Lorenza Pastorino - Academia.edu (original) (raw)

Papers by Lorenza Pastorino

International Journal of Molecular Sciences

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. How... more ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients—and classified by in silico tools as pathogenic, uncertain significance, or benign—using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation i...

Anticancer Research, 2018

Background/Aim: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominantly inherited... more Background/Aim: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominantly inherited disorder characterized by multiple basal cell carcinomas (BCC), odontogenic tumors and various skeletal anomalies. Basaloid follicular hamartomas (BFHs) constitute rare neoplasms that can be detected in sporadic and familial settings as in the Basaloid Follicular Hamartoma Syndrome (BFHS). Although BFHS shares clinical, histopathological and genetic overlapping with the NBCCS, they are still considered two distinctive entities. The aim of our singleinstitution study was the analysis of a cohort of PTCH1mutated patients in order to define clinical and biomolecular relationship between NBCCS and BFHs. Materials and Methods: In our study we evaluated PTCH1 gene-carrier probands affected by NBCCS to detect the incidence of BFHs and their correlation with this rare syndrome. Results: Among probands we recognized 4 patients with BFHs. We found 15 germline PTCH1 mutations, uniformly distributed across the PTCH1 gene. Six of them had familial history of NBCCS, two of them were novel and have not been described previously. Conclusion: NBCCS and BFHS may be the same genetic entity and not two distinctive syndromes. The inclusion of BFH in the NBCCS cutaneous tumor spectrum might be useful for the recognition of misdiagnosed NBCCS cases that could benefit from tailored surveillance strategies. Nevoid basal cell carcinoma syndrome (NBCCS) (OMIM #109400), also known as Gorlin syndrome (GS) is an autosomal-dominant inherited disorder characterized by predisposition to the development of basal cell carcinomas (BCCs) along with various dental, osseous, ophthalmic, and neurological abnormalities (1, 2). A wide variety of benign or malignant tumors can be found in association with these syndromes, such as ovarian fibroma, medulloblastoma, rhabdomyosarcoma, cardiac fibroma and ameloblastoma (1). NBCCS is correlated with PTCH1 germline mutations, but in this hereditary setting the genotype-phenotype correlation is not always present. In fact, gene carriers and family members that present the same PTCH1 germline mutation have a variable skin tumor spectrum phenotype (3, 4) including pigmented BCCs, infundibulocystic BCC, cystic BCCs, Pinkus fibroepithelioma (5), and other benign neoplasms as ovarian fibroma, medulloblastoma, rhabdomyosarcomas, cardiac fibromas and ameloblastoma (1). Basaloid follicular hamartoma (BFH) is a rare benign skin appendage tumor that presents hair follicle differentiation and may be a familial, congenital or acquired condition (6). This tumor appears as small skin-coloured or yellowish or brownish papule and is 471

Cancers, 2020

The contribution of recently established or candidate susceptibility genes to melanoma missing he... more The contribution of recently established or candidate susceptibility genes to melanoma missing heritability has yet to be determined. Multigene panel testing could increase diagnostic yield and better define the role of candidate genes. We characterized 273 CDKN2A/ARF and CDK4-negative probands through a custom-designed targeted gene panel that included CDKN2A/ARF, CDK4, ACD, BAP1, MITF, POT1, TERF2IP, ATM, and PALB2. Co-segregation, loss of heterozygosity (LOH)/protein expression analysis, and splicing characterization were performed to improve variant classification. We identified 16 (5.9%) pathogenic and likely pathogenic variants in established high/medium penetrance cutaneous melanoma susceptibility genes (BAP1, POT1, ACD, MITF, and TERF2IP), including two novel variants in BAP1 and 4 in POT1. We also found four deleterious and five likely deleterious variants in ATM (3.3%). Thus, including potentially deleterious variants in ATM increased the diagnostic yield to about 9%. Incl...

Nature Genetics, 2020

This is a repository copy of Genome-wide association meta-analyses combining multiple risk phenot... more This is a repository copy of Genome-wide association meta-analyses combining multiple risk phenotypes provide insights into the genetic architecture of cutaneous melanoma susceptibility.

The Lancet Child & Adolescent Health, 2019

Journal of the American Academy of Dermatology, Jan 28, 2018

Cyclin dependent kinase inhibitor 2A gene (CDKN2A) germline mutations have recently been associat... more Cyclin dependent kinase inhibitor 2A gene (CDKN2A) germline mutations have recently been associated with poor survival in patients with melanoma. Despite the high mutation rate in our cohort (up to 10% in patients with apparently sporadic melanoma), information on the impact of CDKN2A on survival in this cohort is lacking. To investigate whether poor survival associated with CDKN2A germline mutations was confirmed in a high mutation-prevalence cohort of Italian patients with melanoma undergoing a mutation-based follow-up. A total of 1239 patients with cutaneous melanoma were tested for CDKN2A mutational status and then assigned to a follow-up scheme according not only to family history but also to CDKN2A mutational status, as follow-up intervals were more frequent for CDKN2A germline mutation-positive (MUT) patients. From this cohort, we selected 106 MUT patients (with familial melanoma or apparently sporadic melanoma) and 199 CDKN2A germline mutation-negative (MUT) patients with sp...

