Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Therapeutic Potential of Afatinib in NRG1 Fusion-Driven Solid Tumors: A Case Series.

BACKGROUND: Neuregulin 1 (NRG1) fusions, which activate ErbB signaling, are rare oncogenic drivers in multiple tumor types. Afatinib is a pan-ErbB family inhibitor that may be an effective treatment for NRG1 fusion-driven tumors. PATIENTS AND METHODS: This report summarizes pertinent details, including best tumor response to treatment, for six patients with metastatic NRG1 fusion-positive tumors treated with afatinib. RESULTS: The six cases include four female and two male patients who ranged in age from 34 to 69 years. Five of the cases are patients with lung cancer, including two patients with invasive mucinous adenocarcinoma and three patients with nonmucinous adenocarcinoma. The sixth case is a patient with colorectal cancer. NRG1 fusion partners for the patients with lung cancer were either CD74 or SDC4. The patient with colorectal cancer harbored a novel POMK-NRG1 fusion and a KRAS mutation. Two patients received afatinib as first- or second-line therapy, three patients received the drug as third- to fifth-line therapy, and one patient received afatinib as fifteenth-line therapy. Best response with afatinib was stable disease in two patients (duration up to 16 months when combined with local therapies) and partial response (PR) of >18 months in three patients, including one with ongoing PR after 27 months. The remaining patient had a PR of 5 months with afatinib 40 mg/day, then another 6 months after an increase to 50 mg/day. CONCLUSION: This report reviews previously published metastatic NRG1 fusion-positive tumors treated with afatinib and summarizes six previously unpublished cases. The latter include several with a prolonged response to treatment (>18 months), as well as the first report of efficacy in NRG1 fusion-positive colorectal cancer. This adds to the growing body of evidence suggesting that afatinib can be effective in patients with NRG1 fusion-positive tumors. KEY POINTS: NRG1 fusions activate ErbB signaling and have been identified as oncogenic drivers in multiple solid tumor types. Afatinib is a pan-ErbB family inhibitor authorized for the treatment of advanced non-small cell lung cancer that may be effective in NRG1 fusion-driven tumors. This report summarizes six previously unpublished cases of NRG1 fusion-driven cancers treated with afatinib, including five with metastatic lung cancer and one with metastatic colorectal cancer. Several patients showed a prolonged response of >18 months with afatinib treatment. This case series adds to the evidence suggesting a potential role for afatinib in this area of unmet medical need.

Human bronchial carcinoid tumor initiating cells are targeted by the combination of acetazolamide and sulforaphane.

BACKGROUND: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior. METHODS: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids. RESULTS: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice. CONCLUSIONS: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.

Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

BACKGROUND: The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. KEAP1 silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer. METHODS: We performed a detailed methylation density map of 13 CpGs located into the KEAP1 promoter region by analyzing a set of 25 cell lines from different histologies of lung cancer. The methylation status was assessed using quantitative methylation specific PCR (QMSP) and pyrosequencing, and the performance of the two assays was compared. RESULTS: Hypermethylation at the promoter region of the KEAP1 was detected in one third of cell lines and its effect on the modulation KEAP1 mRNA levels was also confirmed by in vitro 5-Azacytidine treatment on lung carcinoid, small lung cancer and adenocarcinoma cell lines. QMSP and pyrosequencing showed a high rate of concordant results, even if pyrosequencing revealed two different promoter CpGs sub-islands (P1a and P1b) with a different methylation density pattern. CONCLUSIONS: Our results confirm the effect of methylation on KEAP1 transcription control across multiple histologies of lung cancer and suggest pyrosequencing as the best approach to investigate the pattern of CpGs methylation in the promoter region of KEAP1. The validation of this approach on lung cancer patient cohorts is mandatory to clarify the prognostic value of the epigenetic deregulation of KEAP1 in lung tumors.

Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors.

BACKGROUND: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung. METHODS: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment. RESULTS: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids. CONCLUSIONS: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

FOXP1 and TP63 involvement in the progression of myelodysplastic syndrome with 5q- and additional cytogenetic abnormalities.

