Monitoring of RAS mutant clones in plasma of patients with RAS mutant metastatic colorectal cancer

Circulating tumor DNA; Liquid biopsy; Metastatic colorectal cancerADN tumoral circulante; Biopsia liquida; Cáncer colorrectal metastásicoADN tumoral circulant; Biòpsia líquida; Càncer colorectal metastàticPurpose Some patients with histologically confirmed primary mCRC and mutated RAS reported undetectable RAS mutant clones in plasma after receiving anti-VEGF treatment. The aim was to prospectively assess it with its potential therapeutic implications. Methods RAS mutant genes in solid biopsy (before first-line treatment: FOLFOX/CAPOX + bevacizumab) were compared in liquid biopsy (before second-line treatment: panitumumab + FOLFIRI), using Idylla™ system. Discordant results between solid/liquid biopsies were assessed by the next-generation sequencing (NGS) test (solid/liquid biopsies). Results Twenty-three patients were assessed (seven had RAS mutant discrepancies between solid/liquid biopsies). The NGS test confirmed that 3/23 (13%) patients had undetectable RAS mutant clones in liquid biopsy and 3/23 (13%) presented discrepancies in solid biopsy (Idylla™ system vs. NGS test). Conclusion Thirteen percentage of patients had undetectable RAS mutant clones in liquid biopsy after first-line treatment. However, some discrepancies between solid and liquid biopsies have been observed. These results suggest a need to improve accuracy of RAS analyses, especially in solid biopsies.This work was supported by Amgen S.A. Amgen did not have any role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication

Comparison of the Clinical Sensitivity of the Idylla Platform and the OncoBEAM RAS CRC Assay for KRAS Mutation Detection in Liquid Biopsy Samples

Biopsy; KRAS mutation; Colorectal cancerBiòpsia; Mutació KRAS, Càncer colorectalBiopsia; Mutación KRAS, Cáncer colorrectalKRAS mutations are common in colorectal cancer (CRC). In this setting, mutation status determination in circulating-free DNA from blood samples (liquid biopsy) has been shown to be a viable alternative to tissue testing. The objective of this study was to compare the sensitivity of two liquid biopsy methods for detecting KRAS mutations in plasma samples from metastatic CRC patients. Samples with a positive (KRAS-MUT+) result and a mutant allelic fraction (MAF) < 5% according to the OncoBEAM RAS CRC assay were pairly analyzed by the Idylla ctKRAS Mutation Test (n = 116). In a cohort of 71 patients with at least 1 year of follow-up, the progression-free survival (PFS) was determined according to MAF values. Idylla detected KRAS mutations in 81/116 OncoBEAM KRAS-MUT+ samples with MAF < 5% and in 48/79 samples with MAF < 1%. Concordance between OncoBEAM and Idylla significantly improved at higher MAF values. PFS rates at 6 and 12 months tended to be lower in patients with MAF levels between 1% and 5% than in those with levels <1%. OncoBEAM demonstrated greater sensitivity for plasma detection of KRAS mutations than Idylla. Importantly, our data identified a "gray zone" below 1% MAF where Idylla showed reduced KRAS mutation detection, highlighting the importance of an accurate method to provide the mutational status of CRC patients

Biomarker Analysis of the Phase III NALA Study of Neratinib + Capecitabine versus Lapatinib + Capecitabine in Patients with Previously Treated Metastatic Breast Cancer

