Laxmi Silwal-Pandit

TP53 mutation spectrum in breast cancer is subtype specific and has distinct prognostic relevance.

Purpose: In breast cancer, the TP53 gene is frequently mutated and the mutations have been associated with poor prognosis. The prognostic impact of the different types of TP53 mutations across the different molecular subtypes is still poorly understood. Here, we characterize the spectrum and prognostic significance of TP53 mutations with respect to the PAM50 subtypes and integrative clusters (IC). Experimental Design: TP53 mutation status was obtained for 1,420 tumor samples from the METABRIC cohort by sequencing all coding exons using the Sanger method. Results: TP53 mutations were found in 28.3% of the tumors, conferring a worse overall and breast cancer-specific survival [HR = 2.03; 95% confidence interval (CI), 1.65–2.48, P < 0.001], and were also found to be an independent marker of poor prognosis in estrogen receptor-positive cases (HR = 1.86; 95% CI, 1.39–2.49, P < 0.001). The mutation spectrum of TP53 varied between the breast cancer subtypes, and individual alterations showed subtype-specific association. TP53 mutations were associated with increased mortality in patients with luminal B, HER2-enriched, and normal-like tumors, but not in patients with luminal A and basal-like tumors. Similar observations were made in ICs, where mutation associated with poorer outcome in IC1, IC4, and IC5. The combined effect of TP53 mutation, TP53 LOH, and MDM2 amplification on mortality was additive. Conclusion: This study reveals that TP53 mutations have different clinical relevance in molecular subtypes of breast cancer, and suggests diverse roles for TP53 in the biology underlying breast cancer development. Clin Cancer Res; 20(13); 3569–80. ©2014 AACR.

Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

Lymphocyte Invasion in IC10/Basal-Like Breast Tumors Is Associated with Wild-Type TP53

Lymphocytic infiltration is associated with better prognosis in several epithelial malignancies including breast cancer. The tumor suppressor TP53 is mutated in approximately 30% of breast adenocarcinomas, with varying frequency across molecular subtypes. In this study of 1,420 breast tumors, we tested for interaction between TP53 mutation status and tumor subtype determined by PAM50 and integrative cluster analysis. In integrative cluster 10 (IC10)/basal-like breast cancer, we identify an association between lymphocytic infiltration, determined by an expression score, and retention of wild-type TP53. The expression-derived score agreed with the degree of lymphocytic infiltration assessed by pathologic review, and application of the Nanodissect algorithm was suggestive of this infiltration being primarily of cytotoxic T lymphocytes (CTL). Elevated expression of this CTL signature was associated with longer survival in IC10/Basal-like tumors. These findings identify a new link between the TP53 pathway and the adaptive immune response in estrogen receptor (ER)–negative breast tumors, suggesting a connection between TP53 inactivation and failure of tumor immunosurveillance. Implications: The association of lymphocytic invasion of ER-negative breast tumors with the retention of wild-type TP53 implies a novel protective connection between TP53 function and tumor immunosurveillance. Mol Cancer Res; 13(3); 493–501. ©2014 AACR.

TP53 Mutation Spectrum in Smokers and Never Smoking Lung Cancer Patients.

Background: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer. Methods: We analyzed tumor tissue from 394 non-small cell carcinomas including adenocarcinomas (n = 229), squamous cell carcinomas (n = 112), large cell carcinomas (n = 30), and others (n = 23) for mutations in TP53 by the use of Sanger sequencing (n = 394) and next generation sequencing (n = 100). Results: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65%) of mutations among squamous cell carcinomas. Among never-smokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3%) compared to adenocarcinomas (9.1%). Conclusion: TP53 mutation patterns differ between the histological subgroups of lung cancers, and are also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

The sub-cellular localization of WRAP53 has prognostic impact in breast cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.

