Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study.

BACKGROUND: Recent clinical results support the use of new immune checkpoint blockers (ICB), such as anti-PD-1 (e.g. nivolumab and pembrolizumab) and anti-PD-L1 antibodies. Radiological evaluation of ICB efficacy during therapy is challenging due to tumor immune infiltration. Changes of circulating tumor DNA (ctDNA) levels during therapy could be a promising tool for very accurate monitoring of treatment efficacy, but data are lacking with ICB. PATIENTS AND METHODS: This prospective pilot study was conducted in patients with nonsmall cell lung cancer, uveal melanoma, or microsatellite-instable colorectal cancer treated by nivolumab or pembrolizumab monotherapy at Institut Curie. ctDNA levels were assessed at baseline and after 8 weeks (w8) by bidirectional pyrophosphorolysis-activated polymerization, droplet digital PCR or next-generation sequencing depending on the mutation type. Radiological evaluation of efficacy of treatment was carried out by using immune-related response criteria. RESULTS: ctDNA was detected at baseline in 10 out of 15 patients. At w8, a significant correlation (r = 0.86; P = 0.002) was observed between synchronous changes in ctDNA levels and tumor size. Patients in whom ctDNA levels became undetectable at w8 presented a marked and lasting response to therapy. ctDNA detection at w8 was also a significant prognostic factor in terms of progression-free survival (hazard ratio = 10.2; 95% confidence interval 2.5-41, P < 0.001) and overall survival (hazard ratio = 15; 95% confidence interval 2.5-94.9, P = 0.004). CONCLUSION: This proof-of-principle study is the first to demonstrate that quantitative ctDNA monitoring is a valuable tool to assess tumor response in patients treated with anti-PD-1 drugs.

Short-term psychological impact of the BRCA1/2 test result in women with breast cancer according to their perceived probability of genetic predisposition to cancer.

BACKGROUND: The effect of BRCA1/2 gene test result on anxiety, depression, cancer-related thought intrusion or avoidance and perceived control over cancer risk was assessed in breast cancer (BC) patients, according to their perceived probability of genetic predisposition to cancer. METHODS: Two hundred and forty-three (89% response rate) women with BC completed questionnaires after an initial genetic counselling visit (T1), of which 180 (66%) completed questionnaires again after receiving the BRCA1/2 results (T2). The discrepancy between women's perceived probability of cancer genetic predisposition at T1 and the geneticist's computed estimates was assessed. RESULTS: In all, 74% of women received a negative uninformative (NU), 11% a positive BRCA1/2 and 15% an unclassified variant (UV) result. On hierarchical regression analysis, in women with a positive BRCA1/2 result (vs NU or UV), a lower perceived probability of cancer genetic predisposition than objective estimates at T1 predicted lower levels of anxiety at T2 (ß=-0.28; P<0.01), whereas in women receiving a UV result (vs NU or positive BRCA1/2), a lower perceived probability of cancer genetic predisposition than objective estimates at T1 predicted higher levels of anxiety (ß=0.20; P<0.01), depression (ß=0.19; P<0.05) and intrusion (ß=0.18; P<0.05) at T2. CONCLUSION: The type of BRCA1/2 test result differently affects distress according to women's perceived probability of genetic predisposition before testing.

Lack of referral for genetic counseling and testing in BRCA1/2 and Lynch syndromes: a nationwide study based on 240,134 consultations and 134,652 genetic tests.