Cáncer de mama metastásico; Biomarcadores; CapecitabinaCàncer de mama metastàtic; Biomarcadors; CapecitabinaMetastatic breast cancer; Biomarkers; CapecitabinePurpose: Neratinib plus capecitabine (N+C) demonstrated significant progression-free survival (PFS) benefit in NALA (NCT01808573), a randomized phase III trial comparing N+C with lapatinib + capecitabine (L+C) in 621 patients with HER2-positive (HER2+) metastatic breast cancer (MBC) who had received ≥2 prior HER2-directed regimens in the metastatic setting. We evaluated correlations between exploratory biomarkers and PFS. Patients and Methods: Somatic mutations were evaluated by next-generation sequencing on primary or metastatic samples. HER2 protein expression was evaluated by central IHC, H-score, and VeraTag/HERmark. p95 expression (truncated HER2) was measured by VeraTag. HRs were estimated using unstratified Cox proportional hazards models. Results: Four hundred and twenty samples had successful sequencing: 34.0% had PIK3CA mutations and 5.5% had HER2 (ERBB2) mutations. In the combined patient populations, PIK3CA mutations trended toward shorter PFS [wild-type vs. mutant, HR = 0.81; 95% confidence interval (CI), 0.64–1.03], whereas HER2 mutations trended toward longer PFS [HR = 1.69 (95% CI, 0.97–3.29)]. Higher HER2 protein expression was associated with longer PFS [IHC 3+ vs. 2+, HR = 0.67 (0.54–0.82); H-score ≥240 versus <240, HR = 0.77 (0.63–0.93); HERmark positive vs. negative, HR = 0.76 (0.59–0.98)]. Patients whose tumors had higher HER2 protein expression (any method) derived an increased benefit from N+C compared with L+C [IHC 3+, HR = 0.64 (0.51–0.81); H-score ≥ 240, HR = 0.54 (0.41–0.72); HERmark positive, HR = 0.65 (0.50–0.84)], as did patients with high p95 [p95 ≥2.8 relative fluorescence (RF)/mm2, HR = 0.66 (0.50–0.86) vs. p95 < 2.8 RF/mm2, HR = 0.91 (0.61–1.36)]. Conclusions: PIK3CA mutations were associated with shorter PFS whereas higher HER2 expression was associated with longer PFS. Higher HER2 protein expression was also associated with a greater benefit for N+C compared with L+C.This work was supported by Puma Biotechnology Inc. (no grant number is applicable)

First Nationwide Molecular Screening Program in Spain for Patients With Advanced Breast Cancer: Results From the AGATA SOLTI-1301 Study

Anàlisi de seqüències d'ADN; Subtipus PAM50; Genètica molecularAnálisis de secuencias de ADN; Subtipo PAM50; Genética molecularDNA sequence analyses; PAM50 subtype; Molecular geneticBackground: The SOLTI-1301 AGATA study aimed to assess the feasibility of a multi-institutional molecular screening program to better characterize the genomic landscape of advanced breast cancer (ABC) and to facilitate patient access to matched-targeted therapies in Spain. Methods: DNA sequencing of 74 cancer-related genes was performed using FFPE tumor samples in three different laboratories with three different gene panels. A multidisciplinary advisory board prospectively recommended potential targeted treatments. The primary objective was to determine the success of matching somatic DNA alteration to an experimental drug/drug class. Results: Between September 2014 and July 2017, 305 patients with ABC from 10 institutions were enrolled. Tumor sequencing was successful in 260 (85.3%) patients. Median age was 54 (29-80); most tumors were hormone receptor-positive/HER2-negative (74%), followed by triple-negative (14.5%) and HER2-positive (11.5%). Ninety-seven (37%) tumor samples analyzed proceeded from metastatic sites. Somatic mutations were identified in 163 (62.7%) patients, mostly in PIK3CA (34%), TP53 (22%), AKT1 (5%), ESR1 (3%), and ERBB2 (3%) genes. Significant enrichment of AKT1 mutation was observed in metastatic versus primary samples (9% vs. 2%; p=0.01). Genome-driven cancer therapy was recommended in 45% (n=116) of successfully screened patients, 11% (n=13) of whom finally received it. Among these patients, 46.2% had a PFS of ≥6 months on matched therapy. Conclusions: AGATA is the first nationwide molecular screening program carried out in Spain and we proved that implementing molecular data in the management of ABC is feasible. Although these results are promising, only 11% of the patients with genome-driven cancer therapy received it.This study was supported by a grant from Novartis. This study was funded, in part, by the project PI 15/01508, integrated in the Plan Estatal I+D+I and co-funded by Instituto de Salud Carlos III - Subdirección General de Evaluación and European Regional Development Fund (ERDF) (to EC) and by a grant from Mutua Madrileña Foundation (to EC). The decisions and responsibilities of this study were all under the sponsor: SOLTI Breast Cancer Research Group

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer

Neoplasias genitales; Inmunoterapia; Linfocitos TNeoplàsies genitals; Immunoteràpia; Limfòcits TGenital neoplasms; Immunotherapy; T-LymphocytesBackground Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. Methods To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. Results We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1− and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1− or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. Conclusions Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.AG was funded by the Comprehensive Program of Cancer Immunotherapy and Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021), the La Fundació La Marató de TV3 (201919-30; identification number 488/C/2019), the Spanish Ministry of Science and Innovation (PID2020-118529RB-100) and Instituto de Salud Carlos III (CP15/00058). We thank the CERCA Programme/Generalitat de Catalunya for institutional support. JP was supported by the Beatriu de Pinós programme (BP 2018), cofounded by the Agency for Management of University and Research Grants (AGAUR) and European Union's Horizon 2020. HUB-ICO-IDIBELL Biobank received support from Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC)

