Canonical and uncanonical pathogenic germline variants in colorectal cancer patients by next-generation sequencing in a European referral center.

BACKGROUND: Despite increasing use of next-generation sequencing (NGS), data concerning the gain in germline pathogenic variants (PVs) remain scanty, especially with respect to uncanonical ones. We aimed to verify the impact of different cancer predisposition genes (CPGs) on colorectal cancer (CRC) in patients referred for genetic evaluation. MATERIALS AND METHODS: We enrolled for NGS, by Illumina TruSight Cancer panel comprising 94 CPGs, 190 consecutive subjects referred for microsatellite instability (MSI) CRC, polyposis, and/or family history. RESULTS: Overall, 51 (26.8%) subjects carried 64 PVs; PVs coexisted in 4 (7.8%) carriers. PVs in mismatch repair (MMR) genes accounted for one-third of variant burden (31.3%). Four Lynch syndrome patients (20%) harbored additional PVs (HOXB13, CHEK2, BRCA1, NF1 plus BRIP1); such multiple PVs occurred only in subjects with PVs in mismatch syndrome genes (4/20 versus 0/31; P = 0.02). Five of 22 (22.7%) patients with MSI cancers but wild-type MMR genes harbored PVs in unconventional genes (FANCL, FANCA, ATM, PTCH1, BAP1). In 10/63 patients (15.9%) with microsatellite stable CRC, 6 had MUTYH PVs (2 being homozygous) and 4 exhibited uncanonical PVs (BRCA2, BRIP1, MC1R, ATM). In polyposis, we detected PVs in 13 (25.5%) cases: 5 (9.8%) in APC, 6 (11.8%) with biallelic PVs in MUTYH, and 2 (3.9%) in uncanonical genes (FANCM, XPC). In subjects tested for family history only, we detected two carriers (18.2%) with PVs (ATM, MUTYH). CONCLUSION: Uncanonical variants may account for up to one-third of PVs, underlining the urgent need of consensus on clinical advice for incidental findings in cancer-predisposing genes not related to patient phenotype.

Predictors of germline status for hereditary melanoma: 5 years of multi-gene panel testing within the Italian Melanoma Intergroup.

BACKGROUND: The incidence of cutaneous melanoma is increasing in Italy, in parallel with the implementation of gene panels. Therefore, a revision of national genetic assessment criteria for hereditary melanoma may be needed. The aim of this study was to identify predictors of susceptibility variants in the largest prospective cohort of Italian high-risk melanoma cases studied to date. MATERIALS AND METHODS: From 25 Italian centers, we recruited 1044 family members and germline sequenced 940 cutaneous melanoma index cases through a shared gene panel, which included the following genes: CDKN2A, CDK4, BAP1, POT1, ACD, TERF2IP, MITF and ATM. We assessed detection rate according to familial status, region of origin, number of melanomas and presence and type of non-melanoma tumors. RESULTS: The overall detection rate was 9.47% (5.53% analyzing CDKN2A alone), ranging from 5.14% in sporadic multiple melanoma cases (spoMPM) with two cutaneous melanomas to 13.9% in familial cases with at least three affected members. Three or more cutaneous melanomas in spoMPM cases, pancreatic cancer and region of origin predicted germline status [odds ratio (OR) = 3.23, 3.15, 2.43, P < 0.05]. Conversely, age > 60 years was a negative independent predictor (OR = 0.13, P = 0.008), and was the age category with the lowest detection rate, especially for CDKN2A. Detection rate was 19% when cutaneous melanoma and pancreatic cancer clustered together. CONCLUSIONS: Gene panel doubled the detection rate given by CDKN2A alone. National genetic testing criteria may need a revision, especially regarding age cut-off (60) in the absence of strong family history, pancreatic cancer and/or a high number of cutaneous melanomas.

Implementation of preventive and predictive BRCA testing in patients with breast, ovarian, pancreatic, and prostate cancer: a position paper of Italian Scientific Societies.

