Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

A phase 2 study of panitumumab with irinotecan as salvage therapy in chemorefractory KRAS exon 2 wild-type metastatic colorectal cancer patients.

BACKGROUND: Targeted agents are standard treatment for RAS wild-type metastatic colorectal cancer in the first- and second-line settings. This phase 2 study determined the benefit of targeting the epidermal growth factor receptor (EGFR) with panitumumab plus irinotecan in irinotecan-refractory patients. METHODS: KRAS exon-2 wild-type patients failing prior irinotecan received panitumumab (6 mg/kg) and irinotecan (180 mg/m²) every 2 weeks. The primary endpoint was the overall response rate (ORR). Secondary endpoints included safety, progression-free survival (PFS) and overall survival (OS). KRAS exon-2 status was evaluated centrally, along with NRAS, BRAF mutations, epiregulin, amphiregulin, PTEN and EGFR copy number status, and correlated with efficacy. RESULTS: Sixty-one patients were treated. Among the 46 wild-type RAS patients, the ORR was 15.2% (seven partial responses), with median PFS of 3.8 months (95% CI 2.7-4.3) and median OS of 12.5 months (95% CI 6.7-15.9). Wild-type BRAF patients showed a 13.0% response rate. No significant correlations between response and baseline biomarker expression were identified. Common grade 3-4 adverse events were diarrhoea and rash (18.0% each), hypomagnesaemia and asthenia (8.2% each). CONCLUSIONS: The addition of panitumumab to irinotecan as salvage therapy is feasible but has limited activity in irinotecan-refractory metastatic colorectal cancer. No biomarkers predictive of response were identified.

Nasoethmoidal Intestinal-Type Adenocarcinoma Treated with Cetuximab: Role of Liquid Biopsy and BEAMing in Predicting Response to Anti-Epidermal Growth Factor Receptor Therapy.

Sinonasal intestinal-type adenocarcinomas (SNS-ITAC) are very rare tumors that resemble colorectal cancer in many of their pathological and molecular characteristics. Indeed, in most published series, 10%-14% of SNS-ITAC harbor mutations in KRAS. There is no standard systemic treatment in recurrent or metastatic SNS-ITAC, and there is no evidence of the use of any targeted agent in this entity. We present the case of a recurrent nasoethmoidal ITAC informed as RAS and BRAF wild-type by standard real-time polymerase chain reaction methods and treated with first-line cetuximab and irinotecan without response. Circulating tumor cells coupled to highly sensitive DNA analyses unveiled a mutation in KRAS exon 2 codon 12. Subsequent studies in the primary tumor using BEAMing detected a mutation in the same codon, confirming the KRAS mutated status of the tumor, and possibly explaining the absence of treatment response. This case exemplifies how liquid biopsy can aid in the correct and real-time molecular characterization of tumors even in a rare nonmetastatic cancer of the head and neck. KEY POINTS: Sinonasal intestinal type adenocarcinomas (SNS-ITAC) are rare tumors that commonly develop after a prolonged exposure to organic dusts (wood, leather, etc.), and that resemble colorectal cancer in some of their morphological and molecular characteristics.KRAS mutations have been described in 10%-14% in most series. However, its predictive value for guiding treatment decisions with targeted therapies (i.e., anti-epidermal growth factor receptor [EGFR] therapy) has not been defined.The first case of an SNS-ITAC treated with anti-EGFR therapy (cetuximab) is reported. Analysis of DNA from circulating tumor cells (CTCs) unveiled a mutation in KRAS not detected by standard methods in the primary tumor. However, RAS analysis using BEAMing detected a mutation in the primary tumor in the same codon of KRAS originally detected in CTCs, altogether possibly explaining the lack of treatment response.Liquid biopsy may allow for an accurate molecular diagnosis in rare, organ-confined tumors where few therapeutic options exist. Highly sensitive molecular diagnostics may aid in better characterizing rare entities harboring potentially druggable targets.

Trabectedin for reversing platinum resistance and resensitization to platinum in patients with recurrent ovarian cancer.