BACKGROUND: The progression of low-risk del(5q) myelodysplastic syndrome to acute myeloid leukemia is increased when associated with mutations of TP53, or with additional chromosomal abnormalities. However, to date the prognostic impact and molecular consequences of these rearrangements were poorly investigated. Single additional alterations to del(5q) by balanced chromosome rearrangements were rarely found in myelodysplasia. In particular, balanced alterations involving TP63 and FOXP1 genes were never reported in the literature. CASE PRESENTATION: Here we report on a 79-year woman with an aggressive form of myelodysplastic syndrome with del(5q), no TP53 mutation, and a novel complex rearrangement of chromosome 3 in bone marrow cells. Our results revealed that the FOXP1 and TP63 genes were both relocated along chromosome 3. Strikingly, immunohistochemistry analysis showed altered protein levels, disclosing that this rearrangement triggered the expression of FOXP1 and TP63 genes. FOXP1 was also found activated in other patients with myelodysplasia and acute myeloid leukemia, showing that it is an important, recurrent event. CONCLUSIONS: We document an apparent role of FOXP1 and TP63, up to now poorly documented, in the progression of MDS in our patient who is lacking mutations in the TP53 tumor suppressor gene normally associated with poor outcome in myelodysplastic syndrome with 5q-. Finally, our results may suggest a possible broader role of FOXP1 in the pathogenesis and progression of myelodysplasia and acute myeloid leukemia.

B7-H3/CD276 and small-cell lung cancer: What's new?

Immunotherapy revolutionized the treatment landscape of several cancers, including small-cell lung cancer (SCLC), with a huge number of practice-changing trials, and becoming a new frontier for their management. The addition of an anti-PD-L1, atezolizumab or durvalumab, to platinum/etoposide regimen became the standard of care for first-line therapy of extensive-stage (ES)-SCLC with the 12 months median survival exceeded for the first time. Nevertheless, most patients show primary or acquired resistance to anti-PD-L1 therefore new promising therapeutic immune-targets are under clinical investigation in several solid tumors. Among these, B7-H3, also known as CD276, is a member of the B7 family overexpressed in tumor tissues, including SCLC, while showing limited expression in normal tissues becoming an attractive and promising target for cancer immunotherapy. B7-H3 plays a dual role in the immune system during the T-cell activation, acting as a T-cell costimulatory/coinhibitory immunoregulatory protein, and promoting pro-tumorigenic functions such as tumor migration, invasion, metastases, resistance, and metabolism. Immunohistochemistry, flow cytometry, and immunofluorescence were the most used methods to assess B7-H3 expression levels and validate a possible relationship between B7-H3 staining patterns and clinicopathological features in lung cancer patients. To date, there are no clinically available therapeutics/drugs targeting B7-H3 in any solid tumors. The most promising preliminary clinical results have been reported by DS7300a and HS-20093, both are antibody-drug conjugates, that are under investigation in ongoing trials for the treatment of pretreated SCLC. This review will provide an overview of B7-H3 and corresponding inhibitors and the clinical development in the management of SCLC.

Clinical impact of mixed pulmonary carcinoma and carcinoid: the driver from their mono-clonal origin.

The combination of neuroendocrine/non neuroendocrine lung tumors (CNNELT) mentioned in the last edition of the World Health Organization (WHO) of Thoracic Tumors refers to small cell carcinoma (SCLC) or large cell neuroendocrine carcinoma (LCNEC) mixed with any other non-small cell lung carcinoma (NSCLC). Typical Carcinoid (TC)/Atypical Carcinoid (AC) combined with NSCLC is not included among this category. However, case reports of TC/AC combined with NSCLC have been described. We previously reported 2 cases of lung adenocarcinoma (LUA) mixed with carcinoid sharing mutations in both components supporting the hypothesis of a clonal origin. We extended our analysis to other four cases of mixed NSCLC-carcinoid by performing targeted-DNA and RNA-based NGS analysis in both primary and their paired lymph nodes metastasis. In all cases, LUA and AC components shared at least 1 common mutation (KRAS driver mutation p.Gly12Val in cases 1 and 3, AKAP13-RET fusion in case 2, and missense KRAS driver mutation p.Gly12Ala in case 4, reinforcing the hypothesis of a clonal origin. Moreover, the same mutation was detected in the metastasis constituted only by AC (cases 2 and 4). Although it is a rare malignancy in the lung, mixed LUA and TC/AC could be included among the histotypes for which a deep molecular characterization of both components is needed to identify the presence of potential druggable genetic alterations.