Constitutional BRCA1/BRCA2 pathogenic or likely pathogenic variants (PVs) are associated with an increased risk for developing breast and ovarian cancers. Current evidence indicates that BRCA1/2 PVs are also associated with pancreatic cancer, and that BRCA2 PVs are associated with prostate cancer risk. The identification of carriers of constitutional PVs in the BRCA1/2 genes allows the implementation of individual and family prevention pathways, through validated screening programs and risk-reducing strategies. According to the relevant and increasing therapeutic predictive implications, the inclusion of BRCA testing in the routine management of patients with breast, ovarian, pancreatic and prostate cancers represent a key requirement to optimize medical or surgical therapeutic and prevention decision-making, and access to specific anticancer therapies. Therefore, accurate patient selection, the use of standardized and harmonized procedures, and adherence to homogeneous testing criteria, are essential elements to implement BRCA testing in clinical practice. This consensus position paper has been developed and approved by a multidisciplinary Expert Panel of 64 professionals on behalf of the AIOM-AIRO-AISP-ANISC-AURO-Fondazione AIOM-SIAPEC/IAP-SIBioC-SICO-SIF-SIGE-SIGU-SIU-SIURO-UROP Italian Scientific Societies, and a patient association (aBRCAdaBRA Onlus). The working group included medical, surgical and radiation oncologists, medical and molecular geneticists, clinical molecular biologists, surgical and molecular pathologists, organ specialists such as gynecologists, gastroenterologists and urologists, and pharmacologists. The manuscript is based on the expert consensus and reports the best available evidence, according to the current eligibility criteria for BRCA testing and counseling, it also harmonizes with current Italian National Guidelines and Clinical Recommendations.

A phase 2 study of three low-dose intensity subcutaneous bortezomib regimens in elderly frail patients with untreated multiple myeloma.

This phase 2 trial evaluated three low-dose intensity subcutaneous bortezomib-based treatments in patients ⩾75 years with newly diagnosed multiple myeloma (MM). Patients received subcutaneous bortezomib plus oral prednisone (VP, N=51) or VP plus cyclophosphamide (VCP, N=51) or VP plus melphalan (VMP, N=50), followed by bortezomib maintenance, and half of the patients were frail. Response rate was 64% with VP, 67% with VCP and 86% with VMP, and very good partial response rate or better was 26%, 28.5% and 49%, respectively. Median progression-free survival was 14.0, 15.2 and 17.1 months, and 2-year OS was 60%, 70% and 76% in VP, VCP, VMP, respectively. At least one drug-related grade ⩾3 non-hematologic adverse event (AE) occurred in 22% of VP, 37% of VCP and 33% of VMP patients; the discontinuation rate for AEs was 12%, 14% and 20%, and the 6-month rate of toxicity-related deaths was 4%, 4% and 8%, respectively. The most common grade ⩾3 AEs included infections (8-20%), and constitutional (10-14%) and cardiovascular events (4-12%); peripheral neuropathy was limited (4-6%). Bortezomib maintenance was effective and feasible. VP, VCP and VMP regimens demonstrated no substantial difference. Yet, toxicity was higher with VMP, suggesting that a two-drug combination followed by maintenance should be preferred in frail patients.

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene.

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X-chromosome inactivation, thus rendering DNA sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α-Gal-A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α-Gal-A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations.

Morquio A syndrome due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

Morquio A syndrome (MPS IVA) is a recessive lysosomal storage disorder (LSD) caused by mutations in the GALNS gene leading to the deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Patients show a broad spectrum of phenotypes ranging from classical severe type to mild forms. Classical forms are characterized by severe bone dysplasia and usually normal intelligence. So far, more than 170 unique mutations have been identified in the GALNS gene of MPS IVA patients. We report on a Morquio A patient with a classical phenotype who was found to be homozygous for a missense mutation (c.236 G>A; p.Cys79Tyr) in the GALNS gene. This alteration affects the highly conserved p.Cys79 that is transformed into formylglycine, the catalytic residue of the active site. The mutation was present in the proband's mother, but not in the father, whose paternity was confirmed by microsatellite analysis. In order to test the hypothesis of maternal uniparental disomy (UPD), we investigated the segregation of sixteen microsatellite markers from chromosome 16. The results showed a condition of maternal UPD due to an error in meiosis I. Maternal isodisomy of the 16q24 region led to homozygosity for the GALNS mutant allele, causing the patient's disease. These findings allow to add for the first time the LSD Morquio A syndrome to the list of conditions that can be caused by UPD. The possibility of UPD is relevant when giving genetic counseling to couples since the recurrent risk in future pregnancies is dramatically reduced.

Constitutional FLCN mutations in patients with suspected Birt-Hogg-Dubé syndrome ascertained for non-cutaneous manifestations.