AIMS: We evaluated trabectedin in patients with platinum-resistant/refractory and partially platinum-sensitive recurrent ovarian cancer and the outcomes after reintroduction of platinum. METHODS: Twenty-seven patients (platinum-resistant/refractory n = 24/PPS; n = 3) treated with trabectedin were retrospectively analyzed. RESULTS: Trabectedin resulted in an objective response rate (ORR) of 18.2% with a 59.1% of disease control rate (ORR plus stable disease). The median progression-free and overall survival were 3.0 and 21.3 months, respectively. Subsequently, 17 patients were retreated with platinum and yield an ORR of 41.2% and DCR of 47.0%. The median progression-free and overall survival after platinum rechallenge were 5.0 and 14.7 months, respectively. CONCLUSION: Our results suggest that trabectedin may contribute to resensitize tumor cells to platinum rechallenge.

Prospective multicenter real-world RAS mutation comparison between OncoBEAM-based liquid biopsy and tissue analysis in metastatic colorectal cancer.

BACKGROUND: Liquid biopsy offers a minimally invasive alternative to tissue-based evaluation of mutational status in cancer. The goal of the present study was to evaluate the aggregate performance of OncoBEAM RAS mutation analysis in plasma of colorectal cancer (CRC) patients at 10 hospital laboratories in Spain where this technology is routinely implemented. METHODS: Circulating cell-free DNA from plasma was examined for RAS mutations using the OncoBEAM platform at each hospital laboratory. Results were then compared to those obtained from DNA extracted from tumour tissue from the same patient. RESULTS: The overall percentage agreement between plasma-based and tissue-based RAS mutation testing of the 236 participants was 89% (210/236; kappa, 0.770 (95% CI: 0.689-0.852)). Re-analysis of tissue from all discordant cases by BEAMing revealed two false negative and five false positive tumour tissue RAS results, with a final concordance of 92%. Plasma false negative results were found more frequently in patients with exclusive lung metastatic disease. CONCLUSIONS: In this first prospective real-world RAS mutation performance comparison study, a high overall agreement was observed between results obtained from plasma and tissue samples. Overall, these findings indicate that the plasma-based BEAMing assay is a viable solution for rapid delivery of RAS mutation status to determine mCRC patient eligibility for anti-EGFR therapy.

Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging.

BACKGROUND: Accurate measurement of tumor burden in breast cancer disease is essential to improve the clinical management of patients. In this study, we evaluate whether the fluctuations in the fraction of PIK3CA mutant allele correlates with tumor response according to RECIST criteria and tumor markers quantification. METHODS: Eighty six plasma samples were analyzed by digital PCR using Rare Mutation Assays for E542K, E545K and H1047R. Mutant cfDNA and tumor markers CA15-3 and CEA were compared with radiographic imaging. RESULTS: The agreement between PIK3CA mutation status in FFPE samples and circulating tumor DNA (ctDNA) was moderate (K = 0.591; 95% IC = 0.371-0.811). Restricting the analysis to the metastatic patients, we found a good agreement between PIK3CA mutation status assessed in liquid and solid biopsy (K = 0.798 95%; IC = 0.586-1). ctDNA showed serial changes with fluctuations correlating with tumor markers 15.3 and CEA in 7 out of 8 cases with Pearson correlation coefficients ranging from 0.99 to 0.46 and from 0.99 to 0.38 respectively. Similarly, fluctuations in the fraction of PIK3CA mutant allele always correlated with changes in lesion size seen on images, although in two cases it did not correlate with treatment responses as defined by RECIST criteria. CONCLUSION: oncogenic mutation quantification in plasma samples can be useful to monitor treatment outcome. However, it might be limited by tumor heterogeneity in advanced disease and it should be evaluated together with radiographic imaging.

Prognosis of patients with peritoneal metastatic colorectal cancer given systemic therapy: an analysis of individual patient data from prospective randomised trials from the Analysis and Research in Cancers of the Digestive System (ARCAD) database.

