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Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor (PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22–8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. One open question is whether genetics could influence the severity of symptoms. Considering the limited data on cancer patients, we analyzed public data repositories limited to investigate angiotensin-converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) expressions and genetic variants to identify the basis of individual susceptibility to SARS-CoV-2. Gene expression and variant data were retrieved from Tissue Cancer Genome Atlas, Genotype-Tissue Expression, and gnomAD. Differences in gene expression were tested with Mann-Whitney U-test. Allele frequencies of germline variants were explored in different ethnicities, with a special focus on ACE2 variants located in the binding site to SARS-CoV-2 spike protein. The analysis of ACE2 and TMPRSS2 expressions in healthy tissues showed a higher expression in the age class 20 to 59 years (false discovery rate [FDR] < 0.0001) regardless of gender. ACE2 and TMPRSS2 were more expressed in tumors from males than females (both FDR < 0.0001) and, opposite to the regulation in tissues from healthy individuals, more expressed in elderly patients (FDR = 0.005; FDR < 0.0001, respectively). ACE2 and TMPRSS2 expressions were higher in cancers of elderly patients compared with healthy individuals (FDR < 0.0001). Variants were present at low frequency (range 0% to 3%) and among those with the highest frequency, the variant S19P belongs to the SARS-CoV-2 spike protein binding site and it was exclusively present in Africans with a frequency of 0.2%. The mechanisms of ACE2 and TMPRSS2 regulation could be targeted for preventive and therapeutic purposes in the whole population and especially in cancer patients. Further studies are needed to show a direct correlation of ACE2 and TMPRSS2 expressions in cancer patients and the incidence of COVID-19.

Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF , encoding for the cell cycle inhibitors p16 INK4a and p14 ARF , deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM.

// Gianluca Tedaldi 1 , Michela Tebaldi 1 , Valentina Zampiga 1 , Rita Danesi 2 , Valentina Arcangeli 3 , Mila Ravegnani 2 , Ilaria Cangini 1 , Francesca Pirini 1 , Elisabetta Petracci 4 , Andrea Rocca 5 , Fabio Falcini 2 , Dino Amadori 5 and Daniele Calistri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Romagna Cancer Registry, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Department of Medical Oncology, Ospedale Infermi, Rimini, Italy 4 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 5 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy Correspondence to: Daniele Calistri, email: daniele.calistri@irst.emr.it Keywords: hereditary breast and ovarian cancer, multiple-gene panel, next-generation sequencing, bilateral breast cancer, cancer predisposition Received: October 19, 2016 Accepted: March 14, 2017 Published: April 03, 2017 ABSTRACT As new genes predisposing to breast (BC) and ovarian cancer (OC) are constantly emerging, the use of panels of genes analyzed by Next-Generation Sequencing (NGS) is increasing in clinical diagnostics. The identification of a large number of new germline mutations allows for deeper knowledge of cancer predisposition, although raising many questions about patient management. BC and OC patients recruited by our counseling service between 2012-2015 were included in this study. DNA was extracted from peripheral blood and a panel of 94 genes involved in hereditary tumors was analyzed by NGS. Patient clinical features of BC and OC and cancer family history were collected and compared to the patient genetic profile. A total of 255 women were analyzed, 57 of whom had a pathogenic mutation in BRCA1/2 genes, and 17 carried pathogenic mutations in other genes, such as PALB2 , ATM , BRIP1 , RAD51D , MSH6 , PPM1D , RECQL4 , ERCC3 , TSC2 , SLX4 and other Fanconi anemia genes. Patients with a pathogenic mutation in genes other than BRCA1 and BRCA2 showed no significant difference from the BRCA1/2 -mutated carriers with respect to age at diagnosis and clinical features, suggesting that mutations in other genes could pose a high risk of cancer development. These patients had a much higher percentage of bilateral breast cancer (BBC) and a lower rate of OC than BRCA -mutated patients and patients with no pathogenic mutations: as a consequence, the surveillance protocol should be customized to the patient genetic characteristics.