Genomic heterogeneity and efficacy of PI3K pathway inhibitors in patients with gynaecological cancer

Malignitats ginecològiques; Fracció al·lel mutant; PI3KNeoplasias ginecológicas; Fracción alélica mutante; PI3KGynecologic malignancies; Mutant allele fraction; PI3KObjectives Aberrant PI3K/AKT/mTOR activation is common in gynaecological malignancies. However, predictive biomarkers of response to PI3K pathway inhibitors (PAMi) have yet to be identified. Methods We analysed the outcomes of patients with advanced gynaecological cancer with available genomic data, treated with PAMi as single agents or in combination in phase I clinical trials. Clinical relevance of the PIK3CA mutant allele fraction (MAF) was investigated. MAF of each variant was normalised for tumour purity in the sample (adjMAFs) to infer clonality of PIK3CA mutations, defined as clonal (≥0.4) or subclonal (<0.4). Results A total of 50 patients with gynaecological cancer (24 ovarian; 15 endometrial; 11 cervical) with available targeted mutation profiling were selected. PAMi therapy was matched to PIK3CA/PTEN mutation in 30 patients (60%). The overall response rate, median time to progression (mTTP) and clinical benefit rate (CBR) of the entire population were 10% (N=5), 3.57 months (2.57–4.4) and 40% (N=18), respectively. Genotype-matched therapy did not lead to a favourable CBR (OR 0.91, p=1 (0.2–3.7)) or mTTP (3.57 months (2.6–4.4) vs 3.73 months (1.9–13.2); HR 1.41; p=0.29). We did not detect differences in mTTP according to therapy or PIK3CA codon mutation (HR 1.71, p=0.24). Overall, 41% of patients had a TTP ratio (TTP PAMi/TTP on immediately prior or subsequent palliative chemotherapy) ≥1.3, without statistically significant differences according to tumour type (p=0.39), molecular alteration status (p=0.13) or therapy (p=0.54). In univariate analysis, genotype-matched therapy in patients with PIK3CA clonal events was associated with improved mTTP (HR 3.6; p=0.03). Conclusions Our study demonstrates that patients with advanced gynaecological cancer, refractory to standard therapies, achieved meaningful clinical benefit from PAMi. The impact of PI3KCA clonality on response to selected PAMi in patients with gynaecological cancer deserves further investigation

Case Report: A Case Study Documenting the Activity of Atezolizumab in a PD-L1-Negative Triple-Negative Breast Cancer

Biomarcadores; Cáncer de mama; InmunoterapiaBiomarcadors; Càncer de mama; ImmunoteràpiaBiomarkers; Breast cancer; ImmunotherapyThe immune checkpoint inhibitor atezolizumab is approved for PD-L1-positive triple-negative breast cancer (TNBC). However, no activity of atezolizumab in PD-L1-negative TNBC has been reported to date. Here, we present the case study of a woman with TNBC with low tumor infiltrating lymphocytes and PD-L1-negative disease, which achieved a significant response to atezolizumab monotherapy and durable response after the combination of atezolizumab and nab-paclitaxel. The comprehensive genomic analysis that we performed in her tumor and plasma samples revealed high tumor mutational burden (TMB), presence of the APOBEC genetic signatures, high expression of the tumor inflammation signature, and a HER2-enriched subtype by the PAM50 assay. Some of these biomarkers have been shown to independently predict response to immunotherapy in other tumors and may explain the durable response in our patient. Our work warrants further translational studies to identify biomarkers of response to immune checkpoint inhibitors in TNBC beyond PD-L1 expression and to better select patients that will benefit from immunotherapy.This study has received funding from Instituto de Salud Carlos III—PI19/01846 (to AP), Breast Cancer Now—2018NOVPCC1294 (to AP), Breast Cancer Research Foundation-AACR Career Development Awards for Translational Breast Cancer Research 19-20-26-PRAT (to AP), Fundació La Marató TV3 201935-30 (to AP), the European Union’s Horizon 2020 research and innovation programme H2020-SC1-BHC-2018-2020 (to AP), Asociación de Cáncer de Mama Metastásico CMM_CHIARAG19_001 (to AP), Pas a Pas (to AP), Save the Mama (to AP), Fundación Científica Asociación Española Contra el Cáncer AECC_Postdoctoral17-1062 (to FB-M) and INVES19056SANS (to MiS), FERO-ghd 2020 breast cancer award (MS), and Generalitat de Catalunya Peris PhD4MD 2019 SLT008/18/00122 (to NC)

Targeted multiplex proteomics for molecular prescreening and biomarker discovery in metastatic colorectal cancer

