Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation.

Cancer clonal evolution is based on accrual of driving genetic alterations that are expected to cooperate and progressively increase malignancy. Little is known on whether any genetic alteration can hinder the oncogenic function of a coexisting alteration, so that therapeutic targeting of the one can, paradoxically, revive the function of the other. We report the case of a driver oncogene (MET) that is not only bypassed, but also disabled by the mutation of a downstream transducer (BRAF), and reignited by inhibition of the latter. In a metastasis originated from a cancer of unknown primary (CUP), the MET oncogene was amplified eightfold, but unexpectedly, the kinase was dephosphorylated and inactive. As result, specific drugs targeting MET (JNJ-38877605) failed to inhibit growth of xenografts derived from the patient. In addition to MET amplification, the patient harbored, as sole proliferative driver, a mutation hyperactivating BRAF (G469A). Surprisingly, specific blockade of the BRAF pathway was equally ineffective, and it was accompanied by rephosphorylation of the amplified MET oncoprotein and by revived addiction to MET. Mechanistically, MET inactivation in the context of the BRAF-activating mutation is driven through a negative feedback loop involving inactivation of PP2A phosphatase, which in turn leads to phosphorylation on MET inhibitory Ser985. Disruption of this feedback loop allows PP2A reactivation, removing the inhibitory phosphorylation from Ser985 and thereby unleashing MET kinase activity. Evidence is provided for a mechanism of therapeutic resistance to single-oncoprotein targeting, based on reactivation of a genetic alteration functionally dormant in targeted cancer cells.

MET Exon 14 Skipping: A Case Study for the Detection of Genetic Variants in Cancer Driver Genes by Deep Learning.

BACKGROUND: Disruption of alternative splicing (AS) is frequently observed in cancer and might represent an important signature for tumor progression and therapy. Exon skipping (ES) represents one of the most frequent AS events, and in non-small cell lung cancer (NSCLC) MET exon 14 skipping was shown to be targetable. METHODS: We constructed neural networks (NN/CNN) specifically designed to detect MET exon 14 skipping events using RNAseq data. Furthermore, for discovery purposes we also developed a sparsely connected autoencoder to identify uncharacterized MET isoforms. RESULTS: The neural networks had a Met exon 14 skipping detection rate greater than 94% when tested on a manually curated set of 690 TCGA bronchus and lung samples. When globally applied to 2605 TCGA samples, we observed that the majority of false positives was characterized by a blurry coverage of exon 14, but interestingly they share a common coverage peak in the second intron and we speculate that this event could be the transcription signature of a LINE1 (Long Interspersed Nuclear Element 1)-MET (Mesenchymal Epithelial Transition receptor tyrosine kinase) fusion. CONCLUSIONS: Taken together, our results indicate that neural networks can be an effective tool to provide a quick classification of pathological transcription events, and sparsely connected autoencoders could represent the basis for the development of an effective discovery tool.

Whole exome sequencing identifies a germline MET mutation in two siblings with hereditary wild-type RET medullary thyroid cancer.

Whole exome sequencing (WES) was used to investigate two Italian siblings with wild-type RET genotype, who developed medullary thyroid cancers (MTCs) and, later, primary prostate and breast cancers, respectively. The proband's MTC harbored a p.Met918Thr RET mutation; his sister's MTC was RET/RAS wild-type. Both siblings had a germline mutation (p.Arg417Gln) in the extracellular Sema domain of the proto-oncogene MET. Experiments involving ectopic expression of MET p.Arg417Gln in MET-negative T47D breast cancer cells documented the mutant receptor's functionality and its ability to enhance cell migration and invasion. Our findings highlight a possible link between MET germline mutations and MTCs and suggest that MET p. Arg417Gln may promote an invasive malignant phenotype. The possibility that MTC can be driven/co-driven by a MET mutation has potential management implications, since the tyrosine-kinase inhibitor cabozantinib-approved for treating advanced MTCs-is a specific MET inhibitor.

Erratum to: Ockham's razor for the MET-driven invasive growth linking idiopathic pulmonary fibrosis and cancer.

In the original version of this article [1], published on 2 September 2016, the name of author 'Alice Balderacchi' was wrongly displayed. In this Erratum the incorrect name and correct name are shown. The original publication of this article has been corrected.