Evaluation of metabolomic changes during neoadjuvant chemotherapy combined with bevacizumab in breast cancer using MR spectroscopy

IntroductionMetabolomics investigates biochemical processes directly, potentially complementing transcriptomics and proteomics in providing insight into treatment outcome.ObjectivesThis study aimed to use magnetic resonance (MR) spectroscopy on breast tumor tissue to explore the effect of neoadjuvant therapy on metabolic profiles, determine metabolic effects of the antiangiogenic drug bevacizumab, and investigate metabolic differences between responders and non-responders.MethodsBreast tumors from 122 patients were profiled using high resolution magic angle spinning MR spectroscopy. All patients received neoadjuvant chemotherapy, and were randomized to receive bevacizumab or not. Tumors were biopsied prior, during, and after treatment.ResultsPrincipal component analysis showed clear metabolic changes indicating a decline in glucose consumption and a transition to normal breast adipose tissue as an effect of chemotherapy. Partial least squares-discriminant analysis revealed metabolic differences between pathological minimal residual disease patients and pathological non-responders after treatment (accuracy of 77%, p < 0.001), but not before or during treatment. Lower glucose and higher lactate was observed in patients exhibiting a good response (≥90% tumor reduction) compared to those with no response (≤10% tumor reduction) before treatment, while the opposite was observed after treatment. Bevacizumab-receiving and chemotherapy-only patients could not be discriminated at any time point. Linear mixed-effects models revealed a significant interaction between time and bevacizumab for glutathione, indicating higher levels of this antioxidant in chemotherapy-only patients than in bevacizumab receivers after treatment.ConclusionMR spectroscopy showed potential in detecting metabolic response to treatment and complementing other molecular assays for the elucidation of underlying mechanisms affecting pathological response.

TP53 Mutations in Breast and Ovarian Cancer.

Breast and ovarian cancers are the second and fifth leading causes of cancer deaths among women. Both breast and ovarian cancers are highly heterogeneous and are presented with diverse morphology, natural history, and response to therapy. In recent years, international efforts have led to extensive molecular characterization of both breast and ovarian tumors and identified biologically and clinically relevant subtypes of the diseases based on these molecular features. The role of TP53 in tumor initiation and progression is context dependent, and abrogation of the TP53 pathway seems to be essential for the development of basal-like breast cancers and high-grade serous ovarian cancers. These subtypes of breast and ovarian cancer show several genomic similarities including high frequency of TP53 mutation, which seems to be an early, initiating, and driving alteration in these cancer subtypes.

The longitudinal transcriptional response to neoadjuvant chemotherapy with and without bevacizumab in breast cancer

Purpose: Chemotherapy-induced alterations to gene expression are due to transcriptional reprogramming of tumor cells or subclonal adaptations to treatment. The effect on whole-transcriptome mRNA expression was investigated in a randomized phase II clinical trial to assess the effect of neoadjuvant chemotherapy with the addition of bevacizumab. Experimental Design: Tumor biopsies and whole-transcriptome mRNA profiles were obtained at three fixed time points with 66 patients in each arm. Altogether, 358 specimens from 132 patients were available, representing the transcriptional state before treatment start, at 12 weeks and after treatment (25 weeks). Pathologic complete response (pCR) in breast and axillary nodes was the primary endpoint. Results: pCR was observed in 15 patients (23%) receiving bevacizumab and chemotherapy and 8 patients (12%) receiving only chemotherapy. In the estrogen receptor–positive patients, 11 of 54 (20%) treated with bevacizumab and chemotherapy achieved pCR, while only 3 of 57 (5%) treated with chemotherapy reached pCR. In patients with estrogen receptor–positive tumors treated with combination therapy, an elevated immune activity was associated with good response. Proliferation was reduced after treatment in both treatment arms and most pronounced in the combination therapy arm, where the reduction in proliferation accelerated during treatment. Transcriptional alterations during therapy were subtype specific, and the effect of adding bevacizumab was most evident for luminal-B tumors. Conclusions: Clinical response and gene expression response differed between patients receiving combination therapy and chemotherapy alone. The results may guide identification of patients likely to benefit from antiangiogenic therapy. Clin Cancer Res; 23(16); 4662–70. ©2017 AACR.

