Exploring the Role of Immunotherapy-Based Treatments for Advanced Non-Small-Cell Lung Cancer With Novel Driver Alterations.

BACKGROUND: Immunotherapy (IO) single agent or combined with chemotherapy (CT-IO) is the standard treatment for advanced non-small-cell lung cancer (aNSCLC) without driver alterations. IO efficacy in patients with novel driver alterations is not well reported. MATERIALS AND METHODS: Data of aNSCLC patients treated with IO or CT-IO in any line from January 2016 to September 2022 were retrospectively collected. Patients harboring novel driver alterations (m-cohort), including MET exon 14 skipping, BRAF (V600E or atypical), RET rearrangements, HER2 point mutations/exon 20 insertions or uncommon EGFR mutations/EGFR exon 20 insertions, and wild type patients (wt-cohort) were eligible. Clinico-pathological data were extracted from Institutional databases and compared through chi square or Fisher's exact test. Survivals were estimated through Kaplan-Meier method and compared by log-rank test. RESULTS: m-cohort and wt-cohort included 84 and 444 patients, respectively. Progression free survival (PFS) was 5.53 vs. 4.57 months (P= .846) and overall survival (OS) was 25.1 vs. 9.37 months, (P < .0001) for m-cohort compared to wt-cohort. Within the m-cohort, BRAF atypical mutations had the better outcomes (Overall Response Rate [ORR], PFS), targeted agents timing did not affect response to IO and CT-IO had better ORR and disease control rate (DCR) compared to IO single agent (P = .0160 and P = .0152). In the PD-L1≥50% group, first line IO single agent resulted in inferior ORR (P = .027) and PFS (P = .022) in m-cohort compared to wt-cohort. CONCLUSION: IO based treatments seem not detrimental for patients harboring novel driver alteration. Adding CT could improve modest responses to IO alone. Confirmation on larger datasets is required.

Pan-TRK immunohistochemistry as screening tool for NTRK fusions: A diagnostic workflow for the identification of positive patients in clinical practice.

BACKGROUND: Pan-TRK inhibitors Entrectinib and Larotrectinib have been recently approved as tumor-agnostic therapies in NTRK1-2-3 rearranged patients and there is therefore an urgent need to identify reliable and accessible biomarkers for capturing NTRK fusions in the real-world practice. OBJECTIVE: We aim to assess the analytical validity of the recently released pan-TRK assay (Ventana), running a head-to-head comparison between immunohistochemistry and Archer FusionPlex Lung Panel (ArcherDX) that is designed to detect key fusions in 13 genes, also including NTRK1-3. METHODS: Pan-TRK IHC and NGS analysis were conducted on a retrospective/prospective cohort of 124 cancer patients (carcinomas, 93 cases; soft tissue sarcomas, 19; primary central nervous system tumours, 10; and neuroblastomas, 2). FISH data were available in most of the IHC/NGS discordant cases. RESULTS: A comparison between IHC and NGS results was carried out in 117 cases: among 30 pan-TRK positive cases, NTRK rearrangement by NGS was found in 11 (37%), while one of the 87 (1.1%) pan-TRK negative cases (a case of NSCLC) showed a TPM3-NRTK1 rearrangement by NGS. Accordingly, sensitivity and specificity of IHC in predicting NTRK status were 91.7% and 81.9%, respectively, while negative (NPV) and positive predictive value (PPV) were 98.8% and 36.7%, respectively. CONCLUSIONS: These data lead to suggest that IHC with VENTANA pan-TRK antibody can be a reliable screening tool for the identification of patients potentially bearing NTRK rearranged tumours.

A2AR Expression and Immunosuppressive Environment Independent of KRAS and GNAS Mutations in Pseudomyxoma Peritonei.