Ockham's razor for the MET-driven invasive growth linking idiopathic pulmonary fibrosis and cancer.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) identifies a specific lung disorder characterized by chronic, progressive fibrosing interstitial pneumonia of unknown etiology, which lacks effective treatment. According to the current pathogenic perspective, the aberrant proliferative events in IPF resemble those occurring during malignant transformation. MAIN BODY: Receptor tyrosine kinases (RTK) are known to be key players in cancer onset and progression. It has been demonstrated that RTK expression is sometimes also altered and even druggable in IPF. One example of an RTK-the MET proto-oncogene-is a key regulator of invasive growth. This physiological genetic program supports embryonic development and post-natal organ regeneration, as well as cooperating in the evolution of cancer metastasis when aberrantly activated. Growing evidence sustains that MET activation may collaborate in maintaining tissue plasticity and the regenerative potential that characterizes IPF. CONCLUSION: The present work aims to elucidate-by applying the logic of simplicity-the bio-molecular mechanisms involved in MET activation in IPF. This clarification is crucial to accurately design MET blockade strategies within a fully personalized approach to IPF.

The ROR1 pseudokinase diversifies signaling outputs in MET-addicted cancer cells.

MET is a master gene controlling a genetic program driving proliferation, apoptosis protection and invasion. The ROR1 pseudokinase acts as a MET substrate. However, its contribution to MET signaling and MET-dependent biological outcomes remains to be elucidated. By structure-function analysis of ROR1 mutants, we show that ROR1 encompasses two major substrate regions: one is located in the proline-rich domain and is directly phosphorylated by MET; the other resides in the pseudokinase domain and is phosphorylated through intermediate activation of SRC. Differential phosphorylation of these two regions dictates the execution of specific responses: phosphorylation of the ROR1 proline-rich domain by MET-but not phosphorylation of the pseudokinase domain by SRC-is necessary and sufficient to control MET-driven proliferation and protection from apoptosis. Differently, both the proline-rich and the pseudokinase domains mediate cell invasion. Consistent with the role of ROR1 in specifying the functional consequences of MET-dependent signals, ROR1 silencing leads to selective attenuation of only some of the signal transduction pathways sustained by MET. These data enlighten the so far elusive function(s) of pseudokinases and identify a mechanism of biological diversification, based on substrate specificity of oncogenic kinases.

Real-world histopathological approach to malignancy of undefined primary origin (MUO) to diagnose cancers of unknown primary (CUPs).

The aim of this study is to envisage a streamlined pathological workup to rule out CUPs in patients presenting with MUOs. Sixty-four MUOs were classified using standard histopathology. Clinical data, immunocytochemical markers, and results of molecular analysis were recorded. MUOs were histologically subdivided in clear-cut carcinomas (40 adenocarcinomas, 11 squamous, and 3 neuroendocrine carcinomas) and unclear-carcinoma features (5 undifferentiated and 5 sarcomatoid tumors). Cytohistology of 7/40 adenocarcinomas suggested an early metastatic cancer per se. In 33/40 adenocarcinomas, CK7/CK20 expression pattern, gender, and metastasis sites influenced tissue-specific marker selection. In 23/40 adenocarcinomas, a "putative-immunophenotype" of tissue of origin addressed clinical-diagnostic examinations, identifying 9 early metastatic cancers. Cell lineage markers were used to confirm squamous and neuroendocrine differentiation. Pan-cytokeratins were used to confirm the epithelial nature of poorly differentiated tumors, followed by tissue and cell lineage markers, which identified one melanoma. In total, 47/64 MUOs (73.4%) were confirmed CUP. Molecular analysis, feasible in 37/47 CUPs (78.7%), had no diagnostic impact. Twenty CUP patients, mainly with squamous carcinomas and adenocarcinomas with putative-gynecologic-immunophenotypes, presented with only lymph node metastases and had longer median time to progression and overall survival (< 0.001), compared with patients with other metastatic patterns. We propose a simplified histology-driven workup which could efficiently rule out CUPs and identify early metastatic cancer.

MET mutations in cancers of unknown primary origin (CUPs).

Cancer of unknown primary origin (CUP) defines metastatic disease of unknown origin, accounting for 3-5% of all cancers. Growing evidence demonstrates that inappropriate execution of a genetic program named "invasive growth," driven by the MET oncogene, is implicated in the metastatic process. MET activation in cancers is mainly consequent to overexpression, whereas mutations are rarely found. We reasoned that the occurrence of MET somatic mutations might sustain premature occult dissemination of cancer cells, such as that observed in CUPs. We sequenced MET in genomic DNA obtained from 47 early metastatic cancers. By extensive immunohistochemical analysis a primary site was afterward postulated in 24 patients, whereas 23 cases remained of unknown primary (CUPs). MET somatic mutations were found in seven cases, all belonging to the CUP cohort. Mutational incidence (30%) was thus significantly higher than the expected one (4%), in the absence of high mutational background. Several nucleotide changes were novel and clustered either in the kinase domain or in the extracellular semaphorin domain. Mutated receptors were functional and sustained the transformed phenotype, suggesting that MET activating mutations are genetic markers associated with the CUP syndrome.

Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition.

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

Malignant Pleural Mesothelioma: Genetic and Microenviromental Heterogeneity as an Unexpected Reading Frame and Therapeutic Challenge.

Mesothelioma is a malignancy of serosal membranes including the peritoneum, pleura, pericardium and the tunica vaginalis of the testes. Malignant mesothelioma (MM) is a rare disease with a global incidence in countries like Italy of about 1.15 per 100,000 inhabitants. Malignant Pleural Mesothelioma (MPM) is the most common form of mesothelioma, accounting for approximately 80% of disease. Although rare in the global population, mesothelioma is linked to industrial pollutants and mineral fiber exposure, with approximately 80% of cases linked to asbestos. Due to the persistent asbestos exposure in many countries, a worldwide progressive increase in MPM incidence is expected for the current and coming years. The tumor grows in a loco-regional pattern, spreading from the parietal to the visceral pleura and invading the surrounding structures that induce the clinical picture of pleural effusion, pain and dyspnea. Distant spreading and metastasis are rarely observed, and most patients die from the burden of the primary tumor. Currently, there are no effective treatments for MPM, and the prognosis is invariably poor. Some studies average the prognosis to be roughly one-year after diagnosis. The uniquely poor mutational landscape which characterizes MPM appears to derive from a selective pressure operated by the environment; thus, inflammation and immune response emerge as key players in driving MPM progression and represent promising therapeutic targets. Here we recapitulate current knowledge on MPM with focus on the emerging network between genetic asset and inflammatory microenvironment which characterize the disease as amenable target for novel therapeutic approaches.

From Interconnection between Genes and Microenvironment to Novel Immunotherapeutic Approaches in Upper Gastro-Intestinal Cancers-A Multidisciplinary Perspective.

Despite the progress during the last decade, patients with advanced gastric and esophageal cancers still have poor prognosis. Finding optimal therapeutic strategies represents an unmet need in this field. Several prognostic and predictive factors have been evaluated and may guide clinicians in choosing a tailored treatment. Data from large studies investigating the role of immunotherapy in gastrointestinal cancers are promising but further investigations are necessary to better select those patients who can mostly benefit from these novel therapies. This review will focus on the treatment of metastatic esophageal and gastric cancer. We will review the standard of care and the role of novel therapies such as immunotherapies and CAR-T. Moreover, we will focus on the analysis of potential predictive biomarkers such as Modify as: Microsatellite Instability (MSI) and PD-L1, which may lead to treatment personalization and improved treatment outcomes. A multidisciplinary point of view is mandatory to generate an integrated approach to properly exploit these novel antiproliferative agents.

Cancer of Unknown Primary (CUP): genetic evidence for a novel nosological entity? A case report.

Cancer of unknown primary (CUP) is an obscure disease characterized by multiple metastases in the absence of a primary tumor. No consensus has been reached whether CUPs are simply generated from cancers that cannot be detected or whether they are the manifestation of a still unknown nosological entity. Here, we report the complete expression and genetic analysis of multiple synchronous metastases harvested at warm autopsy of a patient with CUP. The expression profiles were remarkably similar and astonishingly singular. The whole exome analysis yielded a high number of mutations present in all metastases (fully shared), additional mutations (partially shared) accumulated one after another in a series, and few private mutations were unique to each metastasis. Surprisingly, the phylogenetic trajectory linking CUP metastases was atypical, depicting a common "stream", sprouting a series of linear "brooks", at variance from the extensive branched evolution observed in metastases from most cancers of known origin. The distinctive genetic and evolutionary features depicted suggest that CUP is a novel nosological entity.

Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies.

Monoclonal antibodies (mAbs) against the extracellular domain of the epidermal growth factor receptor (EGFR) have been introduced for the treatment of metastatic colorectal cancer (mCRC). We have reported recently that increased copy number of the EGFR can predict response to anti-EGFR mAbs and that patients might be selected for treatment based on EGFR copy number. Here, we show that mutations activating the RAS/RAF signaling pathway are also predictive and prognostic indicators in mCRC patients, being inversely correlated with response to anti-EGFR mAbs. In cellular models of CRCs, activation of the RAS signaling pathway by introduction of an activated K-RAS allele (Gly(12)Val) impairs the therapeutic effect of anti-EGFR mAbs. In cancer cells carrying constitutively active RAS, the pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) signaling cascade improves anti-EGFR treatment based on mAbs. These results have implications for the identification of patients who are likely to respond to anti-EGFR treatment. They also provide the rationale for combination therapies, targeted simultaneously to the EGFR and RAS/RAF/MAPK signaling pathways in CRC patients.