Based on nationwide data from the French national cancer institute (INCa), we analyzed the evolution of cancer genetics consultations and testing over time, and the uptake of targeted tests in relatives of families with BRCA1/2 or MMR genes mutation. Genetic testing and consultations for familial high-risk individuals are exclusively funded and monitored by the INCa in France. All nationwide cancer genetics centers reported annually standardized parameters of activity from 2003 to 2011. The analysis included a total of 240,134 consultations and 134,652 genetic tests enabling to identify 32,494 mutation carriers. Referral for hereditary breast and ovarian cancer (HBOC) or colorectal cancer predisposition syndromes represented 59 % (141,639) and 23.2 % (55,698) consultations, respectively. From 2003 to 2011, we found a dramatic and steady increase of tests performed for BRCA1/2 (from 2,095 to 7,393 tests/year, P < 0.0001) but not for MMR genes (from 1,144 to 1,635/year, P = NS). The overall percentage of deleterious mutations identified in the probands tested was 13.8 and 20.9 % in HBOC and Lynch syndromes, respectively. Pooled analysis for BRCA1/2 and Lynch syndrome tests showed an inverse relationship between the percentage of mutation detected and the absolute number of tests performed over the time (overall Cochran-Armitage test for trend: P < 0.001). In families with BRCA1/2 or MMR identified mutations, there was an average number of 2.94 and 3.28 relatives performing targeted tests, respectively. This nationwide study shows a lack of referral and genetic testing in Lynch as compared to HBOC syndromes. Only a third of relatives of a proband with a predisposing mutation performed a targeted test. Enhanced information about benefit of genetic testing should be given to clinicians and patients for Lynch syndrome and relatives of a proband carrying an identified predisposing mutation.

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

BACKGROUND: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. RESULTS: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). CONCLUSIONS: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers.

Breast and ovarian cancer predisposition due to de novo BRCA1 and BRCA2 mutations.

BRCA1 and BRCA2 are the two major genes predisposing to breast and ovarian cancer. Whereas high de novo mutation rates have been demonstrated for several genes, only 11 cases of de novo BRCA1/2 mutations have been reported to date and the BRCA1/2 de novo mutation rate remains unknown. The present study was designed to fill this gap based on a series of 12 805 consecutive unrelated patients diagnosed with breast and/or ovarian cancer who met the inclusion criteria for BRCA1/2 gene analysis according to French guidelines. BRCA1/2 mutations were detected in 1527 (12%) patients, and three BRCA1 mutations and one BRCA2 mutation were de novo. The BRCA1/2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%). Although rare, it may be useful to take the possibility of de novo BRCA1/2 mutation into account in genetic counseling of relatives and to improve the understanding of complex family histories of breast and ovarian cancers.

Review article: non-systemic chemotherapy in the treatment of colorectal cancer-portal vein, hepatic arterial and intraperitoneal approaches.

Loco-regional chemotherapy, an alternative to systemic chemotherapy in the management of colorectal cancer, has been evaluated in both adjuvant and palliative settings. The rationale for loco-regional delivery is to achieve higher dose concentrations of drugs at the tumour site or at the most common sites of tumour recurrence, while limiting systemic exposure and associated toxicity. Adjuvant intraportal chemotherapy and palliative hepa-tic arterial chemotherapy have been most extensively investigated. Intraperitoneal chemotherapy has also been studied as an adjuvant treatment after complete resection of colorectal cancer or cytoreductive surgery in patients with established peritoneal carcinomatosis. The results obtained have been disappointing, and none of these procedures can be considered as a standard therapeutic option today. However, methodological difficulties were encountered in most published studies, and the investigated schedules and doses may not have been optimal. New combinations of cytotoxic drugs and new indications are currently under consideration. Promising results have recently been published for adjuvant intraperitoneal chemotherapy and hepatic arterial chemotherapy following surgical resection of hepatic metastases, but additional well-designed multicentre phase III trials are needed to determine the true benefits of these treatment modalities and to address the issues of cost and quality of life.

Cyclo-oxygenase-2 over-expression in sporadic colorectal carcinoma without lymph node involvement.

