Expectations and psychological issues before genetic counseling: analysis of distress determinant factors.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) and Hereditary Breast and Ovarian Cancer Syndrome (HBOC) are the most common hereditary cancer syndromes in which a genetic test is available. Potential risks associated with testing include psychological harm, emotional distress and insurance problems. METHODS: The aim of the present study is to investigate determinants of distress in a sample of Italian subjects undergoing genetic counseling. Demographic information and psychological distress were assessed by using a self-reported questionnaire and the "Hospital Anxiety and Depression Scale" (HAD), before attending the first counseling session. RESULTS: Of the all subjects referred for the first time to our Center (January 2012-June 2013), a total of 227 were eligible (female/male = 174/53) for the survey, 134 (59%) were oncologic patients and of these, 116 received genetic test (36 for HNPCC and 80 for HBOC). The remaining 93 (41%) were healthy subjects referred for suspected familiar history and of this group, 65 subjects performed predictive test in a family with a known pathogenic mutation (53 for HBOC and 12 for HNPCC). Affected subjects had a significantly higher level of anxiety (p = 0.02) and HAD global score (p = 0.01) than healthy ones. There was no difference in HAD score between individuals testing for different syndromes (p = 0.3). In the affected subgroup, there was a significant linear correlation between the HAD anxiety score and how much subjects perceived their disease as hereditary (p = 0.01). Female and younger subjects had higher levels of anxiety (p = 0.05). Also healthy single subjects show more general distress (p = 0.02) than those with a partner. CONCLUSIONS: Greater level of distress identified on females, single and younger subjects.

Correction to: Expectations and psychological issues before genetic counseling: analysis of distress determinant factors.

[This corrects the article DOI: 10.1186/s13053-020-00142-1.].

BRCA mutations and gastrointestinal cancers: When to expect the unexpected?

BRCA1/2 pathogenic variants are widely known as major risk factors mainly for breast and ovarian cancer, while their role in gastrointestinal (GI) malignancies such as colorectal cancer (CRC), gastric cancer and oesophageal cancer (OeC) is still not well established. The main objective of this review is to summarise the available evidence on this matter. The studies included in the review were selected from PubMed/GoogleScholar/ScienceDirect databases to identify published articles where BRCA1/2 pathogenic variants were assessed either as a risk factor or a prognostic/predictive factor in these malignancies. Our review suggests that BRCA1/2 might have a role as a risk factor for colorectal, gastric and OeC, albeit with differences among these diseases: In particular BRCA1 seems to be much more frequently mutated in CRC whereas BRCA2 appears to be much more closely associated with gastric and OeC. Early-onset cancer seems to be also associated with BRCA1/2 mutations and a few studies suggest a positive prognostic role of these mutations. The assessment of a potentially predictive role of these mutations is hampered by the fact that most patients with these diseases have been treated with platinum compounds, where it is expected that a higher probability of response should be seen. A few clinical trials focused on poly (ADP-ribose) polymerase inhibitors use in GI cancers are currently ongoing.

Lynch syndrome-associated lung cancer: pitfalls of an immunotherapy-based treatment strategy in an unusual tumor type.

Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline alterations in mismatch repair (MMR) genes leading to increased risk of colon cancer as well as other cancer types. Non-small cell lung cancer (NSCLC) is not among typical Lynch syndrome-associated tumors: pembrolizumab, an immune checkpoint inhibitor, is actually approved for the treatment of NSCLC patients and represents a promising treatment option for patients with advanced metastatic MMR-deficient cancer, regardless of tumor origin. This case report describes the clinical presentation and management of a 74-year-old female with a history of rectal adenocarcinoma and ovarian cancer, who has a documented frameshift pathogenic variant in the exon 8 of MSH6 gene and an intronic variant in the BRCA2 gene (classified as a variant of uncertain significance), affected by NSCLC with brain metastases. Despite these premises, the patient was treated with pembrolizumab and she did not benefit from this kind of treatment.

MSH2 missense mutations and HNPCC syndrome: pathogenicity assessment in a human expression system.

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is associated with germline mutations in one of several MisMatch Repair (MMR) genes. An increasing proportion (20-25%) of the reported MSH2 variants consists of single amino-acid substitution with uncertain disease-causing significance. The present study was undertaken to functionally characterize 3 MSH2 nontruncating variants: p.Gly162Arg (c.484G>C), p.Asp167His (c.499G>C) and p.Arg359Ser (c.1077A>T). Missense alterations, were assessed in a human system for expression/stability and for the ability to heterodimerize with MSH6 and correctly localize into the nucleus. Functional assays results were correlated with clinical and genetic features indicative of HNPCC as MicroSatellite-Instability (MSI), abnormalities of MMR gene expression in tumour tissue (IHC) and familial history. p.Gly162Arg and p.Arg359Ser variants showed a clearly decreased expression level of the MutSá complex and were associated with an abnormal subcellular localization pattern, which can be suggestive of an incorrect MSH2/MSH6 heterodimerization. Functional analysis results were supported by MSI and IHC data and by familial cancer history. The subcellular localization assay, performed in a human expression system, classifies as pathogenetic two MSH2 nontruncating alterations providing a useful tool in genetic testing programs.

