Acquired multiple mutations ALK I1171N, L1196M and G1202R mediate lorlatinib resistance in EML4-ALK-rearranged malignant pleural mesothelioma: a case report.

EML4-ALK rearranged malignant pleural mesothelioma (MPM) is rare and its responses to anaplastic lymphoma kinase (ALK) inhibitors, including alectinib and lorlatinib, remain unexplored. In this case report, we describe a patient with EML4-ALK-rearranged stage IIIB MPM who was administered with alectinib and lorlatinib as first-line and fourth-line therapy, respectively. He had remarkable response evaluated as partial response on both regimens lasting approximately 3.5 months on each regimen. His plasma samples were collected during the treatment course and submitted for targeted sequencing to understand the molecular mechanisms of his therapeutic resistance. Sequencing analysis revealed the emergence of ALK I1171N and L1196M at alectinib progression. Meanwhile, ALK I1171N, L1196M, and G1202R mutations were identified at lorlatinib progression, wherein L1196M is confirmed to be in cis to G1202R. We speculate that these multiple mutations synergistically mediated his resistance to both alectinib and lorlatinib. Our report describes the detection of EML4-ALK rearrangement in a patient with MPM who had remarkable therapeutic response with ALK inhibitors. Moreover, our case also revealed acquired mechanisms of lorlatinib resistance mediated by multiple mutations ALK I1171N, L1196M, and G1202R, contributing an incremental step to our understanding of the complexity of acquired resistance mechanisms in sequential ALK inhibitor therapy. The reviews of this paper are available via the supplemental material section.

MTAP immunohistochemistry is an accurate and reproducible surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant pleural mesothelioma.

Ancillary studies facilitate accurate diagnosis of morphologically challenging mesothelial proliferations. The current diagnostic algorithm proceeds from BAP1 immunohistochemistry to CDKN2A fluorescence in situ hybridization. While MTAP immunohistochemistry has recently shown promise as a surrogate for CDKN2A fluorescence in situ hybridization, it has been examined in only a few single-institution studies. Furthermore, there are no published reports on interobserver agreement or interlaboratory reproducibility for MTAP immunohistochemistry. We performed MTAP immunohistochemistry on 20 benign mesothelial lesions and 99 malignant mesotheliomas from five mesothelioma centers in four countries, and each MTAP stain was independently interpreted by four pathologists. CDKN2A fluorescence in situ hybridization data were available for a subset of cases, and a subset of cases was subjected in MTAP immunohistochemistry in multiple laboratories to assess interlaboratory reproducibility. Interobserver agreement in MTAP immunostain interpretation was excellent for all mesothelial lesions (kappa: 0.85) and for malignant mesothelioma cases only (kappa: 0.82). Interlaboratory reproducibility was also excellent (kappa values for paired protocols: 0.77-0.89). MTAP loss by immunohistochemistry was 78% sensitive and 96% specific for CDKN2A homozygous deletion. MTAP immunohistochemistry is a reliable surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant mesothelioma. Interobserver agreement is excellent for interpretation of MTAP staining, and protocols performed in different laboratories yield concordant MTAP staining results. Rare cases with immunohistochemical MTAP loss may retain normal CDKN2A copy number, and the MTAP staining results should be correlated with clinicopathologic findings and other ancillary studies.

Malignant mesothelioma in situ: morphologic features and clinical outcome.

