A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Longitudinal follow-up and performance validation of an mRNA-based urine test (Xpert® Bladder Cancer Monitor ) for surveillance in patients with non-muscle-invasive bladder cancer.

OBJECTIVE: To evaluate the performance of the Xpert Bladder Cancer Monitor (Xpert; Cepheid, Sunnyvale, CA, USA) test as a predictor of tumour recurrence in patients with non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients (n = 429) undergoing surveillance for NMIBC underwent Xpert, cytology, and UroVysion testing. Patients with a positive Xpert and a negative cystoscopy result (positive-negative [PN] group, n = 66) and a control group of double negative patients (negative Xpert and cystoscopy results [NN] group) were followed for 12 months (±90 days). RESULTS: Histology-confirmed recurrences were detected in 58 patients (13.5%). Xpert had an overall sensitivity of 60.3% and a specificity of 76.5%. The sensitivity for high-grade (HG) cancer was 87% with a negative predictive value (NPV) of 99%. Urine cytology showed an overall sensitivity of 23.2% (47.6% sensitivity for HG tumours) and a specificity of 88.3%. In the PN group, 32% (n = 21) developed a recurrence within 12 months, 11 of which were HG tumours. In the NN control group, 14% (n = 9) developed a recurrence and only two were HG tumours. The hazard ratio for developing recurrence in the PN group was 2.68 for all tumours and 6.84 for HG cancer. CONCLUSIONS: The Xpert test has a high sensitivity for detecting the recurrence of cancer and a high NPV for excluding HG cancer. In addition, the data suggest that patients with a positive Xpert assay in the setting of negative cystoscopy are at high risk for recurrence and need close surveillance.

The BRCA1ness signature is associated significantly with response to PARP inhibitor treatment versus control in the I-SPY 2 randomized neoadjuvant setting.

BACKGROUND: Patients with BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments. METHODS: A diagnostic gene expression signature (BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher's exact test, and the relative performance between arms (biomarker × treatment interaction, likelihood ratio p < 0.05) using a logistic model and adjusting for hormone receptor status (HR). RESULTS: We developed a gene expression signature to identify BRCA1-like status. In the I-SPY 2 neoadjuvant setting the BRCA1ness signature associated significantly with response to V-C (p = 0.03), but not in the control arm (p = 0.45). We identified a significant interaction between BRCA1ness and V-C (p = 0.023) after correcting for HR. CONCLUSIONS: A genomic-based BRCA1-like signature was successfully translated to an expression-based signature (BRC1Aness). In the I-SPY 2 neoadjuvant setting, we determined that the BRCA1ness signature is capable of predicting benefit of V-C added to standard chemotherapy compared to standard chemotherapy alone. TRIAL REGISTRATION: I-SPY 2 TRIAL beginning December 31, 2009: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2), NCT01042379 .

Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition.

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

Improved guidance is needed to optimise diagnostics and treatment of patients with thyroid cancer in Europe.

Although thyroid cancer (TC) is generally associated with a favourable prognosis, there are certain high-risk groups with a clear unmet therapeutic need. Unravelling the genomic landscape of TC has recently led to the development of novel effective targeted treatments. To date, these treatments have mostly been evaluated in non-randomised single-arm phase II clinical trials and are consequently non-reimbursed in several countries. Furthermore, most of these agents must be tailored to individual patient molecular characteristics, a context known as personalised cancer medicine, necessitating a requirement for predictive molecular biomarker testing. Existing guidelines, both in Europe and internationally, entail mostly therapeutic rather than molecular testing recommendations. This may reflect ambiguity among experts due to lack of evidence and also practical barriers in availability of the preferred molecular somatic screening and/or targeted treatments. This article reviews existing European recommendations regarding advanced/metastatic TC management with a special focus on molecular testing, and compares findings with real-world practice based on a recent survey involving TC experts from 18 European countries. Significant disparities are highlighted between theory and practice related to variable access to infrastructure, therapies and expertise, together with the insufficient availability of multidisciplinary tumour boards. In particular, practitioners' choice of what, how and when to test is shown to be influenced by the expertise of the available laboratory, the financing source and the existence of potential facilitators, such as clinical trial access. Overall, the need of a collaborative initiative among European stakeholders to develop standardised, accessible molecular genotyping approaches in TC is underscored.

Validation of an mRNA-based Urine Test for the Detection of Bladder Cancer in Patients with Haematuria.