A germline missense mutation in exon 3 of the MSH2 gene in a Lynch syndrome family: correlation with phenotype and localization assay.

Lynch syndrome is caused by germline mutations in any of the MisMatch Repair (MMR) genes. About 37% of MSH2 variants are missense variants causing single amino-acid substitutions. Whether missense variants affect the normal function of MMR proteins is crucial both to provide affected families a more accurate risk assessment and to offer predictive testing to family members. Here we report one family, fulfilling both Amsterdam I and II criteria and Bethesda guidelines, referred to our center for genetic counselling. The proband and some of her relatives have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Also Subcellular Localization Assay and Splice site predictions analyses were used. A germline missense variant of uncertain significance (exon 3, p.Val161Asp) was found in MSH2 gene in proband and in some relatives. The variant was associated with lack of expression of MSH2 protein (DMMR) and MSI-High status in tumour tissues. The localization assay of the MSH2 protein showed an abnormal subcellular localization pattern of the corresponding protein. Finally, splice-site prediction analysis ruled out a potential role of new splice sites as the cause behind the lack of expression of MSH2 protein and we suppose a potential correlation with other forms of post-transcriptional regulation (circular RNAs). The variant here reported shows a high correlation with phenotype and is located in an evolutionary conserved domain. The localization assay also suggest a potential pathogenic role, thus supporting further research on this matter.

A missense germline mutation in exon 7 of the MSH2 gene in a HNPCC family from center-Italy.

INTRODUCTION: Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inherited disease predisposing to the development of colorectal cancers and several other malignancies (endometrium, ovaries, stomach, small bowel, hepatobiliary and urinary tract). HNPCC is caused by germline mutations in any of the MisMatch Repair (MMR) genes. Mutations in MLH1 and MSH2 account for almost 90% of all identified ones. About 15% of mutations identified in MSH2 are missense ones. PATIENTS AND METHODS: We studied one family, fulfilling Amsterdam II criteria, referred to our Center for genetic counselling. The proband, and some of her relatives, have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining and by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: All patients carried the same novel MSH2 germline missense mutation (R359S) in exon 7, which determines the substitution of an Arginine, which is a basic amino acid, with a polar Serine residue (R359S). The mutation was associated with lack of expression of MSH2 protein and high microsatellite instability in tumour tissues. The same mutation has been detected in one healthy relative. CONCLUSIONS: The mutation here reported shows a high correlation with phenotype. The mutation is located in an evolutionary conserved domain. Taken together, our findings suggest evidence that the amino acid substitution can be interpreted as pathogenetic.

Incidence and risk of cardiotoxicity in cancer patients treated with targeted therapies.

BACKGROUND: Cardiotoxicityis a serious side effect of molecularly targeted agents. The purpose of this study was to evaluate the incidence and Relative Risk (RR) of developing all-grade and high-grade cardiotoxicity in patients with solid tumors receiving targeted agents through a revised meta-analysis of available clinical trials. METHODS: The scientific literature regarding cardiotoxicity was extensively analyzed using MEDLINE, PubMed, Embase and Cochrane Central Register of Controlled Trials (CENTRAL). Eligible studies were selected according to PRISMA statement. Summary incidence, RR, and 95% CIs were calculated using random-effects or fixed-effects models based on the heterogeneity of selected studies. RESULTS: Our search yielded a total of 4998 clinical studies; of them, 31 trials were finally considered for this meta-analysis. A total of 28,538 patients were included; 7995 of these patients had breast cancer (28%), 6151 (22%) prostate cancer and 14,392 (50%) were treated for other malignancies. The highest RR of high-grade events was observed with Vandetanib (RR=7.71, 95% CI 1.04-56.99), followed by Ramucirumab (RR=5.0) and Aflibercept (RR=4.1). Grouping by drug category, the highest incidence of high-grade cardiotoxicity was shown by anti-VEGFR-TKIs (RR 5.62, 95% CI 1.49-21.24) and anti-VEGF mAbs/VEGF-trap (RR 1.82, 95% CI 1.24-2.69). Grouping by tumor type, the highest incidence of cardiotoxicity was observed in thyroid cancer (8%), followed by gastric cancer (4%). CONCLUSIONS: Treatment with targeted agents in cancer patients is correlated with a significant increase in the risk of cardiotoxicity. Frequent clinical monitoring should be emphasized when using these and newer biological agents.

