Publications by authors named "Francesca Belardinilli"

22 Publications

  • Page 1 of 1

True conversions from RAS mutant to RAS wild-type in circulating tumor DNA from metastatic colorectal cancer patients as assessed by methylation and mutational signature.

Cancer Lett 2021 Jun 18;507:89-96. Epub 2021 Mar 18.

Cancer Liquid Biopsy Unit, Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena, 324, 00161, Rome, Italy. Electronic address:

The paucity of targeted treatments available in patients with RAS mutant colorectal cancers contributes to the poor prognosis of this patient group compared to those with RAS wild-type disease. Recent liquid biopsy-driven studies have demonstrated that RAS mutant clones might disappear in plasma during the clonal evolution of the disease, opening new unforeseen perspectives for EGFR blockade in these patients. Nevertheless, the lack of detection of RAS mutations in plasma might depend on the low amount of released circulating tumor DNA (ctDNA), making it necessary a more accurate selection of patients with true RAS mutation conversions. In this liquid biopsy-based study, we assessed RAS mutational status in initially RAS-mutant patients at the time of progressive disease from any line of therapy and investigated the incidence of true conversions to plasma RAS wild-type, comparing a colon cancer specific methylation profile with a mutational signature of ctDNA. Globally, considering either mutational panel or methylation profile as reliable tests to confirm or exclude the presence of ctDNA, the percentage of "true RAS converters" was 37.5%. In our series we observed a trend toward a better PFS in patients who received anti-EGFR as second or subsequent treatment lines compared to those who did not.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2021.03.014DOI Listing
June 2021

A multidisciplinary approach for the differential diagnosis between multiple primary lung adenocarcinomas and intrapulmonary metastases.

Pathol Res Pract 2021 Apr 17;220:153387. Epub 2021 Feb 17.

Department of Radiological, Oncological and Pathological Sciences, Sapienza University, Rome, 00161, Italy. Electronic address:

Purpose: The distinction between multiple primary lung cancers (MPLCs) and intrapulmonary metastases has a significant impact on tumor staging and therapeutic choices. Several criteria have been proposed to solve this diagnostic issue, but a definitive consensus is still missing. We tested the efficacy of a combined clinical, histopathological and molecular ("real world") approach for the correct classification of multiple lung tumors in a selected cohort of patients.

Methods: 24 multiple lung tumors with a diagnosis of adenocarcinoma from 10 patients were retrospectively reviewed. Radiological, pathological and clinical information, including follow-up, were integrated with molecular profiling via a routine multigene panel sequencing.

Results: Comprehensive histologic assessment revealed readily distinguishable histologic patterns between multiple tumors suggesting unrelated lesions in 7 cases, in agreement with clinical, radiological and molecular data, thus leading to final diagnosis of MPLCs. In the remaining 3 cases, the differential diagnosis between MPLCs and intrapulmonary metastases was challenging, since the histologic features of the lesions were similar or identical. The final interpretation (2 MPLCs and 1 most likely intrapulmonary metastases) was reached thanks to the integration of all available data, and was confirmed by follow-up.

Conclusions: A multidisciplinary approach including a routinely affordable multigene panel sequencing is a useful tool to discriminate MPLCs from intrapulmonary metastases in multiple lung nodules sharing the adenocarcinoma histotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.prp.2021.153387DOI Listing
April 2021

Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients.

Cancers (Basel) 2020 Dec 4;12(12). Epub 2020 Dec 4.

Liquid Biopsy Unit, Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy.

The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who "convert" to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12123633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761880PMC
December 2020

A novel BRCA2 splice variant identified in a young woman.

Mol Genet Genomic Med 2020 12 7;8(12):e1513. Epub 2020 Nov 7.

Department of Experimental Medicine, University of Roma "La Sapienza", Roma, Italy.

Background: BRCA1/2 VUSs represent an important clinical issue in risk assessment for the breast/ovarian cancer families (HBOC) families. Among them, some occurring within the intron-exon boundary may lead to aberrant splicing process by altering or creating de novo splicing regulatory elements or unmasking cryptic splice site. Defining the impact of these potential splice variants at functional level is important to establish their pathogenic role.