In pseudomyxoma peritonei (PMP), KRAS and GNAS mutations are frequent. We hypothesized that these mutations may contribute to the suppression of antitumor immunity: KRAS may induce GMCSF expression, while GNAS may enhance the expression of cyclic adenosine monophosphate and A2AR signaling. This study aimed to explore possible mechanisms facilitated by KRAS and GNAS mutations for escaping immune surveillance. Additionally, we looked for new potential therapeutic and prognostic targets in this rare disease which is poorly characterized at the molecular level. GM-CSF, A2AR, CD73, CD39, and PD-L1 expression was investigated by immunohistochemistry in 40 PMPs characterized for GNAS and KRAS mutational status. Immune cell populations were studied by immunohistochemistry and nanostring nCounter®. Following the criteria of a prognostic nomogram reported for PMP, we stratified the patients into two different risk groups, with 28 "low-risk" and 12 "high-risk" patients. We observed the expression of GM-CSF (74%); CD39 (37%); CD73 (53%); A2AR (74%); and PD-L1 (16%) which was unrelated to GNAS or KRAS status. The tumor microenvironment showed the presence of CD4+ T cells (86%); CD8+ T cells (27%); CD20+ B (67%); CD15+ cells (86%); and CD163+ M2 macrophages (67%), while CD56+ NK cells were absent. CD163 expression (27%) in PMP tumor cells was associated with poor prognosis. GNAS mutation and A2AR expression were not associated with a specific immune transcriptional signature. However, the expression assay revealed 21 genes associated with prognosis. The "high-risk" patients exhibited worse progression-free survival (HR = 2.3, CI 95%: 1.1-5.1, p = 0.034) and significant downregulation of MET, IL8, PPARG, DTX4, HMGA1, ZIC2, WNT5B, and CCRL2. In conclusion, we documented the presence of immunosuppressive factors such as GM-CSF, A2AR, and PD-L1 in PMP. These factors were not associated with GNAS and KRAS status and could be explored as therapeutic molecular targets. Additionally, a set of potential prognostic biomarkers, including CD163 expression in tumor cells, deserve further investigation.

Molecular Tumor Board as a Clinical Tool for Converting Molecular Data Into Real-World Patient Care.

PURPOSE: The investigation of multiple molecular targets with next-generation sequencing (NGS) has entered clinical practice in oncology, yielding to a paradigm shift from the histology-centric approach to the mutational model for personalized treatment. Accordingly, most of the drugs recently approved in oncology are coupled to specific biomarkers. One potential tool for implementing the mutational model of precision oncology in daily practice is represented by the Molecular Tumor Board (MTB), a multidisciplinary team whereby molecular pathologists, biologists, bioinformaticians, geneticists, medical oncologists, and pharmacists cooperate to generate, interpret, and match molecular data with personalized treatments. PATIENTS AND METHODS: Since May 2020, the institutional MTB set at Fondazione IRCCS Istituto Nazionale Tumori of Milan met weekly via teleconference to discuss molecular data and potential therapeutic options for patients with advanced/metastatic solid tumors. RESULTS: Up to October 2021, among 1,996 patients evaluated, we identified >10,000 variants, 43.2% of which were functionally relevant (pathogenic or likely pathogenic). On the basis of functionally relevant variants, 711 patients (35.6%) were potentially eligible to targeted therapy according to European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets tiers, and 9.4% received a personalized treatment. Overall, larger NGS panels (containing >50 genes) significantly outperformed small panels (up to 50 genes) in detecting actionable gene targets across different tumor types. CONCLUSION: Our real-world data provide evidence that MTB is a valuable tool for matching NGS data with targeted treatments, eventually implementing precision oncology in clinical practice.

Prevalence of BRCA homopolymeric indels in an ION Torrent-based tumour-to-germline testing workflow in high-grade ovarian carcinoma.

Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended.

Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup.

INTRODUCTION: During the last few years it has been shown that an anaplastic T cell lymphoma can develop as a rare and late sequelae of implant-based breast reconstruction. This malignancy was recognized in the 2017 by WHO and named breast implant associated anaplastic large T cell lymphoma (BIA-ALCL). BIA-ALCL usually presents as abundant effusion around the implant, thus, in addition to cytology smears, its diagnosis also requires immunohistochemistry, T cells clonality and cytometry. Due to the increasing attention of clinicians, it is likely that the number of the BIA-ALCL suspected cases will grow in the future, implying the necessity of a reliable and cost-effective diagnostic procedure. METHODS: To achieve this goal, we retrospectively analyzed the results of laboratory investigations performed at our Institute (Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Italy) on 44 effusions obtained from 31 women suspected for BIA-ALCL. RESULTS: Through cytology, eight out of 44 effusions showed the presence of BIA-ALCL cells. Lymphoma cells were than confirmed in seven samples by immunohistochemistry and/or T cell clonality and/or cytometry. Overall, cytology showed 100% sensitivity, 97% specificity and positive and negative predictive values of 87.5% and 100% respectively. Further analyses were particularly useful in effusions showing small percentages of BIA-ALCL cells. Moreover, an extended cytometric profile that can be applied when fast confirmation of the cytologic result is required was also identified. CONCLUSIONS: Our results evidenced a central role of cytopathology in the management of BIA-ALCL suspected effusions and suggested that further laboratory investigations might be applied only in cases showing atypical/activated lymphoid cells through cytology.