Assessing the pathogenicity of MLH1 missense mutations in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with clinical, genetic and functional features.

Assessing the pathogenicity of missense mutations of MLH1 and MSH2 is critical to counsel patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC). Approximately 32% of all MLH1 mutations and 18% of MSH2 mutations are missense variants which often have an uncertain genetic significance. To assess the pathogenicity of four MLH1 missense mutations which were found in five patients with suspected HNPCC, P648S (CCC --> TCC), L559R (CTG --> CGG), K618A (AAG --> GCG), Y646C (TAT --> TGT), we studied their ability to disrupt MLH1 protein function and their relationship with all those clinical, genetic and pathological features which are typical of this syndrome. Our results indicated that the P648S and L559R mutations were probably pathogenic because they disrupted MLH1 protein interaction with its partner PMS2 in vitro and abolished MLH1 expression in HCT116 cells. In addition these variants were associated with features often found in HNPCC patients: in particular high microsatellite instability, occurrence of high grade tumours and, in one case, strong family history. The pathogenicity of the K618A and Y646C mutations was questionable as their correlation with features typical of HNPCC was low and the outcome of the functional analysis was ambiguous. These observations suggested that a clinically usable assessment of the pathogenicity of MLH missense variants can be achieved through the analysis of multiple mutation characteristics among which loss of protein function, occurrence of microsatellite instability and family history seemed to have a predominant role.

Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

BACKGROUND: Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown. Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC. METHODS: 302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence. Patients were classified as mutation-positive or negative according to the genetic testing result. RESULTS: A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46-0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16-0.82, p = 0.0153). CONCLUSIONS: Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future.

Sub-cellular localization analysis of MSH6 missense mutations does not reveal an overt MSH6 nuclear transport impairment.

Nearly one-third of the identified MSH6 germline mutations deal with single amino acid substitutions. For an effective genetic counselling it is necessary to clearly elucidate by functional tools the specific sub-processes underlying the mismatch repair (MMR) misfunctioning in MSH6 non-truncating mutants. Since the MMR repair pathway occurs in the nucleus, we suppose the impairment of MutSα nuclear trafficking to be a possible Lynch syndrome susceptibility causative mechanism. In the present study the MMR status of the tumour, the main clinical features of mutation carriers and population data associated to the MSH6 missense variants, were complemented with computational data about tolerability predictions and amino acid substitution conservation. The selected panel of ten potentially pathogenic MSH6 mutations was analyzed in a homologous expression system for possible deleterious effects on nucleo-cytoplasmic shuttling through the assessment of the sub-cellular localization of the corresponding mutated proteins. Localization analysis results do not reveal an apparent role of MSH6 missense mutations in nuclear import impairment and provide the first hint to exclude the MSH6 nuclear translocation sub-process as a possible causative mechanisms of MMR misfunctioning.

Role of vascular endothelial growth factor (VEGF) and VEGF-R genotyping in guiding the metastatic process in pT4a resected gastric cancer patients.

In radically resected gastric cancer the possibility to predict the site of relapse could be clinically relevant for the selection of post-surgical management. We previously showed that specific tumour integrins genotypes are independently associated with either peritoneal or hematogenous metastases (ITGA and ITGV). Recently VEGF and VEGF-R polymorphisms have been demonstrated to potentially affect tumour angiogenesis and the metastatic process in gastric cancer. We then investigated the role of VEGFs and VEGF-R genotyping in determining either peritoneal carcinosis or hematogenous metastases in radically resected gastric cancer patients. Tumour genotyping for integrins (ITGA and ITGV) was also performed according to our previous findings. Genotyping for VEGF-A, VEGF-C, VEGFR-1,2,3 and ITGA and ITGV was carried out on pT4a radically resected gastric tumours recurring with either peritoneal-only carcinosis or hematogenous metastases. 101 patients fulfilled the inclusion criteria: 57 with peritoneal carcinomatosis only and 44 with hematogenous spread only. At multivariate analysis, intestinal histology and the AC genotype of rs699947 (VEGFA) showed to independently correlate with hematogenous metastases (p = 0.0008 and 0.008 respectively), whereas diffuse histology and the AA genotype of rs2269772 (ITGA) independently correlated with peritoneal-only diffusion (p = <0.0001 and 0.03 respectively). Our results seem to indicate that combining information from genotyping of rs699947 (VEGFA, AC), rs2269772 (ITGA, AA) and tumour histology could allow clinicians to individuate gastric cancer at high risk for recurrence either with peritoneal or hematogenous metastases. The selection tool deriving from this analysis may allow an optimal use of the available treatment strategies in these patients.

