoadjuvant chemotherapy. METHODS: We performed whole exome sequencing of tumor biopsies collected before, at therapy switch, and after treatment with sequential epirubicin and docetaxel monotherapy in 51 out of 109 patients with primary breast cancer, who were included in a prospectively registered, neoadjuvant single-arm phase II trial. RESULTS: There was a profound and differential redistribution of subclones during epirubicin and docetaxel treatment, regardless of therapy response. While truncal mutations and main subclones persisted, smaller subclones frequently appeared or disappeared. Reassessment of raw data, beyond formal mutation calling, indicated that the majority of subclones seemingly appearing during treatment were in fact present in pretreatment breast cancers, below conventional detection limits. Likewise, subclones which seemingly disappeared were still present, below detection limits, in most cases where tumor tissue remained. Tumor mutational burden (TMB) dropped during neoadjuvant therapy, and copy number analysis demonstrated specific genomic regions to be systematically lost or gained for each of the two chemotherapeutics. CONCLUSIONS: Sequential epirubicin and docetaxel monotherapy caused profound redistribution of smaller subclones in primary breast cancer, while early truncal mutations and major subclones generally persisted through treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00496795 , registered on July 4, 2007.
Asunto(s)
Neoplasias de la Mama , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Evolución Clonal , Ciclofosfamida , Docetaxel/uso terapéutico , Epirrubicina , Femenino , Humanos , Terapia Neoadyuvante , Taxoides/efectos adversos , Taxoides/uso terapéuticoRESUMEN
BACKGROUND: The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. RESULTS: In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. CONCLUSIONS: Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.
Asunto(s)
Metilación de ADN/genética , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genética , Epigénesis Genética/genética , Femenino , Humanos , Posmenopausia , Valores de ReferenciaRESUMEN
BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (ß-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P=4.3 × 10-4 and P=7.1 × 10-4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P=.039 and P=.070, respectively) but also the proapoptotic gene PUMA (P=.010 and P=.033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.