Journal of the National Cancer Institute, Jan 4, 2018

The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary t... more The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary tumor syndrome caused by germline pathogenic variants in BAP1 encoding a tumor suppressor associated with uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, and cutaneous BAP1-inactivated melanocytic tumors. However, the full spectrum of tumors associated with the syndrome is yet to be determined. Improved understanding of the BAP1-TPDS is crucial for appropriate clinical management of BAP1 germline variant carriers and their families, including genetic counseling and surveillance for new tumors. We collated germline variant status, tumor diagnoses, and information on BAP1 immunohistochemistry or loss of somatic heterozygosity on 106 published and 75 unpublished BAP1 germline variant-positive families worldwide to better characterize the genotypes and phenotypes associated with the BAP1-TPDS. Tumor spectrum and ages of onset were compared between missense and null var...

Human molecular genetics, Jan 30, 2018

Melanoma heritability is among the highest for cancer and single nucleotide polymorphisms (SNPs) ... more Melanoma heritability is among the highest for cancer and single nucleotide polymorphisms (SNPs) contribute to it. To date, only SNPs that reached statistical significance in genome-wide association studies or few candidate SNPs have been included in melanoma risk prediction models. We compared four approaches for building polygenic risk scores (PRS) using 12,874 melanoma cases and 23,203 controls from Melanoma Meta-Analysis Consortium as a training set, and newly genotyped 3,102 cases and 2,301 controls from the MelaNostrum consortium for validation. We estimated adjusted odds ratios (ORs) for melanoma risk using traditional melanoma risk factors and the PRS with the largest area under the Receiver Operator Characteristics curve (AUC). We estimated absolute risks combining the PRS and other risk factors, with age- and sex-specific melanoma incidence and competing mortality rates from Italy as an example. The best PRS, including 204 SNPs (AUC= 64.4%; 95% CI=63-65.8%), developed usin...

PloS one, 2017

CDKN2A coding region germline variants are associated with pancreatic adenocarcinoma (PC) suscept... more CDKN2A coding region germline variants are associated with pancreatic adenocarcinoma (PC) susceptibility. Recently, we described functional germline 5'UTR CDKN2A variants from melanoma patients affecting the post-transcriptional regulation of p16INK4a mRNA that is dependent, at least in part, on an Internal Ribosome Entry Site (IRES) in the 5'UTR region. Here we describe a 5'UTR c.-201_-198delinsCTTT CDKN2A variant (frequency 0.0028 based on 350 PC patients), which seems to be private to PC, since it has never been found in public databases nor in thousands of melanoma patients tested. Functional analyses confirmed IRES activity of the 5'UTR in BX-PC3 PC cells and revealed a functional impact of the identified variant. Using gene reporter assays we observed reduced translation potential in cells treated with the mTOR inhibitor Torin1, a condition that favors the assessment of IRES activity. At the endogenous gene level we quantified allelic imbalance among polysome-a...

Oncotarget, Jan 19, 2018

Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profil... more Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants.mutations were present in 68% of cases, whilegermline mutations were found in 16 % and p16 loss in tissue was found in 63%.promoter somatic mutations were detected in 38% of cases while the-245T>C polymorphism was found in 51% of cases.mutations were found in 39% ofnegative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a p...

Oncotarget, 2017

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is ... more Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used. Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable. Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid-PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples. Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.

Indian journal of pathology & microbiology

Gorlin-Goltz syndrome (GGS) is an uncommon autosomal dominant inherited disorder which comprises ... more Gorlin-Goltz syndrome (GGS) is an uncommon autosomal dominant inherited disorder which comprises the triad of basal cell carcinomas (BCCs), odontogenic keratocysts, and musculoskeletal malformations. Besides this triad, neurological, ophthalmic, endocrine, and genital manifestations are known to be variable. It is occasionally associated with aggressive BCC and internal malignancies. This report documents a case of GGS with a novel mutation in the PTCH1 gene in an 11-year-old child. The clinical, radiographic, histopathologic and molecular findings of this condition, and treatment are described, and a review of GGS was carried out.