The main gene involved in gastric cancer (GC) predisposition is CDH1, the pathogenic variants of which are associated with diffuse-type gastric cancer (DGC) and lobular breast cancer (LBC). CDH1 only explains a fraction (10&ndash, 50%) of patients suspected of DGC/LBC genetic predisposition. To identify novel susceptibility genes, thus improving the management of families at risk, we performed a multigene panel testing on selected patients. We searched for germline pathogenic variants in 94 cancer-related genes in 96 GC or LBC Italian patients with early-onset and/or family history of GC. We found CDH1 pathogenic variants in 10.4% of patients. In 11.5% of cases, we identified loss-of-function variants in BRCA1, BRCA2, PALB2, and ATM breast/ovarian cancer susceptibility genes, as well as in MSH2, PMS2, BMPR1A, PRF1, and BLM genes. In 78.1% of patients, we did not find any variants with clear-cut clinical significance, however, 37.3% of these cases harbored rare missense variants predicted to be damaging by bioinformatics tools. Multigene panel testing decreased the number of patients that would have otherwise remained genetically unexplained. Besides CDH1, our results demonstrated that GC pathogenic variants are distributed across a number of susceptibility genes and reinforced the emerging link between gastric and breast cancer predisposition.

Discrimination of multifocal primary lung cancers from lung metastases is crucial to allow for an appropriate clinical management. We report here a case of multifocal lung adenocarcinomas with different morphological and molecular patterns. Radical surgery of one lung nodule was performed at the time of diagnosis, and subsequently on two other lung nodules. At the time of distant relapse, biopsy was repeated for molecular characterization. The patient was treated with EGFR tyrosine kinase inhibitor according to the detection of EGFR exon 21 mutation in metastatic sample and in one of the three lung tumors, characterized by lower mutated allele frequency. The progression free survival was three months according to radiological criteria and the treatment was provided for six months, until clinical progression. Following the assessment of EGFR mutations by pyrosequencing, tumor samples were analyzed by a 30-gene next generation sequencing (NGS) panel, allowing to study intra- and inter-tumor heterogeneity and to confirm the three lung tumors as independent. Different molecular profiles of synchronous tumors and identical EGFR, PIK3CA and TP53 mutations in one of three primary lung tumors and the metachronous metastasis were identified. In conclusion, morphological and molecular characterization of multiple lung nodules by NGS may help to define synchronous and metachronous adenocarcinomas, thus affecting surgical indication and systemic treatment. Intratumor heterogeneity may be associated with differential sensitivity to targeted treatment.

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

Male breast cancer (MBC) is a rare tumor, accounting for less than 1% of all breast cancers. In MBC, genetic predisposition plays an important role, however, only a few studies have investigated in depth the role of genes other than BRCA1 and BRCA2. We performed a Next-Generation Sequencing (NGS) analysis with a panel of 94 cancer predisposition genes on germline DNA from an Italian case series of 70 patients with MBC. Moreover, we searched for large deletions/duplications of BRCA1/2 genes through the Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Through the combination of NGS and MLPA, we identified three pathogenic variants in the BRCA1 gene and six in the BRCA2 gene. Besides these alterations, we found six additional pathogenic/likely-pathogenic variants in PALB2, CHEK2, ATM, RAD51C, BAP1 and EGFR genes. From our study, BRCA1 and BRCA2 emerge as the main genes associated with MBC risk, but also other genes seem to be associated with the disease. Indeed, some of these genes have already been implicated in female breast cancer predisposition, but others are known to be involved in other types of cancer. Consequently, our results suggest that novel genes could be involved in MBC susceptibility, shedding new light on their role in cancer development.

Cell-free DNA (cfDNA) is acquiring increasingly importance in oncologic clinical practice, mostly due to its role in predicting the onset of therapy resistance by following the mutation status changes of patients. In this field, high-sensitivity methods like next-generation sequencing (NGS) could help to accurately detect somatic mutations at low frequency. Here, we report some advantages and limitations of NGS approaches for cfDNA mutation analyses with the aim of choosing the most suitable in terms of sensitivity, specificity, data output, costs, and time work.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder occurring at a young age that predisposes individuals to multiple forms of cancer and to a heterogeneous spectrum of malignancies. We describe the clinical history of a patient who had 5 primary malignant cancers and a familiar history consistent with LFS. We analyzed the genomic DNA of the proband and her relatives by next-generation sequencing (NGS) technology using an enrichment protocol for the simultaneous sequencing of 94 genes involved in hereditary cancers. Genetic analysis of the proband revealed a TP53 germline mutation in exon 5 determining a nucleotide alteration at codon 175 (R175H), a hot spot mutation site related to LFS and a reported pathogenic mutation. The proband daughter's and brother's DNA did not carry the TP53 mutation but they had some rare variants in common with the proband, in addition to other variants with a still unclear role. In conclusion, we identified a TP53 mutation in a patient with multiple primary tumors and a family history characterized by a severe susceptibility to cancer. The genetic analysis by targeted NGS led to the identification of the genetic background and to the exclusion of a cancer risk for the family members. Targeted NGS represents an efficient approach for the identification of mutations in families with a heterogeneous phenotype.