BACKGROUND: Patients with peritoneal metastatic colorectal cancer have reduced overall survival compared with patients with metastatic colorectal cancer without peritoneal involvement. Here we further investigated the effect of the number and location of metastases in patients receiving first-line systemic chemotherapy. METHODS: We analysed individual patient data for previously untreated patients enrolled in 14 phase 3 randomised trials done between 1997 and 2008. Trials were included if protocols explicitly pre-specified and solicited for patients with peritoneal involvement in the trial data collection process or had done a formal peritoneum-focused review of individual pre-treatment scans. We used stratified multivariable Cox models to assess the prognostic associations of peritoneal metastatic colorectal cancer with overall survival and progression-free survival, adjusting for other key clinical-pathological factors (age, sex, Eastern Cooperative Oncology Group (ECOG) performance score, primary tumour location [colon vs rectum], previous treatment, and baseline BMI). The primary endpoint was difference in overall survival between populations with and without peritoneal metastases. FINDINGS: Individual patient data were available for 10 553 patients. 9178 (87%) of 10 553 patients had non-peritoneal metastatic colorectal cancer (4385 with one site of metastasis, 4793 with two or more sites of metastasis), 194 (2%) patients had isolated peritoneal metastatic colorectal cancer, and 1181 (11%) had peritoneal metastatic colorectal cancer and other organ involvement. These groups were similar in age, ethnic origin, and use of targeted treatment. Patients with peritoneal metastatic colorectal cancer were more likely than those with non-peritoneal metastatic colorectal cancer to be women (565 [41%] of 1371 vs 3312 [36%] of 9169 patients; p=0·0003), have colon primary tumours (1116 [84%] of 1334 patients vs 5603 [66%]; p<0·0001), and have performance status of 2 (136 [10%] vs 521 [6%]; p<0·0001). We recorded a higher proportion of patients with mutated BRAF in patients with peritoneal-only (eight [18%] of 44 patients with available data) and peritoneal metastatic colorectal cancer with other sites of metastasis (34 [12%] of 289), compared with patients with non-peritoneal metastatic colorectal cancer (194 [9%] of 2230; p=0·028 comparing the three groups). Overall survival (adjusted HR 0·75, 95% CI 0·63-0·91; p=0·003) was better in patients with isolated non-peritoneal sites than in those with isolated peritoneal metastatic colorectal cancer. Overall survival of patients with two of more non-peritoneal sites of metastasis (adjusted HR 1·04, 95% CI 0·86-1·25, p=0.69) and those with peritoneal metastatic colorectal cancer plus one other site of metastasis (adjusted HR 1·10, 95% CI 0·89-1·37, p=0·37) was similar to those with isolated peritoneal metastases. Compared with patients with isolated peritoneal metastases, those with peritoneal metastases and two or more additional sites of metastasis had the shortest survival (adjusted HR 1·40; CI 1·14-1·71; p=0·0011). INTERPRETATION: Patients with peritoneal metastatic colorectal cancer have significantly shorter overall survival than those with other isolated sites of metastases. In patients with several sites of metastasis, poor survival is a function of both increased number of metastatic sites and peritoneal involvement. The pattern of metastasis and in particular, peritoneal involvement, results in prognostic heterogeneity of metastatic colorectal cancer. FUNDING: None.

Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium.

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.

Quality Indicators to Assure and Improve Cancer Care in Spain Using the Delphi Technique.

BACKGROUND: The quality of cancer care has become a priority for health care systems. The goal of this research was to develop a set of evidence-based quality indicators (QIs) for organization, palliative care, and colorectal, breast, and lung cancers for introducing a system of benchmarking in Spain. METHODS: A comprehensive evidence-based literature search was performed to identify potential QIs. An expert panel (the health care quality promotion group) of 9 oncologists identified indicators and evaluated them. A Delphi process involving 58 physicians was used to rank QIs by clinical relevance (validity). The expert panel then evaluated the selected indicators in terms of the feasibility of measuring them in Spanish hospitals, their usefulness for comparisons, their degree of clinical relevance, and their sensitivity to the impact of health care improvements. RESULTS: From the literature review, 99 potential QIs were identified. The Delphi process shortened the list to 72 QIs. A final set of 57 QIs was established by the health care quality promotion group: 12 related to organizational issues, 11 to colorectal cancer, 11 to breast cancer, 12 to lung cancer, and 11 to palliative care. This final set included structure (n=2), process (n=36), and outcome (n=19) indicators. CONCLUSIONS: A set of QIs has been developed using a validated Delphi method, meaning that we can be confident of their validity, feasibility, sensitivity, and acceptability. These QIs are to serve as the basis of a strategy for benchmarking across oncology services in Spanish hospitals and should enable us to assure and improve the quality of cancer care.