The existence of an in situ phase of malignant mesothelioma has long been postulated but until recently has been impossible to prove. Here we describe ten patients with mesothelioma in situ, defined by a single layer of surface mesothelial cells showing loss of BAP1 nuclear immunostaining, no evidence of tumor by imaging and/or by direct examination of the pleura/peritoneum, and no invasive mesothelioma developing for at least 1 year. Nine cases were pleural and one peritoneal. Most patients were biopsied for repeated effusions of unknown etiology; in two patients mesothelioma in situ was found incidentally in lung cancer resections. In addition to surface mesothelium with BAP1 loss, one case had a surface papillary proliferation with BAP1 loss, and two cases had a small (few millimeter) nodule with BAP1 loss. CDKN2A was deleted by FISH in one of eight cases. Methylthioadenosine phosphorylase showed partial loss in the surface mesothelium by immunohistochemistry in three cases. Invasive malignant mesothelioma developed in seven patients with time between biopsy and invasive disease from 12 to 92 (median 60) months. Invasive mesothelioma has not developed in the other three patients at 12, 57, and 120 months, but the latter patient, who has pleural plaques, still has repeated pleural effusions, probably representing a so-called "benign asbestos effusion." We conclude that mesothelioma in situ, as diagnosed using the criteria outlined above, is associated with a high risk of developing invasive mesothelioma, but typically over a relatively protracted time, so that curable interventions maybe possible.

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition.

Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

The mTOR Signaling Pathway Is Associated With the Prognosis of Malignant Pleural Mesothelioma After Multimodality Therapy.

BACKGROUND/AIM: We performed multimodality therapy comprising preoperative chemotherapy, extrapleural pneumonectomy (EPP), and radiation therapy for patients with malignant pleural mesothelioma (MPM). Although multimodality therapy resulted in good prognosis, further improvement is required. Therefore, herein, we analysed the prognostic factors using surgical specimens and searched for suitable molecular targets to improve the prognosis after multidisciplinary treatment. PATIENTS AND METHODS: Forty-six patients with MPM underwent multimodality therapy. Paraffin-embedded surgical samples were used for immunohistochemistry to evaluate the expression of phosphorylated (p-) AKT, extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and S6 ribosomal protein (S6RP). RESULTS: On univariate and multivariate analyses, significant differences were observed according to the histological type, pathological stage, and p-mTOR expression rate. CONCLUSION: The prognosis of MPM is affected by p-mTOR expression, suggesting that molecular-targeted treatment might be used during multimodal therapy for MPM.

Phase 1 Dose-Escalation Study of Pegylated Arginine Deiminase, Cisplatin, and Pemetrexed in Patients With Argininosuccinate Synthetase 1-Deficient Thoracic Cancers.

Purpose Pegylated arginine deiminase (ADI-PEG 20) depletes essential amino acid levels in argininosuccinate synthetase 1 (ASS1) -negative tumors by converting arginine to citrulline and ammonia. The main aim of this study was to determine the recommended dose, safety, and tolerability of ADI-PEG 20, cisplatin, and pemetrexed in patients with ASS1-deficient malignant pleural mesothelioma (MPM) or non-small-cell lung cancer (NSCLC). Patients and Methods Using a 3 + 3 + 3 dose-escalation study, nine chemotherapy-naïve patients (five MPM, four NSCLC) received weekly ADI-PEG 20 doses of 18 mg/m2, 27 mg/m2, or 36 mg/m2, together with pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 which were given every three weeks (maximum of six cycles). Patients achieving stable disease or better could continue ADI-PEG 20 monotherapy until disease progression or withdrawal. Adverse events were assessed by Common Terminology Criteria for Adverse Events version 4.03, and pharmacodynamics and immunogenicity were also evaluated. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 for NSCLC and by modified RECIST criteria for MPM. Results No dose-limiting toxicities were reported; nine of 38 reported adverse events (all grade 1 or 2) were related to ADI-PEG 20. Circulating arginine concentrations declined rapidly, and citrulline levels increased; both changes persisted at 18 weeks. Partial responses were observed in seven of nine patients (78%), including three with either sarcomatoid or biphasic MPM. Conclusion Target engagement with depletion of arginine was maintained throughout treatment with no dose-limiting toxicities. In this biomarker-selected group of patients with ASS1-deficient cancers, clinical activity was observed in patients with poor-prognosis tumors. Therefore, we recommend a dose for future studies of weekly ADI-PEG 20 36 mg/m2 plus three-weekly cisplatin 75 mg/m2 and pemetrexed 500 mg/m2.

Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain.