Birt-Hogg-Dubé syndrome (BHDS) is characterized by a clinical triad including cutaneous hamartomas originating from hair follicles, lung cysts/pneumothorax, and kidney tumors. Inactivating mutations of the tumor suppressor gene FLCN are identified in most families with BHDS. Usually, patients are referred for genetic examination by dermatologists because of the presence of typical multiple skin tumors with or without additional symptoms. However, because of phenotypic variability and incomplete penetrance, the clinical presentation of BHDS is not yet fully defined. Criteria for genetic testing and diagnosis that take into account variable manifestations have recently been proposed by the European BHD Consortium. We sequenced the FLCN gene coding region in a series of 19 patients selected for kidney and/or lung manifestations. Overall, FLCN mutations were found in 9 of 19 (47%) families and were detected only in probands who had either >2 components of the clinical triad or a single component (renal or pulmonary) along with a family history of another main BHDS manifestation. Typical cutaneous lesions were present only in 8 of 21 FLCN mutation carriers aged >20 years identified in the mutation-positive families. In addition, we provide clinical and molecular evidence that parotid oncocytoma, so far reported in six BHDS cases, is associated with this condition, based on the observation of a patient with bilateral parotid involvement and marked reduction of the wild-type FLCN allele signal in tumor DNA. Overall, the results obtained in this study contribute to the definition of the phenotypic characteristics that should be considered for BHDS diagnosis and FLCN mutation testing.

Lenalidomide, melphalan, prednisone and thalidomide (RMPT) for relapsed/refractory multiple myeloma.

This multicenter, open-label, non-comparative phase II trial evaluated the safety and efficacy of salvage therapy with lenalidomide, melphalan, prednisone and thalidomide (RMPT) in patients with relapsed/refractory multiple myeloma (MM). Oral lenalidomide (10 mg/day) was administered on days 1-21, and oral melphalan (0.18 mg/kg) and oral prednisone (2 mg/kg) on days 1-4 of each 28-day cycle. Thalidomide was administered at 50 mg/day or 100 mg/day on days 1-28; six cycles were administered in total. Maintenance included lenalidomide 10 mg/day on days 1-21, until unacceptable adverse events or disease progression. Aspirin (100 mg/day) was given as thromboprophylaxis. A total of 44 patients with relapsed/refractory MM were enrolled and 75% achieved at least a partial response (PR), including 32% very good PR (VGPR) and 2% complete response (CR). The 1-year progression-free survival (PFS) was 51% and the 1-year overall survival (OS) from study entry was 72%. Grade 4 hematologic adverse events included neutropenia (18%), thrombocytopenia (7%) and anemia (2%). Grade 3 non-hematologic adverse events were infections (14%), neurological toxicity (4.5%) and fatigue (7%). No grade 3/4 thromboembolic events or peripheral neuropathy were reported. In conclusion, RMPT is an active salvage therapy with good efficacy and manageable side effects. This study represents the basis for larger phase III randomized trials.

Malignant extra-adrenal pheochromocytoma caused by an SDHB intronic variation leading to a 54-bp deletion in exon 4.

In the last few years several papers have reported on the association between mutations of the genes encoding the structural (SDHC, SDHD) and catalytic (SDHB) subunits of succinate dehydrogenase and the occurrence of hereditary pheochromocytomas/paragangliomas (Pheo/PGL) syndromes. We diagnosed a malignant extraadrenal Pheo in a 38-yr-old man with abdominal lesions; many areas of increased uptake at octreoscan scintigraphy in the skeleton indicated metastatic disease. We then approached genetic analysis through the screening of the SDHB, SDHC, and SDHD genes. Here we report a heterozygous G>A transversion at position +1 of intron 4 of SDHB gene. To clarify this mutation we performed cDNA analysis by RT-PCR and we assume that the splice site mutation in intron 4 abolishes the consensus splice donor sequence leading to an in-frame deletion of 18 amino acid. This finding indicates once again that SDHB mutations could predispose to malignant Pheo.

Frequency of constitutional MSH6 mutations in a consecutive series of families with clinical suspicion of HNPCC.