Methods: Genomic DNA was extracted from peripheral blood sample of a young woman affected with breast cancer belonging to a HBOC family and the entire coding regions of the BRCA1 and BRCA2 genes were amplified using the Ion AmpliSeq BRCA1 and BRCA2 Panel. The BRCA2 c.682-2delA variant has been characterized by RT-PCR analysis performed on mRNA extracted from blood and lymphoblastoid cell line.

Results: We demonstrated that a novel BRCA2 c.682-2delA variant at the highly conserved splice consensus site in intron 8 disrupts the canonical splice acceptor site generating a truncated protein as predicted by several bioinformatics tools. Segregations analysis in the family and LOH performed on proband breast cancer tissue further confirmed its classification as pathogenic variant.

Conclusion: Combining different methodologies, we characterized this new BRCA2 variant and provided findings of clinical utility for its classification as pathogenic variant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mgg3.1513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767566PMC
December 2020

Clinical Multigene Panel Sequencing Identifies Distinct Mutational Association Patterns in Metastatic Colorectal Cancer.

Front Oncol 2020 7;10:560. Epub 2020 May 7.

Department of Molecular Medicine, University La Sapienza, Rome, Italy.

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2020.00560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221020PMC
May 2020

PIK3CA somatic mutation in sinonasal teratocarcinosarcoma.

Auris Nasus Larynx 2021 Jun 8;48(3):530-534. Epub 2020 May 8.

Department of Molecular Medicine, Sapienza University, Viale Regina Elena 291 Rome, Italy. Electronic address:

Sinonasal Teratocarcinosarcoma (SNTCS) is a rare and histologically heterogeneous tumor of uncertain origin and unknown molecular pathogenesis. Its location and aggressiveness, with frequent recurrences, high rate for metastasis and short mean survival, make SNTCS a tumor highly difficult to treat. Thus, the identification of underlying genetic changes could potentially provide successful adjuvant or alternative precision medicine treatment options for patients with this tumor. We report here a 55-year-old male with a naso-ethmoidal SNTCS that invaded right maxillary sinus, orbital cavity and cranial anterior fossa and that was treated with surgery followed by radiotherapy and chemotherapy in which we evaluated the mutational profile by multigene panel sequencing. Tumor and adjacent normal mucosa were screened for hotspots and targeted regions of 22 cancer related genes by multigene panel sequencing. The analysis revealed a somatic pathogenic mutations in the PIK3CA gene (p.His1047Leu) and a germline alteration in the DDR2 gene (p.Pro476Leu) whose oncogenic function is considered unknown. This study suggests the involvement of PIK3CA gene mutation in SNTCS tumorigenesis highlighting a potential target for individualized molecular therapy for patients with this tumor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.anl.2020.03.006DOI Listing
June 2021

SMO-M2 mutation does not support cell-autonomous Hedgehog activity in cerebellar granule cell precursors.

Sci Rep 2019 12 23;9(1):19623. Epub 2019 Dec 23.

Department of Molecular Medicine, University La Sapienza, 00161, Rome, Italy.

Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-56057-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928071PMC
December 2019

Identification of novel large genomic rearrangements by a computational algorithm of amplicon-based Next-Generation Sequencing data.

PeerJ 2019 15;7:e7972. Epub 2019 Nov 15.

Department of Molecular Medicine, University of Roma "La Sapienza", Roma, Italy.

Background: Genetic testing for germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients.

Methods: We first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel LGRs was obtained via long-range PCR and direct sequencing of the DNA products.

Results: In our cohort, the newly defined DQ-based algorithm detected 3/3 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6-99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6-99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel deletions, involving exons 16-17, 21-22 and 20, have been characterized.

Conclusions: Our study defined a DQ-based algorithm to identify LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.7972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859874PMC
November 2019

Circulating Tumor Cells in Right- and Left-Sided Colorectal Cancer.

Cancers (Basel) 2019 Jul 24;11(8). Epub 2019 Jul 24.