The aim of present study is to elucidate autophagic mechanism of tanshinone I (Tan I) in H28 and H2452 mesothelioma cells. Herein, Tan I exerted cytotoxicity with autophagic features of autophagy protein 5 (ATG5)/ microtubule-associated protein 1A/1B-light chain 3II (LC3 II) activation, p62/sequestosome 1 (SQSTM1) accumulation and increased number of LC3II punctae, acridine orange-stained cells and autophagic vacuoles. However, 3-methyladenine (3MA) and NH4Cl increased cytotoxicity in Tan I treated H28 cells. Furthermore, autophagy flux was enhanced in Tan I-treated H28 cells transfected by RFP-GFP-LC3 constructs, with colocalization of GFP-LC3 punctae with LAMP1 or Lysotracker. Interestingly, C-terminal UBA domain is required for Tan 1 induced aggregation of p62 in H28 cells. Notably, Tan I upregulated CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring protein-1 (IRE1) and p-c-Jun N-terminal kinase (p-JNK), but silencing of IRE1 or p62 and JNK inhibitor SP600125 blocked the LC3II accumulation in Tan I-treated H28 cells. Overall, these findings demonstrate that Tan I exerts antitumor activity through a compromise between apoptosis and p62/SQSTM1-dependent autophagy via activation of JNK and IRE 1 in malignant mesothelioma cells.

Analysis of expression of PTEN/PI3K pathway and programmed cell death ligand 1 (PD-L1) in malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) frequently express elevated AKT/mTOR activity. Previous reports in gliomas, colon, breast and prostate cancer suggest that PTEN/PI3K pathway may be important for the induction of PD-L1 expression. This study explored the expression of PTEN/PI3K pathway and PD-L1 in MPM and its relationship with the patient́s prognosis MATERIAL AND METHODS: Twenty seven consecutive MPM patients were reviewed. Formalin-fixed, paraffin-embedded tissue biopsies were used for immunohistochemical analysis of PTEN/PI3K pathway and PD-L1 RESULTS: Expression of PTEN, mTOR, pAKT, p4EBP1, peif4E, pS6 and FOXO3a was found in 88.5%, 92.3%, 78.3%, 38.5%, 100%, 52.2% and 100% of tumors and PD-L1 in 23%. We found a significant correlation between pAKT, FOXO3a and PD-L1 expression and longer overall survival (p <0.05). We did not identify significant association between the level of PD-L1 expression and alterations in PI3K pathway CONCLUSIONS: This study shows PTEN/PI3K pathway and PD-L1 in MPM are frequently activated. Our results suggests that there is not association between PD-L1 and the involvement of the PI3K pathway in MPM.

Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery.

OBJECTIVES: Survival and response to platinum-based induction chemotherapy are heterogeneous among patients with malignant pleural mesothelioma. The aim of the present study was to assess the prognostic role of DNA repair markers, such as excision repair cross-complementation group 1 and ribonucleotide reductase M1, in multimodally treated patients with malignant pleural mesothelioma. METHODS: Tumor tissue of a malignant pleural mesothelioma cohort (n = 107) treated with platinum/gemcitabine (n = 46) or platinum/pemetrexed (n = 61) induction chemotherapy followed by extrapleural pneumonectomy was assembled on a tissue microarray. Immunohistochemical expression of excision repair cross-complementation group 1 (nuclear) and ribonucleotide reductase M1 (nuclear and cytoplasmic) was assessed for its prognostic impact (association with overall survival or freedom from recurrence). RESULTS: Patients with high nuclear ribonucleotide reductase M1 expression before chemotherapy showed significantly longer freedom from recurrence (P = .03). When specifically analyzed in the subgroup of patients receiving platinum/gemcitabine followed by extrapleural pneumonectomy, high nuclear ribonucleotide reductase M1 was associated with prolonged freedom from recurrence (P = .03) and overall survival (P = .02). Low excision repair cross-complementation group 1 expression in prechemotherapy tumor tissues was associated with significantly longer freedom from recurrence (P = .04). Nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 were independent prognosticators of freedom from recurrence in addition to pT stage in multivariate analysis. CONCLUSIONS: In the present study, nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 expression were identified as independent prognosticators for freedom from recurrence of malignant pleural mesothelioma in patients undergoing induction chemotherapy followed by extrapleural pneumonectomy.