Molecular signatures reflecting microenvironmental metabolism and chemotherapy-induced immunogenic cell death in colorectal liver metastases

BACKGROUND Metastatic colorectal cancer (CRC) is associated with highly variable clinical outcome and response to therapy. The recently identified consensus molecular subtypes (CMS1-4) have prognostic and therapeutic implications in primary CRC, but whether these subtypes are valid for metastatic disease is unclear. We performed multi-level analyses of resectable CRC liver metastases (CLM) to identify molecular characteristics of metastatic disease and evaluate the clinical relevance. METHODS In this ancillary study to the Oslo-CoMet trial, CLM and tumor-adjacent liver tissue from 46 patients were analyzed by profiling mutations (targeted sequencing), genome-wide copy number alteration (CNAs), and gene expression. RESULTS Somatic mutations and CNAs detected in CLM were similar to reported primary CRC profiles, while CNA profiles of eight metastatic pairs suggested intra-patient divergence. A CMS classifier tool applied to gene expression data, revealed the cohort to be highly enriched for CMS2. Hierarchical clustering of genes with highly variable expression identified two subgroups separated by high or low expression of 55 genes with immune-related and metabolic functions. Importantly, induction of genes and pathways associated with immunogenic cell death (ICD) was identified in metastases exposed to neoadjuvant chemotherapy (NACT). CONCLUSIONS The uniform classification of CLM by CMS subtyping may indicate that novel class discovery approaches need to be explored to uncover clinically useful stratification of CLM. Detected gene expression signatures support the role of metabolism and chemotherapy in shaping the immune microenvironment of CLM. Furthermore, the results point to rational exploration of immune modulating strategies in CLM, particularly by exploiting NACT-induced ICD.Background Metastatic colorectal cancer (CRC) is associated with highly variable clinical outcome and response to therapy. The recently identified consensus molecular subtypes (CMS1-4) have prognostic and therapeutic implications in primary CRC, but whether these subtypes are valid for metastatic disease is unclear. We performed multi-level analyses of resectable CRC liver metastases (CLM) to identify molecular characteristics of metastatic disease and evaluate the clinical relevance. Methods In this ancillary study to the Oslo-CoMet trial, CLM and tumor-adjacent liver tissue from 46 patients were analyzed by profiling mutations (targeted sequencing), genome-wide copy number alteration (CNAs), and gene expression. Results Somatic mutations and CNAs detected in CLM were similar to reported primary CRC profiles, while CNA profiles of eight metastatic pairs suggested intra-patient divergence. A CMS classifier tool applied to gene expression data, revealed the cohort to be highly enriched for CMS2. Hierarchical clustering of genes with highly variable expression identified two subgroups separated by high or low expression of 55 genes with immune-related and metabolic functions. Importantly, induction of genes and pathways associated with immunogenic cell death (ICD) was identified in metastases exposed to neoadjuvant chemotherapy (NACT). Conclusions The uniform classification of CLM by CMS subtyping may indicate that novel class discovery approaches need to be explored to uncover clinically useful stratification of CLM. Detected gene expression signatures support the role of metabolism and chemotherapy in shaping the immune microenvironment of CLM. Furthermore, the results point to rational exploration of immune modulating strategies in CLM, particularly by exploiting NACT-induced ICD.

Assessment of TP53 Polymorphisms and MDM2 SNP309 in Pre-Menopausal Breast Cancer Risk.

Germline polymorphic variants in cancer predisposition genes such as TP53 have been shown to impact the risk of premenopausal cancer. Accordingly, the aim of this study was to assess the spectrum of polymorphisms in TP53 and its negative regulatory gene, MDM2 (SNP309:T>G) in patients with premenopausal breast cancer. Our findings in a cohort of 40 female patients demonstrate no significant correlation between the studied polymorphisms and risk of premenopausal breast cancer. Although one polymorphism is found in high frequency in this cohort (rs1800372:A>G, 9.0%), it was not associated with the risk of developing cancer before the age of 35 years in an extended cohort of 1,420 breast cancer cases. Functional studies of the rs1800372:A>G polymorphic allele reveal that it does not affect p53 transactivation function. Further study of variants or mutations in other cancer susceptibility genes is warranted to refine our understanding of the germline contribution to premenopausal breast cancer susceptibility.