BACKGROUND: Cyclo-oxygenase-2 over-expression has been reported in most advanced human colorectal cancers. AIMS: To assess the prevalence of cyclo-oxygenase-2 over-expression in non-advanced colorectal cancers, to investigate the correlation between cyclo-oxygenase-2 status and tumour clinicopathological features and molecular phenotype, and to determine the impact of cyclo-oxygenase-2 status on long-term clinical outcome. METHODS: Sixty-one patients who had undergone surgery for colorectal cancer without lymph node involvement were evaluated retrospectively. Cyclo-oxygenase-2 expression was determined by immunohistochemistry. The tumour replication error phenotype was assessed by amplification of the two microsatellites, BAT-25 and BAT-26. RESULTS: Thirty-six tumours were classified as cyclo-oxygenase-2 positive and 25 as cyclo-oxygenase-2 negative. No correlation was found between cyclo-oxygenase-2 over-expression and clinicopathological features or molecular phenotype. Cyclo-oxygenase-2 over-expression was an independent predictor of a poor prognosis. Indeed, the relative risk of tumour recurrence or death for patients with cyclo-oxygenase-2-positive tumours was 2.13 times that of patients with cyclo-oxygenase-2-negative tumours (P=0.008; 95% confidence interval, 1.22-3.73). This difference remained significant when post-operative deaths were censored in the multivariate analysis (P=0.014). CONCLUSION: Cyclo-oxygenase-2 over-expression is not associated with tumour phenotype, but is indicative of a poorer clinical outcome in patients with non-advanced colorectal carcinoma.

Molecular mechanisms involved in the antiproliferative effect of two COX-2 inhibitors, nimesulide and NS-398, on colorectal cancer cell lines.

BACKGROUND: Cyclooxygenase (COX)-2 is up-regulated in most colorectal cancers. Chronic use of non-steroidal anti-inflammatory drugs, which target cyclooxygenases, have been shown to reduce the risk of these cancers. However, the mechanisms underlying this protective effect remain unclear. AIMS: The aim of our study was to characterize the effects of two COX-2 selective inhibitors, NS-398 and nimesulide, on colorectal cancer cell proliferation, and to describe the molecular mechanisms involved. MATERIALS AND METHODS: HT-29 and SW-1116 cell lines were cultured with either NS-398 or nimesulide. Cell proliferation was assessed by staining DNA with crystal violet. Cell cycle repartition and apoptosis were analysed by flow cytometry. The expression of COX-1 and COX-2. and of two cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1, was analysed by Western blotting and RT-PCR. RESULTS: Both drugs dose-dependently inhibited cell proliferation and induced G1 cell cycle blockade. HT-29 cells were more sensitive to both drugs than SW-1116 cells. p21Cip1 and p27Kip1 were induced on both cell lines. Concomitant induction of p21Cip1 mRNA indicates transcriptional modulation, whereas induction of p27Kip1 only at the protein level suggests post-translational modulation. CONCLUSION: NS-398 and nimesulide inhibit colorectal cell proliferation through induction of p21Cip1 and p27Kip1.

[Standards, options and recommendations for tumor markers in colorectal cancer]. / Standards, Options et Recommandations: marqueurs tumoraux sériques dans les cancers du côlon.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the French National Federation of Comprehensive Cancer Centers (FNCLCC), the 20 French Cancer Centers and specialists from French Public University or General Hospitals, and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome of cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To define, according to the definitions of the Standards, Options and Recommendations project, the characteristics of the main tumor markers in colorectal cancer and their potential role in the management of patients with this malignancy. METHODS: Data were identified by searching Medline and the personal reference lists of members of the expert groups. Once the guidelines were defined, the document was submitted for review to 117 independent reviewers, and to the medical committees of the 20 French Cancer Centers. RESULTS: The main recommendations for the tumor markers in colorectal cancer are: 1) The carcinoembryonic antigen (CEA) is the reference serum marker (standard). 2) All the analyses for a given patient must be performed in the same laboratory, using the same technique (standard, expert agreement). 3) CEA or CA 19-9 should not be used for screening or diagnosis (standard, level of evidence B2). 4) High initial serum concentration of CEA is of bad predictive value (standard, level of evidence C). CEA is an independent prognostic factor of survival in colorectal cancers with lymph node metastases (standard, level of evidence B2). 5) CEA is the most sensitive biological parameter for the screening of hepatic metastases (standard, level of evidence B2). 6) CEA serum concentration before palliative chemotherapy is an independent prognostic factor of survival (standard, level of evidence B2). The combination of CEA assay with imagery techniques and clinical examination can help monitor the response to palliative chemotherapy (standard), in particular in non measurable disease (standard, expert agreement). 7) In 65% of the cases, CEA is the first indicator of relapse (standard, level of evidence B2). CEA is the choice marker for monitoring patients with colorectal cancer (standard, level of evidence B2). 8) A sustained biological follow-up including CEA assay can be used to predict the operability of recurring tumors (standard, level of evidence B2). Nevertheless, no survival advantage has been shown (standard).