BRCA1 Alternative splicing landscape in breast tissue samples.

BACKGROUND: BRCA1 is a key protein in cell network, involved in DNA repair pathways and cell cycle. Recently, the ENIGMA consortium has reported a high number of alternative splicing (AS) events at this locus in blood-derived samples. However, BRCA1 splicing pattern in breast tissue samples is unknown. Here, we provide an accurate description of BRCA1 splicing events distribution in breast tissue samples. METHODS: BRCA1 splicing events were scanned in 70 breast tumor samples, 4 breast samples from healthy individuals and in 72 blood-derived samples by capillary electrophoresis (capillary EP). Molecular subtype was identified in all tumor samples. Splicing events were considered predominant if their relative expression level was at least the 10% of the full-length reference signal. RESULTS: 54 BRCA1 AS events were identified, 27 of them were annotated as predominant in at least one sample. Δ5q, Δ13, Δ9, Δ5 and ▼1aA were significantly more frequently annotated as predominant in breast tumor samples than in blood-derived samples. Predominant splicing events were, on average, more frequent in tumor samples than in normal breast tissue samples (P = 0.010). Similarly, likely inactivating splicing events (PTC-NMDs, Non-Coding, Δ5 and Δ18) were more frequently annotated as predominant in tumor than in normal breast samples (P = 0.020), whereas there were no significant differences for other splicing events (No-Fs) frequency distribution between tumor and normal breast samples (P = 0.689). CONCLUSIONS: Our results complement recent findings by the ENIGMA consortium, demonstrating that BRCA1 AS, despite its tremendous complexity, is similar in breast and blood samples, with no evidences for tissue specific AS events. Further on, we conclude that somatic inactivation of BRCA1 through spliciogenic mutations is, at best, a rare mechanism in breast carcinogenesis, albeit our data detects an excess of likely inactivating AS events in breast tumor samples.

Renal cell cancer metastases to esophagus and stomach successfully treated with radiotherapy and pazopanib.

Renal cell cancer has been rarely reported as a cause of gastric or esophageal metastases. They usually present with gastrointestinal bleeding and most cases have been managed surgically or endoscopically. We report the case of a 38-year-old man with a 4-year history of metastatic renal cell carcinoma admitted to the emergency room with melena and anemia. At endoscopy, three esophageal polypoid lesions (middle and distal thirds) and a 7 cm mass in the gastric fundus were identified. Biopsy revealed esophageal mucosa infiltrated by renal cell carcinoma. Radiotherapy was administered (30 Gy in 10 fractions), followed by pazopanib, with excellent tolerance and without new bleeding episodes. Computed tomography scan showed complete disappearance of the esophageal and fundic lesions at 3 months follow-up. Twenty-four months after being initiated on pazopanib, there is no radiological evidence of disease. This is the first reported case showing complete remission of gastric and esophageal metastases after treatment with radiotherapy and pazopanib.

Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data.

BACKGROUND: We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data. METHODS: We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics. FINDINGS: 17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p<0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p<0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p<0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p<0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p<0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p<0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p<0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p<0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model. INTERPRETATION: These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. FUNDING: Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

Capillary electrophoresis analysis of conventional splicing assays: IARC analytical and clinical classification of 31 BRCA2 genetic variants.

Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c.7617+1G>A, and c.8954-5A>G), and 27 analytical Class-2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806-14T>C) is a BRCA2 splicing quantitative trait locus.

About 1% of the breast and ovarian Spanish families testing negative for BRCA1 and BRCA2 are carriers of RAD51D pathogenic variants.