Molecular screening with liquid biopsy for anti-EGFR retreatment in metastatic colorectal cancer: preliminary data from the randomized phase 2 PARERE trial.

Background: Retreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening. Methods: Patients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing. Results: A total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients. Conclusions: This is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples.

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics. Methods: Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex© Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]. Results: The combination of FusionPlex© Solid tumor with ArcherDX® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens. Conclusions: Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

Management of BRCA Tumour Testing in an Integrated Molecular Tumour Board Multidisciplinary Model.

Tumour testing of the BRCA1/2 genes is routinely performed in patients with different cancer histological subtypes. To accurately identify patients with tumour-detected germline pathogenic variants (PVs) is a relevant issue currently under investigation. This study aims at evaluating the performance of the tumour-to-germline diagnostic flowchart model defined at our Institutional Molecular Tumour Board (MTB). Results from tumour BRCA sequencing of 641 consecutive unselected cancer patients were discussed during weekly MTB meetings with the early involvement of clinical geneticists for appropriate referral to genetic counselling. The overall tumour detection rate of BRCA1/2 PVs was 8.7% (56/641), ranging from 24.4% (31/127) in high-grade ovarian cancer to 3.9% (12/304) in tumours not associated with germline BRCA1/2 PVs. Thirty-seven patients with PVs (66%) were evaluated by a clinical geneticist, and in 24 of them (64.9%), germline testing confirmed the presence of the PV in blood. Nine of these patients (37.5%) were not eligible for germline testing according to the criteria in use at our institution. Cascade testing was subsequently performed on 18 relatives. The tumour-to-germline diagnostic pipeline, developed in the framework of our institutional MTB, compared with guideline-based germline testing following genetic counselling, proved to be effective in identifying a higher number of germline BRCA PVs carriers.

ALK Inhibitors in Patients With ALK Fusion-Positive GI Cancers: An International Data Set and a Molecular Case Series.

PURPOSE: In GI cancers, anaplastic lymphoma kinase (ALK) rearrangements are extremely less frequent than in non-small-cell lung cancer but may be important to offer personalized strategies of treatment in selected patients. Data about the activity and efficacy of ALK inhibitors (ALKi) in GI cancers are scarce. MATERIALS AND METHODS: We assembled a clinical and molecular international data set of pretreated patients with metastatic or nonresectable cancers of GI primary tumor origin with documented ALK rearrangement treated with at least one line of ALKi. Measurable disease as per RECIST 1.1 was required for response analysis. RESULTS: Primary tumor sites were distributed as follows: 5 (38%) pancreas, 3 (23%) right colon, and 1 (8%) for each one of gastric, duodenal, rectal, left colon, and biliary tract sites. Seven patients (54%) were treated with alectinib, 5 (38%) with crizotinib, and 1 (8%) with entrectinib. After disease progression, five patients (38%) received a subsequent ALKi treatment line, and at the time of data cutoff date, treatment was still ongoing in two patients. Five of 12 evaluable patients (41%) achieved a partial response to first-line ALKi, five patients (41%) had stable disease, and 2 (17%) had progressive disease. No complete responses were registered. At a median follow-up of 39.6 months (interquartile range: 19.8-59.5), the median progression-free survival was 5.0 months (95% CI, 3.68 to no response) and the median overall survival was 9.3 months (95% CI, 5.46 to no response). CONCLUSION: Treatment with ALKi provides remarkable responses and clinical benefit in pretreated patients with ALK fusion-positive GI malignancies. Despite the rarity, ALK rearrangements represent an important therapeutic target in individual pretreated patients with GI solid tumors. Further work providing prospective clinical validation of this target is needed.