Clinical significance of soluble CD26 in malignant pleural mesothelioma.

There is no established single diagnostic marker for malignant pleural mesothelioma (MPM). CD26 is a 110 kDa, multifunctional, membrane-bound glycoprotein that has dipeptidyl peptidase IV (DPPIV) enzyme activity. The aim of this study was to evaluate the clinical significance of soluble CD26 (sCD26) in patients with MPM. The study included 80 MPM patients, 79 subjects with past asbestos exposure (SPE), and 134 patients with other benign pleural diseases (OPD) that were included as a control group. sCD26 levels and DPPIV activity in serum and/or pleural fluid were determined using an ELISA kit. Serum sCD26 levels and DPPIV enzyme activity in patients with MPM were significantly decreased compared with those in the SPE group (P = 0.000). The level of serum sCD26 was significantly decreased in patients with advanced stages of MPM compared with those with earlier stages (P = 0.047). The median OS of patients with MPM who had higher DPPIV enzyme activity was significantly longer than that of those with lower DPPIV enzyme activity (P = 0.032). The sCD26 levels in the pleural fluid of MPM patients with an epithelioid subtype were significantly increased compared with the OPD cohort (P = 0.012). Moreover, DPPIV enzyme activity in the pleural fluid of patients with MPM with an epithelioid subtype were significantly increased compared with those in the OPD cohort (P = 0.009). Patients with MPM who had lower specific DPPIV activity, determined as DPPIV/sCD26, showed significantly prolonged survival compared with those with higher specific DPPIV activity (P = 0.028). Serum sCD26 and DPPIV enzyme activity appear to be useful biomarkers for differentiating patients with MPM from SPE. The sCD26 levels or DPPIV enzyme activity in pleural fluid appear to be biomarkers in patients with an epithelioid subtype of MPM. DPPIV activity in serum or pleural fluid appears to be predictive for the prognosis of patients with MPM.

CK2α, over-expressed in human malignant pleural mesothelioma, regulates the Hedgehog signaling pathway in mesothelioma cells.

BACKGROUND: The Hedgehog (Hh) signaling pathway has been implicated in stem cell maintenance and its activation is aberrant in several types of cancer including mesothelioma. Protein kinase CK2 affects several cell signaling pathways through the mechanism of phosphorylation. METHODS: Protein and mRNA levels of CK2α and Gli1 were tested by quantitative RT-PCR and immunohistochemistry staining in mesothelioma samples and cell lines. Down-regulated Gli1 expression and transcriptional activity were demonstrated by RT-PCR, Western blot and luciferase reporter assay. RESULTS: In this study, we show that CK2α is over-expressed and a positive regulator of Hegdehog/Gli1 signaling in human malignant pleural mesothelioma. First of all, we found that the mRNA levels of CK2α and Gli1 were broadly elevated and correlated (n = 52, r = 0.401, P < 0.05), compared with LP9 (a normal mesothelial cell line). We then investigated their expression at the protein level, and found that all the 7 mesothelioma cell lines tested showed positive staining in CK2α and Gli1 immunohistochemistry. Correlation analysis by Pearson test for CK2α and Gli1 expression in the 75 mesothelioma tumors and the 7 mesothelioma cell lines showed that the two protein expression was significantly correlated (n = 82, r = 0.554, P < 0.01). Furthermore, we demonstrated that Gli1 expression and transcriptional activity were down-regulated after CK2α was silenced in two mesothelioma cell lines (H28 and H2052). CK2α siRNA also down-regulated the expression of Hh target genes in these cell lines. Moreover, treatment with a small-molecule CK2α inhibitor CX-4945 led to dose-dependent inhibition of Gli1 expression and transcriptional activity. Conversely, forced over-expression of CK2α resulted in an increase in Gli1 transcriptional activity in H28 cells. CONCLUSIONS: Thus, we report for the first time that over-expressed CK2α positively regulate Hh/Gli1 signaling in human mesothelioma.