A large majority of constitutional mutations in hereditary non-polyposis colorectal cancer (HNPCC) are because of the MHL 1 or MSH 2 genes. In a lower fraction of cases, another gene of the mismatch repair (MMR) machinery, MSH6, may be responsible. Families with MSH6 mutations are difficult to recognize, as microsatellite instability (MSI) may not be detectable and immunohistochemistry (IHC) may give ambiguous results. In the present study, we proposed (i) to determine the frequency of MSH6 mutations in a selected population of colorectal cancer patients obtained from a tumor registry, (ii) to assess whether IHC is a suitable tool for selecting and identifying MSH6 mutation carriers. One hundred neoplasms of the large bowel from suspected HNPCC families were analyzed for MSI (BAT 25 and BAT 26 markers) and immunohistochemical expression of the MSH6 protein. We found on 12 tumors (from different families) showing instability or lack of MSH6 expression. Among these, four potentially pathogenic MSH6 mutations were detected (del A at 2984; del TT at 3119; del AGG cod 385; and del CGT cod 1242) by direct gene sequencing. These represented 12.9% of all families with constitutional mutations of the DNA MMR genes. Thus, some 5% of all HNPCC families are featured by constitutional mutation of the MSH6 gene. This appears, however, as a minimum estimate; routine use of IHC and the study of large numbers of individuals and families with little or no evidence of Lynch syndrome might reveal that mutation of this gene account for a large fraction of HNPCC.

Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

A mononucleotide markers panel to identify hMLH1/hMSH2 germline mutations.

Hereditary NonPolyposis Colorectal Cancer (Lynch syndrome) is an autosomal dominant disease caused by germline mutations in a class of genes deputed to maintain genomic integrity during cell replication, mutations result in a generalized genomic instability, particularly evident at microsatellite loci (Microsatellite Instability, MSI). MSI is present in 85-90% of colorectal cancers that occur in Lynch Syndrome. To standardize the molecular diagnosis of MSI, a panel of 5 microsatellite markers was proposed (known as the "Bethesda panel"). Aim of our study is to evaluate if MSI testing with two mononucleotide markers, such as BAT25 and BAT26, was sufficient to identify patients with hMLH1/hMSH2 germline mutations. We tested 105 tumours for MSI using both the Bethesda markers and the two mononucleotide markers BAT25 and BAT26. Moreover, immunohistochemical evaluation of MLH1 and MSH2 proteins was executed on the tumours with at least one unstable microsatellite, whereas germline hMLH1/hMSH2 mutations were searched for all cases showing two or more unstable microsatellites. The Bethesda panel detected more MSI(+) tumors than the mononucleotide panel (49.5% and 28.6%, respectively). However, the mononucleotide panel was more efficient to detect MSI(+) tumours with lack of expression of Mismatch Repair proteins (93% vs 54%). Germline mutations were detected in almost all patients whose tumours showed MSI and no expression of MLH1/MSH2 proteins. No germline mutations were found in patients with MSI(+) tumour defined only through dinucleotide markers. In conclusion, the proposed mononucleotide markers panel seems to have a higher predictive value to identify hMLH1 and hMSH2 mutation-positive patients with Lynch syndrome. Moreover, this panel showed increased specificity, thus improving the cost/effectiveness ratio of the biomolecular analyses.

SDH mutations in patients affected by paraganglioma syndromes: a personal experience.

Mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are frequently involved in the development of neural crest-derived (NCD) tumors, such as pheochromocytomas (PHEOs) or paragangliomas (PGLs). In this study we report the results of sequencing analysis in leukocyte DNA of patients affected by PHEO/PGL who turned out to be SDH mutation carriers. A nonsense germline heterozygous mutation (Q109X) was found in the exon 4 of the SDHD gene in the index cases of six unrelated families affected by PHEO/PGL. Haplotype analysis showed the presence of a founder effect. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumors, variably associated or not with PGLs or PHEOs. A novel missense SDHD variant, T112I, was also found in one of our families. A new missense G106D mutation, involving a highly conserved amino acid, was found in two sisters affected by bilateral glomus tumors. A P81L mutation associated with abdominal and head and neck PGL was detected in three families. A G12S variant of the SDHD gene was found in one patient affected by a PHEO. The finding of this variant in 3 of 100 control subjects suggests that it is a polymorphism and not a mutation. A novel IVS2-1G>T variant was found at intron 2 of SDHD gene in one patient affected by a glomus tumor. All the tumors associated with SDHD mutations were benign. Conversely, the only mutation we found in SDHB gene (IVS3+1G>A) was associated with a malignant PHEO.

A genetic model for determining MSH2 and MLH1 carrier probabilities based on family history and tumor microsatellite instability.