RAD51D mutations have been recently identified in breast (BC) and ovarian cancer (OC) families. Although an etiological role in OC appears to be present, the association of RAD51D mutations and BC risk is more unclear. We aimed to determine the prevalence of germline RAD51D mutations in Spanish BC/OC families negative for BRCA1/BRCA2 mutations. We analyzed 842 index patients: 491 from BC/OC families, 171 BC families, 51 OC families and 129 patients without family history but with early-onset BC or OC or metachronous BC and OC. Mutation detection was performed with high-resolution melting, denaturing high-performance liquid chromatography or Sanger sequencing. Three mutations were found in four families with BC and OC cases (0.82%). Two were novel: c.1A>T (p.Met1?) and c.667+2_667+23del, leading to the exon 7 skipping and one previously described: c.674C>T (p.Arg232*). All were present in BC/OC families with only one OC. The c.667+2_667+23del cosegregated in the family with one early-onset BC and two bilateral BC cases. We also identified the c.629C>T (p.Ala210Val) variant, which was predicted in silico to be potentially pathogenic. About 1% of the BC and OC Spanish families negative for BRCA1/BRCA2 are carriers of RAD51D mutations. The presence of several BC mutation carriers, albeit in the context of familial OC, suggests an increased risk for BC, which should be taken into account in the follow-up and early detection measures. RAD51D testing should be considered in clinical setting for families with BC and OC, irrespective of the number of OC cases in the family.

Limited family structure and triple-negative breast cancer (TNBC) subtype as predictors of BRCA mutations in a genetic counseling cohort of early-onset sporadic breast cancers.

Early-onset diagnosis is an eligibility criterion for BRCA1 and BRCA2 (BRCA) testing in sporadic breast cancer patients. Limited family structure has been proposed as a predictor of BRCA mutation status in this group of patients. An overwhelming amount of data supports a strong association between BRCA1 mutations and triple-negative breast cancer (TNBC). Here, we analyze the feasibility of using limited family structure and TNBC as predictors of BRCA mutation status in early-onset breast cancer patients attending genetic counseling units. We have conducted the study in a cohort of sporadic early-onset (≤35 years) breast cancer patients (N = 341) previously selected for BRCA genetic testing in Academic Hereditary Cancer Clinics from Spain. A retrospective review of medical records available at the time of risk assessment allowed us classifying patients according to family structure and TNBC. In addition, BRCAPRO score was calculated for all patients. Association between categorical variables was investigated using the Fisher's exact test. Binary Logistic Regression Analysis was used for multivariate analysis. Limited family structure (OR 3.61, p = 0.013) and TNBC (OR 3.14, p = 0.013) were independent predictors of BRCA mutation status. Mutation prevalence in the subgroup of patients with at least one positive predictor was 14%, whereas it dropped to 3% in non-TNBCs with adequate family history (OR 5.31, 95% CI 1.38-23.89, p = 0.006). BRCAPRO correctly discerned between limited and adequate family structures. Limited family structure and TNBC are feasible predictors of BRCA mutation status in sporadic early-onset (≤35 years) breast cancer patients attending genetic counseling units. The low prevalence of mutations observed in non-TNBCs with adequate family structure suggests that this subgroup of patients might be excluded from genetic testing.

Prognostic value of liver metastases in colorectal cancer treated by systemic therapy: An ARCAD pooled analysis.