Overexpression and promoter mutation of the TERT gene in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a very aggressive tumor with no known curative treatment. Better knowledge of the molecular mechanisms of mesothelial carcinogenesis is required to develop new therapeutic strategies. MPM, like all cancer cells, needs to maintain telomere length to prevent senescence. Previous studies suggested that the telomere lengthening mechanism in MPM is based mainly on telomerase activity. For this reason, we focused on the key catalytic enzyme, TERT (telomerase reverse transcriptase), by analyzing its gene expression in MPM and by studying the mechanism underlying its upregulation. We used our large collection of MPM composed of 61 MPM in culture and 71 frozen MPM tumor samples. Evaluation of TERT mRNA expression by quantitative RT-PCR showed overexpression in MPM in culture compared with normal mesothelial cells, and in MPM tumor samples compared with normal pleura. We identified a 'hot spot' of mutations in the TERT gene core promoter in both MPM in culture and in MPM tumor samples with an overall frequency of 15%. Furthermore, data clearly identified mutation in the TERT promoter as a mechanism of TERT mRNA upregulation in MPM. In contrast, gene copy number amplification was not associated with TERT overexpression. Then, we analyzed the clinicopathological, etiological and genetic characteristics of MPM with mutations in the TERT promoter. TERT promoter mutations were more frequent in MPM with sarcomatoid histologic subtype (P<0.01), and they were frequently associated with CDKN2A gene inactivation (P=0.03). In conclusion, a subgroup of MPM presents TERT promoter mutations, which lead to TERT mRNA upregulation. This is the first recurrent gain-of-function oncogenic mutations identified in MPM.

Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma.

BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. METHODS: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. RESULTS: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. CONCLUSIONS: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.

Histone deacetylase inhibitors as potential therapeutic agents for the treatment of malignant mesothelioma.

Histone deacetylase inhibitors (HDACIs) represent one of the most promising, recently developed classes of anticancer agents already approved by the U.S. FDA. The effectiveness of these new drugs has currently being explored in a variety of cancer cell lines, in vitro, animal models, in vivo, as well as in clinical trials. Malignant mesothelioma (MM) is a rare aggressive malignancy with a median overall survival of 12 months when the current chemotherapy regimen, cisplatin-pemetrexed, is applied. This disappointing overall survival has encouraged the experimental use of novel pharmaceutical agents, including HDACIs. In this aspect, the present review is aimed to summarize the existing data regarding the potential utility of HDACIs as therapeutic targets for the treatment of MM. Taking into consideration the research investigations so far, both in vitro and in vivo studies have documented encouraging results. Promising results are also being expected by ongoing clinical trials that concern combination of chemotherapy with HDACIs against MM.

Carbonic anhydrase IX in malignant pleural mesotheliomas: a potential target for anti-cancer therapy.

Malignant mesothelioma is a neoplasm deriving from mesothelial cells, which line the body cavities. The most common type is malignant pleural mesothelioma (MPM), which is a locally aggressive malignancy with poor prognosis. To improve both the clinical diagnostics and treatment it is necessary to identify novel molecular targets which are characteristic for MPM. Although carbonic anhydrase (CA) enzymes have been linked to pH regulation and spread of cancer cells, they have not been thoroughly studied in MPM specimens. We investigated by immunohistochemistry the expression of CA isozymes II, VII, IX, and XII in a series of 27 histological MPM tumor samples. CA IX was absent in the normal lung alveolar cells, whereas it was abundantly expressed in the normal pleural mesothelium and malignant mesothelioma cells. CA VII also showed weak or moderate reactions in several cases of mesotheliomas. Neither high expression of CA VII nor CA IX did correlate significantly with the survival of the patients. The very high expression of CA IX in MPM suggests that it could represent a novel molecular target for cancer research applications.