Temozolomide Followed by Combination With Low-Dose Ipilimumab and Nivolumab in Patients With Microsatellite-Stable, O6-Methylguanine-DNA Methyltransferase-Silenced Metastatic Colorectal Cancer: The MAYA Trial.

PURPOSE: This is a multicenter, single-arm phase II trial evaluating the efficacy and safety of an immune-sensitizing strategy with temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab in patients with microsatellite-stable (MSS) and O6-methylguanine-DNA methyltransferase (MGMT)-silenced metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients with pretreated mCRC were centrally prescreened for MSS status and MGMT silencing (ie, lack of MGMT expression by immunohistochemistry plus MGMT methylation by pyrosequencing). Eligible patients received two priming cycles of oral temozolomide 150 mg/sqm once daily, days 1-5, once every 4 weeks (first treatment part) followed, in absence of progression, by its combination with ipilimumab 1 mg/kg once every 8 weeks and nivolumab 480 mg once every 4 weeks (second treatment part). The primary end point was the 8-month progression-free survival (PFS) rate calculated from enrollment in patients who started the second treatment part, with ≥ 4 out of 27 subjects progression-free by the 8-month time point as decision rule. RESULTS: Among 716 prescreened patients, 204 (29%) were molecularly eligible and 135 started the first treatment part. Among these, 102 (76%) were discontinued because of death or disease progression on temozolomide priming, whereas 33 patients (24%) who achieved disease control started the second treatment part and represented the final study population. After a median follow-up of 23.1 months (interquartile range, 14.9-24.6 months), 8-month PFS rate was 36%. Median PFS and overall survival were 7.0 and 18.4 months, respectively, and overall response rate was 45%. Grade 3-4 immune-related adverse events were skin rash (6%), colitis (3%), and hypophysitis (3%). No unexpected adverse events or treatment-related deaths were reported. CONCLUSION: The MAYA study provided proof-of-concept that a sequence of temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab may induce durable clinical benefit in MSS and MGMT-silenced mCRC.

Abiraterone Acetate in Patients With Castration-Resistant, Androgen Receptor-Expressing Salivary Gland Cancer: A Phase II Trial.

PURPOSE: The activity of androgen-deprivation therapy (ADT) in androgen receptor-positive (AR+) salivary gland carcinomas (SGCs) has been established in the past few years. Second-line treatment in castration-resistant patients is still unknown. We investigated the activity of abiraterone acetate as second-line treatment in ADT-resistant, AR+ patients with SGC. METHODS: This was a single-institution phase II trial. A two-stage Simon's design was applied. The primary end point was confirmed objective response rate. Secondary end points were disease control rate, safety, progression-free survival, and overall survival. Patients were eligible when the following criteria were met: histologic diagnosis of AR-overexpressing SGC, measurable disease according to RECIST 1.1, clinical and/or radiologic progression on ADT, suppressed serum testosterone, and no limits for the number of previous chemotherapy lines. All patients received abiraterone 1 g daily plus prednisone 10 mg and luteinizing hormone-releasing hormone agonist until progression or unacceptable toxicities. RESULTS: From 2015 to 2019, 24 AR+ patients with SGC (23 men; median age 65.8 years) were treated within the study. The overall response rate was 21% (5 partial responses), with a disease control rate of 62.5%. The median duration of response was 5.82 months. Median progression-free survival was 3.65 months (95% CI, 1.94 to 5.89), and median overall survival was 22.47 months (95% CI, 6.74 to not reached). Objective response to previous ADT did not correlate with the activity of abiraterone. Adverse events (AEs) were recorded in 22 cases (92%) with grade 3 AEs in six patients (25%): fatigue (two), flushing (one), supraventricular tachycardia (one), and two non-drug-related AEs. No drug-related grade 4 or 5 AEs were recorded. CONCLUSION: Abiraterone plus luteinizing hormone-releasing hormone agonist is active and safe as a second-line option in AR-expressing, castration-resistant SGC.

Rationale and Study Design of the PARERE Trial: Randomized phase II Study of Panitumumab Re-Treatment Followed by Regorafenib Versus the Reverse Sequence in RAS and BRAF Wild-Type Chemo-Refractory Metastatic Colorectal Cancer Patients.