Department of Molecular Medicine, Circulating tumor cells Unit, Sapienza University of Rome, 00161 Rome, Italy.

Molecular alterations are not randomly distributed in colorectal cancer (CRC), but rather clustered on the basis of primary tumor location underlying the importance of colorectal cancer sidedness. We aimed to investigate whether circulating tumor cells (CTC) characterization might help clarify how different the patterns of dissemination might be relative to the behavior of left- (LCC) compared to right-sided (RCC) cancers. We retrospectively analyzed patients with metastatic CRC who had undergone standard baseline CTC evaluation before starting any first-line systemic treatment. Enumeration of CTC in left- and right-sided tumors were compared. The highest prognostic impact was exerted by CTC in left-sided primary cancer patients, even though the lowest median number of cells was detected in this subgroup of patients. CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTC from distal compared to proximal primary tumors. Most CTC in RCC patients exhibited an apoptotic pattern. CTC in left-sided colon cancer patients exhibit a predominant mesenchymal phenotype. This might imply a substantial difference in the biology of proximal and distal cancers, associated with different patterns of tumor cells dissemination. The poor prognosis of right-sided CRC is not determined by the hematogenous dissemination of tumor cells, which appears to be predominantly a passive shedding of non-viable cells. Conversely, the subgroup of poor-prognosis left-sided CRC is reliably identified by the presence of mesenchymal CTC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11081042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721440PMC
July 2019

Next-generation sequencing of and genes for rapid detection of germline mutations in hereditary breast/ovarian cancer.

PeerJ 2019 22;7:e6661. Epub 2019 Apr 22.

Department of Experimental Medicine, University of Roma "La Sapienza", Roma, Italy.

Background: Conventional methods used to identify and germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment.

Methods: We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set).

Results: By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis.

Discussion: Our experience strongly supports that the Ion Torrent PGM technology in and germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.6661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482939PMC
April 2019

A Simplified Genomic Profiling Approach Predicts Outcome in Metastatic Colorectal Cancer.

Cancers (Basel) 2019 01 27;11(2). Epub 2019 Jan 27.

Department of Molecular Medicine, University La Sapienza, 00161 Rome, Italy.

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on and/or genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of , the expected response is much worse compared to patients with exclusive mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11020147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406354PMC
January 2019

Transient Disappearance of RAS Mutant Clones in Plasma: A Counterintuitive Clinical Use of EGFR Inhibitors in RAS Mutant Metastatic Colorectal Cancer.

Cancers (Basel) 2019 Jan 4;11(1). Epub 2019 Jan 4.

Department of Molecular Medicine, Sapienza University of Rome, V.le Regina Elena 324, 00161 Rome, Italy.

Genomic studies performed through liquid biopsies widely elucidated the evolutionary trajectory of RAS mutant clones under the selective pressure of EGFR inhibitors in patients with wild type RAS primary colorectal tumors. Similarly, the disappearance of RAS mutant clones in plasma has been more recently reported in some patients with primary RAS mutant cancers, supporting for the first time an unexpected negative selection of RAS mutations during the clonal evolution of mCRC. To date, the extent of conversion to RAS wild type disease at the time of progression has not been clarified yet. As a proof of concept, we prospectively enrolled mCRC patients progressing under anti-VEGF based treatments. Idylla™ system was used to screen RAS mutations in plasma and the wild type status of RAS was further confirmed through IT-PGM (Ion Torrent Personal Genome Machine) sequencing. RAS was found mutant in 55% of cases, retaining the same plasma mutation as in the primary tumor at diagnosis, while it was found wild-type in 45%. Four patients testing negative for RAS mutations in plasma at the time of progression of disease (PD) were considered eligible for treatment with EGFR inhibitors and treated accordingly, achieving a clinical benefit. We here propose a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care of mutant RAS colorectal cancer patients through the delivery of a further line of therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11010042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357143PMC
January 2019

MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors.

Cell Death Dis 2018 08 30;9(9):895. Epub 2018 Aug 30.