BACKGROUND: The liver is the most frequent site of metastases in colorectal cancer (CRC). This study aimed to assess the response rate and survival outcomes in metastatic CRC patients with non-liver metastases (NLM) compared to those with liver metastases (LM) across different lines of treatment. METHODS: A total of 17,924 mCRC patients included in 26 trials from the ARCAD CRC database were analyzed. The analysis was conducted based on the presence or absence of LM across different treatment groups: chemotherapy (CT) alone, CT + anti-VEGF, CT + anti-EGFR in KRAS wild-type tumors, within the first-line (1 L) and second-line (2 L), and patients enrolled in third-line (≥3 L) trials treated with trifluridine/tipiracil or regorafenib or placebo. The endpoints were overall survival (OS), progression-free survival (PFS), and overall response rate (ORR). RESULTS: Out of the 17,924 patients, 14,066 had LM (30.6 % with only liver involvement and 69.4 % with liver and other metastatic sites), while 3858 patients had NLM. In the CT alone and CT + anti-VEGF subgroups, NLM patients showed better OS and PFS in the 1 L and 2 L settings. However, in the CT + anti-EGFR 1 L and 2 L subgroups, there was no significant difference in OS and PFS between NLM and LM patients. In the ≥ 3 L subgroups, better OS and PFS were observed in NLM patients. ORRs were higher in LM patients than in NLM patients across all cohorts treated in the 1 L and only in the anti-EGFR cohort in the 2 L. CONCLUSION: LM is a poor prognostic factor for mCRC increasing from 1 L to ≥ 3 L except for patients in 1 L and 2 L receiving CT+anti-EGFR. These data justify using LM as a stratification factor in future trials for patients with unresectable mCRC.

Circulating tumor cells following first chemotherapy cycle: an early and strong predictor of outcome in patients with metastatic breast cancer.

We investigated the prognostic significance of circulating tumor cells (CTCs) determined immediately before the second cycle of chemotherapy in patients with metastatic breast cancer (MBC). The CTC counts were taken at baseline, before the first cycle of chemotherapy (CTC-0), and on day 21 before commencing the second cycle of chemotherapy (CTC-21) in consecutive MBC patients. The study's primary objectives were to analyze relationships between CTC-21 count and overall survival (OS). Based on the current literature, the CTC measurements were dichotomized as 0-4 versus ≥ 5 CTCs. Of 117 patients recruited, 99 were evaluable. Patients with 0-4 CTCs on day 21 had a significantly better OS than those with ≥ 5 CTCs (median OS: 38.5 months vs. 8.7 months). They also had a significantly better progression-free survival (PFS; median: 9.4 months vs. 3.0 months) and clinical benefit rate (77% vs. 44%). The OS of patients whose baseline CTCs were ≥ 5 but dropped to <5 on day 21 was apparently similar to those who had <5 CTCs at baseline. In a Cox regression analysis, CTC-21 was the only independent variable significantly predicting OS and PFS. Our data indicate that CTCs determined immediately before the second cycle of chemotherapy is an early and strong predictor of treatment outcome in MBC patients.

Results of a Phase II Trial Testing the Resensitization With Trabectedin in Platinum-resistant Ovarian Cancer.

BACKGROUND/AIM: In patients with advanced platinum-resistant ovarian cancer we prospectively evaluated whether trabectedin could resensitize the tumor cells to platinum rechallenge. PATIENTS AND METHODS: Upon progression to platinum-based chemotherapy, trabectedin was administered as a 3-hour infusion every three weeks and subsequently crossed over to carboplatin/carboplatin-based combinations. The primary endpoints comprised objective response rate (ORR) and time to progression after trabectedin (TTP Trab). Secondary endpoints included ORR following platinum post-trabectedin, the growth modulation index (GMI) assessed as the ratio of successive TTP to platinum, given after (TTP2) and before (TTP1) trabectedin, quality of life (QoL), and ancillary translational studies. RESULTS: Ten patients with platinum-resistant ovarian cancer from a single institution were treated with trabectedin, one of whom achieved a partial response (PR) reaching the ORR of 10% and six had stable disease (SD) for a disease control rate (DCR) of 70%. After the treatment with platinum post-trabectedin, one patient achieved a PR and two had SD, attaining a rate of resensitization to platinum of 37.5%. The median TTP with trabectedin treatment was 15.0 weeks, while eight patients who received platinum post-trabectedin had the median TTP2 of 19.9 weeks. One patient reached the threshold of GMI >1 (12.5%) as indicator of clinical benefit. QoL of patients was not deteriorated with trabectedin. Predictive biomarkers of response to trabectedin and/or re-exposure to platinum could not be identified. CONCLUSION: Although trabectedin did not achieve a wide resensitization to platinum in this heavily pretreated platinum-resistant population, a significant number of patients attained disease control.