Clinical and Molecular Traits of a Novel SPECC1L-ALK Fusion in a Patient with Advanced Non-Small Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) fusions account for 5-7% of non-small cell lung cancer (NSCLC) patients, the therapeutic approaches for which have significantly evolved in the last few years. However, the response to target therapies remains heterogeneous, partially due to the many different ALK fusion variants reported to date. Rare fusion variants have also been discovered, but their role in influencing responses to ALK inhibitors (ALKis) remains poorly elucidated. Laboratory investigation at both the tissue and protein levels, and a molecular profile by next-generation sequencing (NGS) were performed on a lung biopsy sample from one patient with poorly differentiated adenocarcinoma. An in silico prediction model using ColabFold software v1.5.5 was used to model and predict the entire structure of the chimeric aberrant ALK protein. Here, we report a case of a former smoker, a 60-year-old man, diagnosed with NSCLC and undergoing ALK translocation. He received first-, second- and third-generation ALK protein inhibitors (ALKis), showing a clinical benefit for about 4 years. IHC analysis and the molecular examination of the tissue biopsy indicated a positive staining for ALK and a novel ALK gene fusion variant, involving the sperm antigen with calponin homology and coiled-coil domain 1-like (SPECC1L) gene with an unreported breakpoint in exon 7. The novel SPECCL1::ALK fusion was identified using Anchored Multiplex PCR (AMP)-NGS technology and was predicted to retain the Pkinase_Tyr domain at the carboxy-terminal region of the resulting chimeric protein. To the best of our knowledge, this is the first case of an ALK-positive NSCLC patient carrying the SPECC1L exon 7 fusion breakpoint and one of the few reports about clinical outcomes related to SPECC1L::ALK fusion. The in silico hypothesized biological role of this newly identified fusion variant corroborates the observed clinical response to multiple ALKis. The molecular findings also reinforce the utility of AMP-based NGS technology as a valuable tool for the identification of rare chromosomal events that may be related to the variability of patient outcomes to different ALKis treatments.

Effects of KEAP1 Silencing on NRF2 and NOTCH Pathways in SCLC Cell Lines.

The KEAP1/NRF2 pathway is a master regulator of several redox-sensitive genes implicated in the resistance of tumor cells against therapeutic drugs. The dysfunction of the KEAP1/NRF2 system has been correlated with neoplastic patients' outcomes and responses to conventional therapies. In lung tumors, the growth and the progression of cancer cells may also involve the intersection between the molecular NRF2/KEAP1 axis and other pathways, including NOTCH, with implications for antioxidant protection, survival of cancer cells, and drug resistance to therapies. At present, the data concerning the mechanism of aberrant NRF2/NOTCH crosstalk as well as its genetic and epigenetic basis in SCLC are incomplete. To better clarify this point and elucidate the contribution of NRF2/NOTCH crosstalk deregulation in tumorigenesis of SCLC, we investigated genetic and epigenetic dysfunctions of the KEAP1 gene in a subset of SCLC cell lines. Moreover, we assessed its impact on SCLC cells' response to conventional chemotherapies (etoposide, cisplatin, and their combination) and NOTCH inhibitor treatments using DAPT, a γ-secretase inhibitor (GSI). We demonstrated that the KEAP1/NRF2 axis is epigenetically controlled in SCLC cell lines and that silencing of KEAP1 by siRNA induced the upregulation of NRF2 with a consequent increase in SCLC cells' chemoresistance under cisplatin and etoposide treatment. Moreover, KEAP1 modulation also interfered with NOTCH1, HES1, and DLL3 transcription. Our preliminary data provide new insights about the downstream effects of KEAP1 dysfunction on NRF2 and NOTCH deregulation in this type of tumor and corroborate the hypothesis of a cooperation of these two pathways in the tumorigenesis of SCLC.