Department of Molecular Medicine, University La Sapienza, 00161, Rome, Italy.

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-018-0924-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117286PMC
August 2018

Effective treatment of a platinum-resistant cutaneous squamous cell carcinoma case by EGFR pathway inhibition.

Mol Clin Oncol 2018 Jul 21;9(1):30-34. Epub 2018 May 21.

Medical Oncology Sant'Andrea Hospital, I-00189 Rome, Italy.

Cutaneous squamous cell carcinoma (cSCC) is the second most common type of non-melanoma skin cancer. Platinum-based regimens have been an integral part of palliative care for patients with locally advanced or metastatic disease. There is no evidence of efficacy for later lines of chemotherapy and no targeted therapy has been introduced as 'standard of care'. Here we report on the case of an elderly cSCC patient, resistant to conventional therapy, however successfully treated with anti-epidermal growth factor receptor (EGFR) agent (Cetuximab) in addition to a daily dose of Curcumin phospholipid. The patient responded to treatment and experienced no recurrence for 11 months with only minor skin-related toxicity. To our knowledge, this is the first report of clinical evidence that an anti EGFR targeted therapy with a daily oral dose of Curcumin phospholipid is well tolerated and results in a highly effective disease control in a heavily pretreated cSCC patient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mco.2018.1634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031894PMC
July 2018

Coexistence of three EGFR mutations in an NSCLC patient: A brief report.

Int J Biol Markers 2018 Jun 1:1724600818782200. Epub 2018 Jun 1.

1 Dipartimento di Medicina Molecolare, Sapienza Università di Roma, Roma, Italia.

Background: The epidermal growth factor receptor (EGFR) represents a molecular target for tyrosine kinase inhibitors for non-small cell lung cancer (NSCLC) patients with a mutation in the EGFR gene. Mutations of the EGFR gene that occur at a single position in NSCLC tissue are found as single, whereas two or more mutations on the same allele are poorly detected and investigated.

Patient And Methods: We investigated the presence of the EGFR gene mutations in tumor tissue by Sanger sequencing and ion torrent sequencing in an NSCLC patient at Stage IV of disease.

Results: We found the presence of three coexisting mutations on the EGFR gene-two of which on exon 21 are present on the same allele, and the third, on exon 20, was analyzed by Sanger sequencing of the peripheral blood lymphocytes. The patient staged as cT4N0M1c (Stage IV) and started afatinib 40 mg daily 8 months ago, showing a clinical benefit.

Conclusion: In this report we describe the case of an NSCLC patient harboring three coexisting mutations on the EGFR gene, two of which are present on the same allele. This mutation pattern may represent, for patient progeny, a genetic risk of cancer development. Therefore it should be possible to obtain screening guidelines to improve the risk calculation for lung cancer susceptibility in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1724600818782200DOI Listing
June 2018

Evaluation of Polygenic Determinants of Non-Alcoholic Fatty Liver Disease (NAFLD) By a Candidate Genes Resequencing Strategy.

Sci Rep 2018 02 27;8(1):3702. Epub 2018 Feb 27.

Departments of Internal Medicine and Medical Specialties, "Sapienza" University, Rome, Italy.

NAFLD is a polygenic condition but the individual and cumulative contribution of identified genes remains to be established. To get additional insight into the genetic architecture of NAFLD, GWAS-identified GCKR, PPP1R3B, NCAN, LYPLAL1 and TM6SF2 genes were resequenced by next generation sequencing in a cohort of 218 NAFLD subjects and 227 controls, where PNPLA3 rs738409 and MBOAT7 rs641738 genotypes were also obtained. A total of 168 sequence variants were detected and 47 were annotated as functional. When all functional variants within each gene were considered, only those in TM6SF2 accumulate in NAFLD subjects compared to controls (P = 0.04). Among individual variants, rs1260326 in GCKR and rs641738 in MBOAT7 (recessive), rs58542926 in TM6SF2 and rs738409 in PNPLA3 (dominant) emerged as associated to NAFLD, with PNPLA3 rs738409 being the strongest predictor (OR 3.12, 95% CI, 1.8-5.5, P < 0.001). A 4-SNPs weighted genetic risk score value >0.28 was associated with a 3-fold increased risk of NAFLD. Interestingly, rs61756425 in PPP1R3B and rs641738 in MBOAT7 genes were predictors of NAFLD severity. Overall, TM6SF2, GCKR, PNPLA3 and MBOAT7 were confirmed to be associated with NAFLD and a score based on these genes was highly predictive of this condition. In addition, PPP1R3B and MBOAT7 might influence NAFLD severity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-21939-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829219PMC
February 2018