BACKGROUND: In patients with haematuria, a fast, noninvasive test with high sensitivity (SN) and negative predictive value (NPV), which is able to detect or exclude bladder cancer (BC), is needed. A newly developed urine assay, Xpert Bladder Cancer Detection (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC. OBJECTIVE: To validate the performance of Xpert in patients with haematuria. DESIGN, SETTING, AND PARTICIPANTS: Voided precystoscopy urine specimens were prospectively collected at 22 sites from patients without prior BC undergoing cystoscopy for haematuria. Xpert, cytology, and UroVysion procedures were performed. Technical validation was performed and specificity (SP) was determined in patients without BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion. RESULTS AND LIMITATIONS: We included 828 patients (mean age 64.5 yr, 467 males, 401 never smoked). Xpert had an SN of 78% (95% confidence interval [CI]: 66-87) overall and 90% (95% CI: 76-96) for high-grade (HG) tumours. The NPV was 98% (95% CI: 97-99) overall. The SP was 84% (95% CI: 81-86). In patients with microhaematuria, only one HG patient was missed (NPV 99%). Xpert had higher SN and NPV than cytology and UroVysion. Cytology had the highest SP (97%). In a separate SP study, Xpert had an SP of 89% in patients with benign prostate hypertrophy and 92% in prostate cancer patients. CONCLUSIONS: Xpert is an easy-to-use, noninvasive test with improved SN and NPV compared with cytology and UroVysion, representing a promising tool for identifying haematuric patients with a low likelihood of BC who might not need to undergo cystoscopy. PATIENT SUMMARY: Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help identify (micro)haematuria patients with a very low likelihood to have bladder cancer.

Prospective Validation of an mRNA-based Urine Test for Surveillance of Patients with Bladder Cancer.

BACKGROUND: A fast, noninvasive test with high sensitivity (SN) and a negative predictive value (NPV), which is able to detect recurrences in bladder cancer (BC) patients, is needed. A newly developed urine assay, Xpert Bladder Cancer Monitor (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC. OBJECTIVE: To validate Xpert characteristics in patients previously diagnosed with non-muscle-invasive BC. DESIGN, SETTING, AND PARTICIPANTS: Voided precystoscopy urine samples were prospectively collected at 22 sites. Xpert, cytology, and UroVysion were performed. If cystoscopy was suspicious for BC, a histologic examination was performed. Additionally, technical validation was performed and specificity was determined in patients without a history or clinical evidence of BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion. RESULTS AND LIMITATIONS: Of the eligible patients, 239 with a history of BC had results for all assays. The mean age was 71 yr; 190 patients were male, 53 never smoked, and 64% had previous intravesical immunotherapy (35%) or chemotherapy (29%). Forty-three cases of recurrences occurred. Xpert had overall SN of 74% (95% confidence interval [CI]: 60-85) and 83% (95% CI: 64-93) for high-grade (HG) tumors. The NPV was 93% (95% CI: 89-96) overall and 98% (95% CI: 94-99) for HG tumors. Specificity was 80% (95% CI: 73-85). Xpert SN and NPV were superior to those of cytology and UroVysion. Specificity in non-BC individuals (n=508) was 95% (95% CI: 93-97). CONCLUSIONS: Xpert has an improved NPV compared with UroVysion and cytology in patients under follow-up for BC. It represents a promising tool for excluding BC in these patients, reducing the need for cystoscopy. PATIENT SUMMARY: Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help optimize the follow-up of recurrent bladder cancer patients.

BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.

PURPOSE: Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population. EXPERIMENTAL DESIGN: In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression-based subgroups and characterized pathway activation. RESULTS: We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers. CONCLUSIONS: We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104-15. ©2016 AACR.

Neoadjuvant tamoxifen synchronizes ERα binding and gene expression profiles related to outcome and proliferation.

Estrogen receptor alpha (ERα)-positive breast cancers are frequently treated with tamoxifen, but resistance is common. It remains elusive how tamoxifen resistance occurs and predictive biomarkers for treatment outcome are needed. Because most biomarker discovery studies are performed using pre-treatment surgical resections, the effects of tamoxifen therapy directly on the tumor cell in vivo remain unexamined. In this study, we assessed DNA copy number, gene expression profiles and ERα/chromatin binding landscapes on breast tumor specimens, both before and after neoadjuvant tamoxifen treatment. We observed neoadjuvant tamoxifen treatment synchronized ERα/chromatin interactions and downstream gene expression, indicating that hormonal therapy reduces inter-tumor molecular variability. ERα-synchronized sites are associated with dynamic FOXA1 action at these sites, which is under control of growth factor signaling. Genes associated with tamoxifen-synchronized sites are capable of differentiating patients for tamoxifen benefit. Due to the direct effects of therapeutics on ERα behavior and transcriptional output, our study highlights the added value of biomarker discovery studies after neoadjuvant drug exposure.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential.

Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10-15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination-mediated DNA repair and deficiency results in genomic instability. BRCA1-mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1-like or non-BRCA1-like. BRCA1 mutation, promoter methylation, BRCA1-like status and genome-wide expression data was determined for 112 TN breast cancer samples with long-term follow-up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1-like and non-BRCA1-like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty-five percent of tumors classified as BRCA1-like. The functions of genes significantly up-regulated in BRCA1-like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1-like (P < 0.05), while PIK3CA was frequently mutated in non-BRCA1-like tumors (P < 0.05). A significant association with worse prognosis was evident for patients with BRCA1-like tumors (adjusted HR = 3.32, 95% CI = 1.30-8.48, P = 0.01). TN tumors can be further divided into two major subgroups, BRCA1-like and non-BRCA1-like with different mutation and expression patterns and prognoses. Based on these molecular patterns, subgroups may be more sensitive to specific targeted agents such as PI3K or PARP inhibitors.

The consensus molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-ß activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

MammaPrint molecular diagnostics on formalin-fixed, paraffin-embedded tissue.

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.