Lung-Seeking Metastases.

Metastases from different cancer types most often affect the lung parenchyma. Moreover, the lungs are among the most frequent sites of growth of metastatic masses of uncertain/unknown lineage of origin. Thus, with regards to pulmonary neoplastic parenchymal nodules, the critical issue is to determine if they are IN the lung or OF the lung. In this review, we highlight the clinical, instrumental and molecular features which characterize lung metastases, mainly focusing on recently advancing and emerging concepts regarding the metastatic niche, inflammation, angiogenesis, immune modulation and gene expression. A novel issue is related to the analysis of biomechanical forces which cooperate in the expansion of tumor masses in the lungs. We here aim to analyze the biological, genetic and pathological features of metastatic lesions to the lungs, here referred to as site of metastatic growth. This point should be a crucial part of the algorithm for a proper diagnostic and therapeutic approach in the era of personalized medicine.

Erratum: Stella, G.M. et al. Brain Metastases from Lung Cancer: Is MET an Actionable Target? Cancers 2019, 11, 271.

The authors wish to make the following corrections to this paper [...].

Brain Metastases from Lung Cancer: Is MET an Actionable Target?

The process of metastatic dissemination begins when malignant cells start to migrate and leave the primary mass. It is now known that neoplastic progression is associated with a combination of genetic and epigenetic events. Cancer is a genetic disease and this pathogenic concept is the basis for a new classification of tumours, based precisely on the presence of definite genetic lesions to which the clones are addicted. Regarding the scatter factor receptors MET and Recepteur d'Origin Nantais (RON), it is recognised that MET is an oncogene necessary for a narrow subset of tumours (MET-addicted) while it works as an adjuvant metastogene for many others. This notion highlights that the anti-MET therapy can be effective as the first line of intervention in only a few MET-addicted cases, while it is certainly more relevant to block MET in cases of advanced neoplasia that exploit the activation of the invasive growth program to promote dissemination in other body parts. Few data are instead related to the role played by RON, a receptor homologous to MET. We have already demonstrated an implication of MET and RON genes in brain metastases from lung cancer. On this basis, the aim of this work is to recapitulate and dissect the molecular basis of metastatic brain dissemination from lung cancer. The latter is among the big killers and frequently gives rise to brain metastases, most often discovered at diagnosis. Molecular mechanisms leading to tumour spread to the brain are mostly unknown and in turn these tragic cases are still lacking effective therapies. Based on previously published data from our group, we aim to summarise and analyse the pathogenic mechanisms leading to activation of the scatter factor receptor in brain metastatic lesions of lung primaries, from the point of view of replacing the currently used empirical treatment with a more targeted approach.

Pemetrexed-loaded nanoparticles targeted to malignant pleural mesothelioma cells: an in vitro study.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth. METHODS: MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry). RESULTS: GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization. CONCLUSION: GNP-HCPe treatment displays in vitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy.

PIK3CA cancer mutations display gender and tissue specificity patterns.

The occurrence of oncogenic alleles can display striking tissue specificity. For example KRAS mutations are very frequent in pancreatic cancers but relatively rare in melanomas. The opposite is true for BRAF mutations. Somatic mutations in the gene encoding for the phosphatidylinositol 3-kinase (PI3KCA) catalytic subunit, PIK3CA, occur at high frequency in many solid cancers. We have examined whether PI3K oncogenic mutations (exons 9 and 20) might exhibit gender and/or tissue specificity. By examining large cohorts of breast and colorectal cancers affecting both men and women we found that the pattern of PIK3CA mutations is distinctive. In colorectal cancers, PIK3CA (but not KRAS, APC, or TP53) mutations display a gender bias occurring at higher frequencies in women. We also found that male breast cancers display PIK3CA mutations at an overall frequency similar to that observed in female breast tumors. In male breast cancers, however, PIK3CA mutations are found mainly in exon 20. We conclude that PI3KCA mutations affecting exons 9 and 20 display gender- and tissue-specific patterns, thus suggesting that the different amino acid changes could exert distinct functional effects on the oncogenic properties of this enzyme. Furthermore, we propose that sexual dimorphisms and tissue specific factors might directly or indirectly influence the occurrence of PI3KCA cancer alleles.

RON tyrosine kinase mutations in brain metastases from lung cancer.

RON mutations might identify actionable targets in highly aggressive lung tumours http://ow.ly/RTUp30hSBX6.