Optimizing the identification of risk-relevant mutations by multigene panel testing in selected hereditary breast/ovarian cancer families.

Cancer Med 2018 01 22;7(1):46-55. Epub 2017 Dec 22.

Department of Molecular Medicine, University La Sapienza, V.le R. Elena 291, Rome, 00161, Italy.

The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence-based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger-sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2-negative families to be subjected to the BROCA-Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss-of-heterozygosity and further molecular studies. We identified loss-of-function mutations in 6 out 20 high-risk families, 5 of which occurred on BRCA1, CHEK2 and ATM and are esteemed to be risk-relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre-organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk-relevant alleles impacting on the clinical management of their carriers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cam4.1251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773970PMC
January 2018

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production.

Oncoimmunology 2017;6(3):e1290037. Epub 2017 Feb 10.

Department of Experimental Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2017.1290037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384385PMC
February 2017

Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer.

Clin Biochem 2015 Sep 11;48(13-14):908-10. Epub 2015 Apr 11.

Department of Molecular Medicine, University La Sapienza, V.le R. Elena, 291, 00161 Rome, Italy. Electronic address:

Objectives: Treatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing.

Design And Methods: Formalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method.

Results: The proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future.

Conclusion: The IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.clinbiochem.2015.04.003DOI Listing
September 2015

Novel and recurrent BRCA2 mutations in Italian breast/ovarian cancer families widen the ovarian cancer cluster region boundaries to exons 13 and 14.

Breast Cancer Res Treat 2014 Dec 14;148(3):629-35. Epub 2014 Nov 14.

Department of Experimental Medicine, University La Sapienza, v. le R. Elena 324, 00161, Rome, Italy.

Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10549-014-3196-zDOI Listing
December 2014

Vemurafenib and panitumumab combination tailored therapy in BRAF-mutated metastatic colorectal cancer: a case report.

Cancer Biol Ther 2014 Jul 22;15(7):826-31. Epub 2014 Apr 22.

Neuromed IRCCS; Pozzilli, Italy; Department of Molecular Medicine; Center for Life Nanoscience@Sapienza; Italian Institute of Technology and Istituto Pasteur; Fondazione Cenci-Bolognetti; Sapienza University of Rome; Rome, Italy.

As the knowledge on cancer genetic alterations progresses, it fosters the need for more personalized therapeutic intervention in modern cancer management. Recently, mutations in KRAS, BRAF, and PIK3CA genes have emerged as important mechanisms of resistance to EGFR-targeted therapy in metastatic colorectal cancer (mCRC). Here we report the first case of a mCRC patient whose disease had progressed on standard lines of treatment and for which we devised a personalized therapeutic approach consisting of vemurafenib (Zelboraf) and panitumumab (Vectibix), based on the following molecular profile: BRAF(V600E)-mutant, amplified EGFR (double positive) and WT KRAS, WT PIK3CA, not-amplified HER2 (triple negative). This new combination therapy was well tolerated and resulted in a strong control of the disease. In particular, the vemurafenib-panitumumab combination appears to limit the typical toxicity of single agents, since no cutaneous toxic effects typically associated with vemurafenib were observed. Here we report the first clinical evidence that the combination of an anti-EGFR (panitumumab) and an inhibitor of BRAF(V600E) (vemurafenib) is well tolerated and results in a strong disease control in an extensively pretreated mCRC patient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cbt.28878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100983PMC
July 2014