Human genetics, Jan 23, 2016

The risk of pancreatic cancer (PC) is increased in melanoma-prone families but the causal relatio... more The risk of pancreatic cancer (PC) is increased in melanoma-prone families but the causal relationship between germline CDKN2A mutations and PC risk is uncertain, suggesting the existence of non-CDKN2A factors. One genetic possibility involves patients having mutations in multiple high-risk PC-related genes; however, no systematic examination has yet been conducted. We used next-generation sequencing data to examine 24 putative PC-related genes in 43 PC patients with and 23 PC patients without germline CDKN2A mutations and 1001 controls. For each gene and the four pathways in which they occurred, we tested whether PC patients (overall or CDKN2A+ and CDKN2A- cases separately) had an increased number of rare nonsynonymous variants. Overall, we identified 35 missense variants in PC patients, 14 in CDKN2A+ and 21 in CDKN2A- PC cases. We found nominally significant associations for mismatch repair genes (MLH1, MSH2, MSH6, PMS2) in all PC patients and for ATM, CPA1, and PMS2 in CDKN2A- PC...

Journal of the American Academy of Dermatology, 2016

Background: Multiple primary melanoma (MPM), in concert with a positive family history, is a pred... more Background: Multiple primary melanoma (MPM), in concert with a positive family history, is a predictor of cyclin-dependent kinase (CDK) inhibitor 2A (CDKN2A) germline mutations. A rule regarding the presence of either 2 or 3 or more cancer events (melanoma and pancreatic cancer) in low or high melanoma incidence populations, respectively, has been established to select patients for genetic referral. Objective: We sought to determine the CDKN2A/CDK4/microphthalmia-associated transcription factor mutation rate among Italian patients with MPM to appropriately direct genetic counseling regardless of family history. Methods: In all, 587 patients with MPM and an equal number with single primary melanomas and control subjects were consecutively enrolled at the participating centers and tested for CDKN2A, CDK4, and microphthalmia-associated transcription factor. Results: CDKN2A germline mutations were found in 19% of patients with MPM versus 4.4% of patients with single primary melanoma. In familial MPM cases the mutation rate varied from 36.6% to 58.8%, whereas in

Pigment cell & melanoma research, Jan 18, 2015

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulato... more Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (9 of which were novel) in the CDKN2A 5'UTR with independent approaches, which included mono and bicistronic reporter assays, western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.-27del23,c.-93-91delAGG) were classified as causal mutations (score ≥3), along with the c.-21C>T,c.-34G>T and c.-56G>T, which had already been studied by a subset of assays. The novel c.-42T>A as well as the previously described c.-67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.-14C>T,c.-20A>G,…

Endocrine, 2015

The optimal method for BRAF mutation detection remains to be determined despite advances in molec... more The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

Pigment Cell & Melanoma Research, 2013

A French and an Australian study have recently identified a rare germline functional variant in t... more A French and an Australian study have recently identified a rare germline functional variant in the microphthalmia-associated transcription factor (MITF) (E318K) that predisposes to familial and sporadic melanoma and to renal cell carcinoma (RCC), showing a new link between two tumour types with different risk factors and between deregulated sumoylation and cancer. The aim of this study was to test the prevalence of the MITF E318K mutation in 667 Italian melanoma patients. We observed significant associations between histological subtypes and family cancer history. Carriers exhibited a nearly threefold higher risk of developing melanoma compared with controls. Carriers were also more likely to have developed multiple primary melanomas (6.40-fold), compared with wt patients. Carriers with a personal and/or family history of pancreatic cancer and kidney cancer had a nearly 31- and eightfold higher risk of developing melanoma compared with wt patients. Our findings further support MITF as a medium-penetrance melanoma susceptibility gene, highlight a potential association with histological subtypes and suggest that MITF may predispose to pancreatic cancer.

OncoTargets and Therapy, 2015

BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy a... more BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. More recently, the combination of BRAF inhibitor dabrafenib with MEK inhibitor trametinib has shown improved progression-free survival, compared to dabrafenib monotherapy, in a Phase II study and has received approval by the US Food and Drug Administration. However, even when treated with the combination, most patients develop mechanisms of acquired resistance, and some of them do not achieve tumor regression at all, because of intrinsic resistance to therapy. Along with the development of BRAF inhibitors, immunotherapy made an important step forward: ipilimumab, an anti-CTLA-4 monoclonal antibody, was approved for the treatment of metastatic melanoma; anti-PD-1 agents achieved promising results in Phase I/II trials, and data from Phase III studies will be ready soon. The availability of such drugs, which are effective regardless of BRAF status, has made the therapeutic approach more complex, as first-line treatment with BRAF inhibitors may not be the best choice for all BRAF-mutated patients. The aim of this paper is to review the systemic therapeutic options available today for patients affected by BRAF V600-mutated metastatic melanoma, as well as to summarize the mechanisms of resistance to BRAF inhibitors and discuss the possible strategies to overcome them. Moreover, since the molecular analysis of tumor specimens is now a pivotal and decisional factor in the treatment strategy of metastatic melanoma patients, the advances in the molecular detection techniques for the BRAF V600 mutation will be reported.