Monitoring EGFR-lung cancer evolution: a possible beginning of a "methylation era" in TKI resistance prediction.

The advances in scientific knowledge on biological therapies of the last two decades have impressively oriented the clinical management of non-small-cell lung cancer (NSCLC) patients. The treatment with tyrosine kinase inhibitors (TKIs) in patients harboring Epidermal Growth Factor Receptor (EGFR)-activating mutations is dramatically associated with an improvement in disease control. Anyhow, the prognosis for this selected group of patients remains unfavorable, due to the innate and/or acquired resistance to biological therapies. The methylome analysis of many tumors revealed multiple patterns of methylation at single/multiple cytosine-phosphate-guanine (CpG) sites that are linked to the modulation of several cellular pathways involved in cancer onset and progression. In lung cancer patients, ever increasing evidences also suggest that the association between DNA methylation changes at promoter/intergenic regions and the consequent alteration of gene-expression signatures could be related to the acquisition of resistance to biological therapies. Despite this intriguing hypothesis, large confirmatory studies are demanded to consolidate and finalize many preliminary observations made in this field. In this review, we will summarize the available knowledge about the dynamic role of DNA methylation in EGFR-mutated NSCLC patients.

Robust Performance of the Novel Research-Use-Only Idylla GeneFusion Assay Using a Diverse Set of Pathological Samples with a Proposed 1-Day Workflow for Advanced NSCLC Evaluation.

A range of different techniques are available for predictive biomarker testing for non-small-cell lung cancer (NSCLC) clinical management. International guidelines suggest next-generation sequencing (NGS) as the preferred procedure, but other reverse transcriptase-polymerase chain reaction (RT-PCR)-based methods are rapidly evolving. In this study, we evaluated the reliability and accuracy of the IdyllaTM GeneFusion assay, a rapid and fully automated platform able to simultaneously detect ALK, ROS1, RET and NTRK1/2/3 and MET ex14 skipping mutations and compared its performance with routine reference methods. The cohort included thirty-seven NSCLCs plus two parotid gland carcinomas, previously characterized for the above alterations through either IHC, FISH, RT-PCR or NGS. In 36 of 39 cases, the Idylla GeneFusion assay and the reference methods were concordant (overall agreement: 92.3%). Tumor sections stored at room temperature for up to 60 days and 17 cases older than 2 years were successfully characterized. Our results suggest that the Idylla GeneFusion assay is a reliable tool to define gene fusion status and may be a valuable stand-alone diagnostic test when time efficiency is needed or NGS is not feasible.

Rapid EGFR evaluation from used H&E, IHC and FISH diagnostic slides with the Idylla platform.

AIMS: Diagnostic tumour samples are mandatory for morphologic and molecular diagnosis of non-small cell lung cancer (NSCLC) to establish the best therapeutic approach. In the presence of small tumour tissue sample, the pathologist needs to make responsible choices to achieve a correct diagnosis and save material for subsequent molecular evaluations. Nevertheless, in some instances, the diagnostic process can lead to tissue depletion. The automated Idylla epidermal growth factor receptor (EGFR) mutation test has been developed to rapidly process formalin-fixed paraffin-embedded (FFPE) pathologic material, without previous DNA extraction. This study aimed to test whether this platform is suitable for the reuse of H&E, immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) diagnostic slides. METHODS: A training set of 19 FFPE tissues with known EGFR status was revaluated on H&E slides. Fourteen of them were also tested using IHC and FISH treated specimens. An additional series of 25 H&E, IHC or FISH slides of NSCLC cases tested for EGFR mutation at an external institution was blindly assessed as a validation cohort. RESULTS: Combining the two sets, 32 of 32 classical ex19dels and p.L858R were correctly identified. Three uncommon mutations (p.G719X, p.L861Q and ex20ins) were also detected. Four discrepancies were related to rare ex19del/ins not included in the Idylla list of detectable mutations. Two p.T790M variants were missed on one FFPE and two H&E slides but were detected using IHC and FISH sections from the same FFPE blocks. CONCLUSIONS: The Idylla EGFR mutation test is highly reliable using differently treated tumour specimens and should be validated in larger studies.