Bevacizumab in Combination With Oxaliplatin-Based Chemotherapy As First-Line Therapy in Metastatic Colorectal Cancer: A Randomized Phase III Study.

PURPOSE: To evaluate the efficacy and safety of bevacizumab when added to first-line oxaliplatin-based chemotherapy (either capecitabine plus oxaliplatin [XELOX] or fluorouracil/folinic acid plus oxaliplatin [FOLFOX-4]) in patients with metastatic colorectal cancer (MCRC). PATIENTS AND METHODS: Patients with MCRC were randomly assigned, in a 2 × 2 factorial design, to XELOX versus FOLFOX-4, and then to bevacizumab versus placebo. The primary end point was progression-free survival (PFS). RESULTS: A total of 1,401 patients were randomly assigned in this 2 × 2 analysis. Median progression-free survival (PFS) was 9.4 months in the bevacizumab group and 8.0 months in the placebo group (hazard ratio [HR], 0.83; 97.5% CI, 0.72 to 0.95; P = .0023). Median overall survival was 21.3 months in the bevacizumab group and 19.9 months in the placebo group (HR, 0.89; 97.5% CI, 0.76 to 1.03; P = .077). Response rates were similar in both arms. Analysis of treatment withdrawals showed that, despite protocol allowance of treatment continuation until disease progression, only 29% and 47% of bevacizumab and placebo recipients, respectively, were treated until progression. The toxicity profile of bevacizumab was consistent with that documented in previous trials. CONCLUSION: The addition of bevacizumab to oxaliplatin-based chemotherapy significantly improved PFS in this first-line trial in patients with MCRC. Overall survival differences did not reach statistical significance, and response rate was not improved by the addition of bevacizumab. Treatment continuation until disease progression may be necessary in order to optimize the contribution of bevacizumab to therapy.

Optimal maintenance strategy following FOLFOX plus anti-EGFR induction therapy in patients with RAS wild type metastatic colorectal cancer: An individual patient data pooled analysis of randomised clinical trials.

BACKGROUND: Anti-EGFR antibodies plus doublet chemotherapy is the standard of care in RAS/BRAF wild-type metastatic colorectal cancer (mCRC). No phase-3 level of evidence is available to guide treatment de-escalation after anti-EGFR-based first-line. Several randomised clinical trials investigated de-intensification strategies with 5-fluorouracil/leucovorin (5-FU/LV) and/or anti-EGFR. METHODS: We performed an individual patient data pooled analysis of Valentino, Panama, MACRO-2, COIN-B trials including RAS wild-type mCRC patients who received first-line therapy with FOLFOX plus panitumumab or cetuximab followed by pre-specified maintenance strategy. Only patients who started maintenance according to the assigned arm were included. Patients were categorised by type of maintenance (i.e. 5-FU/LV, anti-EGFR or 5-FU/LV + anti-EGFR). Progression-free survival (PFS) and overall survival (OS) were calculated from the start of maintenance; toxicity was evaluated for the maintenance treatment period. RESULTS: A total of 518 patients were included in the pooled analysis. Overall, 123, 185 and 210 patients received maintenance with 5-FU/LV, anti-EGFR, 5-FU/LV + anti-EGFR, respectively. Median PFS was 5.6, 6.0 and 9.0 (P = 0.009) and OS was 25.7, 24.0 and 28.0 months (P = 0.134) in 5-FU/LV, anti-EGFR and 5-FU/LV + anti-EGFR arms, respectively. Monotherapy maintenance (either 5-FU/LV or anti-EGFR) was inferior to combination in terms of PFS (hazard ratios [HR] 1.26, P = 0.016) and non-significantly trending also in OS (HR 1.20, P = 0.111). An increase of overall any grade and grade ≥ 3 AEs and selected AEs was reported in combination compared to either 5-FU/LV or anti-EGFR arms. CONCLUSIONS: This pooled analysis including four randomised phase II supports the use of 5-FU/LV plus anti-EGFR as the preferred maintenance regimen. Data provide rational for a more individualised maintenance treatment approach based on tumour and patients features.