Mutation-predicting models can be useful when deciding on the genetic testing of individuals at risk and in determining the cost effectiveness of screening strategies at the population level. The aim of this study was to evaluate the performance of a newly developed genetic model that incorporates tumor microsatellite instability (MSI) information, called the AIFEG model, and in predicting the presence of mutations in MSH2 and MLH1 in probands with suspected hereditary non-polyposis colorectal cancer. The AIFEG model is based on published estimates of mutation frequencies and cancer penetrances in carriers and non-carriers and employs the program MLINK of the FASTLINK package to calculate the proband's carrier probability. Model performance is evaluated in a series of 219 families screened for mutations in both MSH2 and MLH1, in which 68 disease-causing mutations were identified. Predictions are first obtained using family history only and then converted into posterior probabilities using information on MSI. This improves predictions substantially. Using a probability threshold of 10% for mutation analysis, the AIFEG model applied to our series has 100% sensitivity and 71% specificity.

Phenotype variability of neural crest derived tumours in six Italian families segregating the same founder SDHD mutation Q109X.

BACKGROUND: Mutations in genes coding for the mitochondrial complex II succinate dehydrogenase (SDH) subunits cause familial neural crest derived (NCD) tumours. METHODS: Index cases from six apparently unrelated families affected by NCD tumours were analysed for mutations in the SDHB, SDHC, and SDHD genes. RESULTS: The same nonsense germline heterozygous mutation (Q109X) in exon 4 of the SDHD gene was found in each of the six families. Overall, 43 heterozygotes were identified. These were evaluated for the presence of NCD tumours through radiological examination of the neck, thorax, and abdomen, and measurement of urinary metanephrines and plasma chromogranin A. A novel missense SDHD variant, T112I, which did not segregate with the Q109X mutation and was not associated with phenotypic manifestations, was observed in one of the families. Microsatellite analysis showed a common haplotype in all individuals heterozygous for the Q109X mutation, indicating a founder effect. Overall, 18 heterozygotes were clinically affected by at least one NCD tumour. Every affected patient inherited the germline mutation from the father, confirming SDHD maternal genomic imprinting. Penetrance of the paternally inherited mutation progressively increased from 33% to 83% at 30 and 60 years, respectively. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumours variably associated or not with paragangliomas or phaeochromocytomas. Loss of heterozygosity was observed in tumour cells isolated by laser capture microdissection. CONCLUSIONS: This study shows that a single founder SDHD mutation is present in an area of central Italy and that this mutation is associated with widely variable interfamilial and intrafamilial expressivity.

Microsatellite instability is not related to response to cisplatin-based chemotherapy in cervical cancer.

This study investigates whether microsatellite instability (MSI) due to defects of the mismatch repair (MMR) system could be associated with response to cisplatin-based neoadjuvant chemotherapy (NACT) and if cisplatin exposure could select MSI-positive cell clones in cervical cancer. Microsatellite analysis was performed by polymerase chain reactions using six microsatellite markers, while hMLH1 protein expression was investigated by immunohistochemistry. We found that 1 tumor out of 20 (5%) NACT-responding patients and 1 tumor out of 18 (6%) nonresponding patients showed MSI. The analysis of tumor specimens collected after NACT revealed no change in the banding pattern as compared to each corresponding pre-NACT tumor at each locus tested. hMLH1 staining was observed in at least > or =80% of cells in all tumors examined except the two exhibiting MSI. Our data showed that MSI due to defects of the MMR system seems not to play a crucial role in the biology of human cervical cancer cells and that MSI seems not to be related to response to chemotherapy. Moreover, cisplatin exposure did not seem to select for MMR-deficient tumor clones in cervical cancer.

A novel microdeletion syndrome with loss of the MSH2 locus and hereditary non-polyposis colorectal cancer.

Constitutional chromosome deletions can predispose to the development of cancer with the phenotypic characteristics of inherited cancer syndromes, when the deleted region encompasses a tumour suppressor gene. Examples of such conditions are represented by the cytogenetic deletions associated with retinoblastoma, Wilms tumour and familial adenomatous polyposis. So far, no constitutional deletions involving the genes implicated in hereditary non-polyposis colorectal cancer (HNPCC) have been identified. This may be at least partially because of the lack of distinctive phenotypic manifestations in HNPCC. We describe the first case of a constitutional microdeletion associated with HNPCC. Suspicion of a microdeletion was prompted by the association of mental retardation, postnatal growth deficiency, minor congenital anomalies and early onset (37 years) sporadic colon cancer. The patient was found to harbour a microdeletion within chromosome 2p16-p21, including the MSH2 gene. Since there are very few reports of deletions of the 2p16-p21 region, our observation sets the grounds for the definition of a novel multiple congenital anomaly/mental retardation/cancer microdeletion syndrome.