[Towards an allotype of second generation colon cancer]. / Vers un allélotype de deuxième génération du cancer colique.

A subset of genetic alterations distinguishes two groups of colon cancers. In the first group instability of microsatellite loci due to a defective DNA mismatch repair system is observed. The second group is characterized by recurrent losses of chromosome regions, frequently associated with hyperploidization. We have developed a technique which enables a fine description of allelic losses in this second group of tumours. The typing of 278 loci in 47 hyperploid colon cancers has provided information for an average of 160 loci per tumour. The high frequency of allelic losses on chromosomes 17, 18 and 5 was confirmed thus validating our methodological approach. Several additional chromosome segments were observed lost in over 40% of the cases, suggesting that tumour suppressor genes may map within these regions. Further technical development should contribute to the identification of these genes.

[Localization of a tumor suppressor gene distal to D22S270 in colorectal cancers]. / Localisation d'un gène suppresseur de tumeur distal à D22S270 dans les cancers colorectaux.

Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.

[Clinical significance of BRAF mutations in colorectal cancer]. / Intérêt clinique des mutations de BRAF dans le cancer colorectal.

BRAF mutations, present in 5 to 10% of colorectal cancers, have a proved oncogenic effect which is linked to their implication in the RAS/MAPK intracellular signalling pathway and they occurred at early stage of colorectal carcinogenesis. Many studies have therefore assessed their clinical significance as diagnostic and prognostic marker in colorectal cancers. More recently, their location downstream to EGFR and KRAS in the RAS/MAPK pathway have led to their evaluation as predictive marker of resistance to anti-EGFR monoclonal antibodies. This article aims to review the role of BRAF mutations in the diagnostic strategy of Lynch syndrome, their prognostic value in colorectal cancers and their potential value as predictive marker of resistance to anti-EGFR antibodies.

Contribution of CDKN2A/P16 ( INK4A ), P14 (ARF), CDK4 and BRCA1/2 germline mutations in individuals with suspected genetic predisposition to uveal melanoma.

Uveal melanoma arises from melanocytes of the uveal tract (iris, ciliary body and choroid) and represents the most common intraocular malignancy in adults. Some rare clinical situations (young age at diagnosis, bilateral or multifocal forms, association with cutaneous malignant melanoma and/or familial aggregations of melanomas) are suggestive of genetic susceptibility. The aim of this study was to evaluate the contribution of CDKN2A/P16INK4A, P14ARF and CDK4 gene germline mutations in a series of patients with uveal melanoma recruited in a single institution with a clinical presentation indicative of genetic predisposition. Molecular analyses were proposed to 36 patients and were performed in 25 cases. The contribution of BRCA1/2 gene germline mutations in patients with uveal melanoma and a personal and/or family history of breast/ovarian cancers was also evaluated. Molecular analysis of BRCA1/2 genes was proposed to 35 patients and was performed in 25 patients. No deleterious germline mutation was identified in either group of patients. These results indicate that the CDKN2A/P16INK4A, P14ARF, CDK4 genes are not responsible for the vast majority of genetic susceptibility to uveal melanoma. They also suggest that one case of uveal melanoma in a family with a history of breast cancer is not sufficient to justify BRCA1/2 genetic testing when the classical criteria for molecular analysis are not present. International studies are ongoing in melanoma-prone families in an attempt to identify uveal melanoma susceptibility loci and genes.