Biomarcadores del cáncer; Cáncer metástico colorrectal; Terapias experimentalesCancer biomarkers; Colorectal metastatic cancer; Experimental therapiesBiomarcadors del càncer; Càncer metastàtic colorrectal; Teràpies experimentalsProtein biomarkers are widely used in cancer diagnosis, prognosis, and prediction of treatment response. Here we introduce the use of targeted multiplex proteomics (TMP) as a tool to simultaneously measure a panel of 54 proteins involved in oncogenic, tumour suppression, drug metabolism and resistance, in patients with metastatic colorectal cancer (mCRC). TMP provided valuable diagnostic information by unmasking an occult neuroendocrine differentiation and identifying a misclassified case based on abnormal proteins phenotype. No significant differences in protein levels between unpaired primary and metastatic samples were observed. Four proteins were found differentially expressed in KRAS-mutant as compared to wild-type tumours (overexpressed in mutant: KRAS, EGFR; overexpressed in wild-type: TOPO1, TOP2A). Survival analyses revealed the association between mesothelin expression and poor overall survival, whereas lack of PTEN protein expression associated with lower progression-free survival with anti-EGFR-based therapy in the first-line setting for patients with RAS wild-type tumour. Finally, outlier analysis identified putative targetable proteins in 65% of patients lacking a targetable genomic alteration. Our data show that TMP constitutes a promising, novel molecular prescreening tool in mCRC to identify protein expression alterations that may impact on patient outcomes and more precisely guide patient eligibility to clinical trials with novel targeted experimental therapies

High levels of chromosomal aberrations negatively associate with benefit to checkpoint inhibition in NSCLC

Immunotherapy; Lung neoplasms; Tumor biomarkersImmunoteràpia; Càncer de pulmó; Biomarcadors tumoralsInmunoterapia; Cáncer de pulmón; Biomarcadores tumoralesBackground Immune checkpoint inhibitors (ICIs) targeting the programmed cell death 1/programmed death-ligand 1 axis have transformed the management of advanced non-small cell lung cancer (NSCLC). However, many patients do not benefit from this type of treatment, and thus several molecular biomarkers of benefit have been explored. The value of somatic copy number alterations (SCNAs) burden remains elusive. Patients and methods We assembled a cohort of 109 patients with NSCLC treated with ICIs and available tumor samples. We performed shallow whole-genome sequencing on 89 patients to determine genome-wide SCNAs and targeted gene expression analysis on 63 patients to study immune infiltration. We analyzed SCNAs burden in different ways (ie, the fraction of the genome altered or number of events) and studied their association with ICIs benefit based on survival analysis. We correlated SCNAs burden and immune infiltration on 35 patients of our cohort and on patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA). Results High SCNAs burden, computed in diverse ways, is negatively associated with ICIs progression-free survival (PFS), with the fraction of the genome altered (FGA) by arm and chromosome events showing the strongest association with PFS (p=0.002) (n=77). Nevertheless, we found differences in SCNAs across some clinicopathological features (sample site origin). A multivariate analysis adjusted for relevant characteristics showed that the FGA of arm and chromosome alterations was strongly associated with PFS (HR=2.21, p=3.3 x 10−5). Finally, we confirmed that SCNAs burden negatively correlates with tumor immune infiltration (n=35), although this correlation was not found for the males studied. Similar results were observed in the TCGA cohort. Conclusions SCNAs burden is a potential biomarker of benefit to ICIs in patients with NSCLC, although there appear to be some nuances worth consideration. Further studies will be needed to establish its role as a biomarker of benefit to ICIs.This work was supported by Merck Healthcare KGaA, Darmstadt, Germany (Grant for Oncology Innovation to the Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain), Fundación Cientifica Asociación Española Contra el Cancer-AECC (grant number GCB14142170 to EF); the Catalan Government/AGAUR (2017–SGR–1738 to EF). Merck Healthcare KGaA reviewed the manuscript for medical accuracy only before journal submission

pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation

Cancer; Mechanisms of diseaseCàncer; Mecanismes de la malaltiaCáncer; Mecanismos de la enfermedadThe human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.Work in the Abad lab is supported by VHIO, Fero Foundation, La Caixa Foundation, Asociación Española Contra el Cancer (AECC), La Mutua Foundation and by grants from the Spanish Ministry of Science and Innovation (SAF2015-69413-R; RTI2018-102046-B-I00). M.A. was recipient of a Ramón y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2013-14747). O.B. is recipient of a FPI-AGAUR fellowship from Generalitat de Catalunya. We also acknowledge funding from grant PGC2018-094091-B-I00 from the Spanish Government