The role of LDH serum levels in predicting global outcome in HCC patients undergoing TACE: implications for clinical management.

In many tumor types serum lactate dehydrogenase (LDH) levels is an indirect marker of tumor hypoxia, neo-angiogenesis and worse prognosis. However data about hepatocellular carcinoma (HCC) are lacking in the clinical setting of patients undergoing transarterial-chemoembolization (TACE) in whom hypoxia and neo-angiogenesis may represent a molecular key to treatment failure. Aim of our analysis was to evaluate the role of LDH pre-treatment levels in determining clinical outcome for patients with HCC receiving TACE. One hundred and fourteen patients were available for our analysis. For all patients LDH values were collected within one month before the procedure. We divided our patients into two groups, according to LDH serum concentration registered before TACE (first: LDH≤450 U/l 84 patients; second: LDH>450 U/l 30 patients). Patients were classified according to the variation in LDH serum levels pre- and post-treatment (increased: 62 patients vs. decreased 52 patients). No statistically significant differences were found between the groups for all clinical characteristics analyzed (gender, median age, performance status ECOG, staging systems). In patients with LDH values below 450 U/l median time to progression (TTP) was 16.3 months, whereas it was of 10.1 months in patients above the cut-off (p = 0.0085). Accordingly median overall survival (OS) was 22.4 months and 11.7 months (p = 0.0049). In patients with decreased LDH values after treatment median TTP was 12.4 months, and median OS was 22.1 months, whereas TTP was 9.1 months and OS was 9.5 in patients with increased LDH levels (TTP: p = 0.0087; OS: p<0.0001). In our experience, LDH seemed able to predict clinical outcome for HCC patients undergoing TACE. Given the correlation between LDH levels and tumor angiogenesis we can speculate that patients with high LDH pretreatment levels may be optimal candidates for clinical trial exploring a multimodality treatment approach with TACE and anti-VEGF inhibitors in order to improve TTP and OS.

BRCA1 expression in triple negative sporadic breast cancers.

OBJECTIVE: To study how to identify patients with "triple negative" sporadic breast cancers (BCs) having BRCA1 silenced or down-regulated due to epigenetic BRCA1 inactivation. STUDY DESIGN: We selected, from our database, patients diagnosed with BC between 1995 and 2001 with tumors exhibiting the "triple negative" phenotype. "Triple positive" tumors were used as controls. BRCA1 protein expression was determined by immunohistochemistry. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing on genomic DNA were used to assess BRCA1 promoter methylation. BRCA1 m-RNA expression analysis was conducted by real-time PCR. RESULTS: Forty-four triple negative and 68 controls (triple positive) were eligible for our analysis. BRCA1 promoter methylation was present in 31.8% of triple negative and in 20.6% of triple positive cases. BRCA1 was inactivated (absent BRCA1 m-RNA expression and lack of BRCA1 protein) in 21.4% of tumors with BRCA1 promoter methylation, as compared with 6% of non-methylated ones (p = 0.0453). CONCLUSION: BRCA1 inactivation due to promoter methylation could play an important role in some sporadic BC cases. Patients with this signature could represent the basis for prospective studies aiming to compare clinical response to different drugs.

CAT25 is a mononucleotide marker to identify HNPCC patients.

Mismatch repair mutations are the cause of generalized genomic instability and are particularly evident at microsatellite loci, which is known as microsatellite instability (MSI). MSI is present in 85% to 90% of colorectal cancers and occurs in hereditary non-polyposis colorectal cancer (HNPCC). The National Cancer Institute recommends the "Bethesda panel" for MSI screening. Recently, a novel T(25) mononucleotide marker was described, termed CAT25. This microsatellite marker displays a quasi-monomorphic pattern in normal tissues. The aim of our study was to evaluate the performance of CAT25 in HNPCC patients and to compare its reliability with the results of the Bethesda panel. We tested 55 tumor tissues from HNPCC patients using both the Bethesda panel and the CAT25 mononucleotide marker. One hundred healthy blood donors were used as controls. The CAT25 microsatellite was found to be altered in all 13 colorectal cancers classified as MSI-H using the standard Bethesda panel. Colorectal tumors that showed a stable Bethesda pattern did not show altered CAT25 repeats. Additionally, CAT25 showed a monomorphic allele pattern in all tissue samples. In our series, the concordance between the Bethesda panel and CAT25 in identifying colorectal cancers with high MSI reached 100%. Our results suggest that the CAT25 microsatellite represents a sensitive and specific marker for MSI and could be, at least, included in the panel of markers for the identification of HNPCC patients.