Correction: Next-generation multimodality of nutrigenomic cancer therapy: sulforaphane in combination with acetazolamide actively target bronchial carcinoid cancer in disabling the PI3K/Akt/mTOR survival pathway and inducing apoptosis.

[This corrects the article DOI: 10.18632/oncotarget.28011.].

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing.

DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the "methylation_analysis" plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

Detection of ALK fusion variants by RNA-based NGS and clinical outcome correlation in NSCLC patients treated with ALK-TKI sequences.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) fusions identify a limited subset of non-small cell lung cancer (NSCLC) patients, whose therapeutic approach have been radically changed in recent years. However, diagnostic procedures and clinical-radiological responses to specific targeted therapies remain heterogeneous and intrinsically resistant or poor responder patients exist. METHODS: A total of 290 patients with advanced NSCLC defined as ALK+ by immunohistochemistry (IHC) and/or fluorescent in situ hybridisation (FISH) test and treated with single or sequential multiple ALK inhibitors (ALKi) from 2011 to 2017 have been retrospectively retrieved from a multicentre Italian cancer network database. In 55 patients with enough leftover tumour tissue, specimens were analysed with both targeted and customised next generation sequencing panels. Identified fusion variants have been correlated with clinical outcomes. RESULTS: Of the 55 patients, 24 received crizotinib as first-line therapy, 1 received ceritinib, while 30 received chemotherapy. Most of the patients (64%) received ALKi in sequence. An ALK fusion variant was identified in 73% of the cases, being V3 variant (E6A20) the most frequent, followed by V1 (E13A20) and more rare ones (e.g. E6A19). In three specimens, four new EML4-ALK fusion breakpoints have been reported. Neither fusion variants nor brain metastases were significantly associated with overall survival (OS), while it was predictably longer in patients receiving a sequence of ALKi. The presence of V1 variant was associated with progression-free survival (PFS) improvement when crizotinib was used (p = 0.0073), while it did not affect cumulative PFS to multiple ALKi. CONCLUSION: Outcomes to sequential ALKi administration were not influenced by fusion variants. Nevertheless, in V1+ patients a prolonged clinical benefit was observed. Fusion variant identification by NGS technology may add relevant information about rare chromosomal events that could be potentially correlated to worse outcomes.

High RAD51 mRNA expression characterize estrogen receptor-positive/progesteron receptor-negative breast cancer and is associated with patient's outcome.

Mutations in DNA double-strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)-positive/progesteron receptor (PR)-negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER-positive/PR-negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER-positive/PR-negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Identification of a novel RUNX2 gene mutation in an Italian family with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare, well-defined skeletal disorder with autosomal dominant inheritance and complete penetrance. Although it involves the whole skeletal system, the main clinical manifestations of CCD are malformations of the skull and clavicles, which lead to a typical appearance of the face and shoulders. Dental aspects are particularly evident and often eruption difficulties are the first indication for the patient, who does not present any other problem. It has been established that insufficiency of the RUNX-related transcription factor, the core-binding factor alpha 1 (CBFA1) protein, causes CCD. This protein is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. CBFA1 protein is encoded by the RUNX2 gene located on chromosome 6p21. The molecular characterization of the novel RUNX2 gene mutation c.580 + 1G > A in an Italian family (a 27-year-old female, her 54-year-old mother and 24-year-old sister) affected by the typical CCD phenotype, which was proven to alter splicing of the RUNX2 messenger RNA, underscoring the contribution of novel altered splicing mechanism to the aetiology of this disease is presented.