BACKGROUND: A recent phase II randomized Japanese study reported better survival with regorafenib followed at progression by cetuximab ± irinotecan compared with the reverse standard sequence in chemo-refractory and anti-EGFR-naïve, RAS wild-type (wt) mCRC patients. Nowadays the use of anti-EGFR antibodies is more frequently anticipated to the first-line of therapy especially in patients with left-sided RAS/BRAF wt tumours. However, retrospective analyses and phase II single-arm trials showed promising activity of re-using anti-EGFRs in metastatic colorectal cancer (mCRC) patients who previously achieved benefit from a first-line anti-EGFR-based treatment. Post-hoc analyses of these trials revealed that the detection of RAS mutations in circulating tumour DNA (ct-DNA) at the time of re-treatment may be useful to identify resistant patients. PATIENTS AND METHODS: PARERE (NCT04787341) is a prospective, open label, multicentre phase II study in which 214 RAS/BRAF wt chemo-refractory mCRC patients with previous benefit from first-line anti-EGFR-based treatment and RAS/BRAF wt ct-DNA in the liquid biopsy collected at the time of inclusion will be randomized in a 1:1 ratio to receive panitumumab followed after progression by regorafenib versus the reverse sequence. Primary endpoint is overall survival. Secondary endpoints are 1st-progression free-survival (PFS), 2nd-PFS, time to failure strategy, objective response rate, and safety. AIM OF THE STUDY: The aim of this study is to validate the role of anti-EGFR retreatment and its proper placement in the therapeutic route of mCRC patients selected according to the analysis of ct-DNA in liquid biopsy. Results are expected at the end of 2023.

Successful treatment of triple EGFR mutation T785A/L861Q/H297_E298 with afatinib.

Patients with non-small cell lung cancer (NSCLC) and uncommon epidermal growth factor receptor (EGFR) mutation are characterized by high heterogeneity, and globally considered to have a worse prognosis than patients with the two common mutations; exon 19 deletion, and exon 21 L858R. Nevertheless, some uncommon mutations do confer sensitivity to tyrosine kinase inhibitors (TKIs) which is comparable with common mutations. In particular, some compound EGFR mutations seem to be characterized by a favorable prognosis. Unfortunately, the rarity of complex EGFR mutations results in difficult clinical decision-making. Herein, to the best of our knowledge, we report the first case of an NSCLC patient with an EGFR triple mutation containing T785A/L861Q/H297_E298 who was successfully treated with afatinib.

LKB1 Down-Modulation by miR-17 Identifies Patients With NSCLC Having Worse Prognosis Eligible for Energy-Stress-Based Treatments.

INTRODUCTION: Preclinical models recently unveiled the vulnerability of LKB1/KRAS comutated NSCLC to metabolic stress-based treatments. Because miR-17 is a potential epigenetic regulator of LKB1, we hypothesized that wild-type LKB1 (LKB1WT) NSCLC with high miR-17 expression may be sensitive to an energetic stress condition, and eligible for metabolic frailties-based therapeutic intervention. METHODS: We took advantage of NSCLC cell lines with different combinations of KRAS mutation and LKB1 deletion and of patient-derived xenografts (PDXs) with high (LKB1WT/miR-17 high) or low (LKB1WT/miR-17 low) miR-17 expression. We evaluated LKB1 pathway impairment and apoptotic response to metformin. We retrospectively evaluated LKB1 and miR-17 expression levels in tissue specimens of patients with NSCLC and PDXs. In addition, a lung cancer series from The Cancer Genome Atlas data set was analyzed for miR-17 expression and potential correlation with clinical features. RESULTS: We identified miR-17 as an epigenetic regulator of LKB1 in NSCLC and confirmed targeting of miR-17 to LKB1 3' untranslated region by luciferase reporter assay. We found that miR-17 overexpression functionally impairs the LKB1/AMPK pathway. Metformin treatment prompted apoptosis on miR-17 overexpression only in LKB1WT cell lines, and in LKB1WT/miR-17 high PDXs. A retrospective analysis in patients with NSCLC revealed an inverse correlation between miR-17 and LKB1 expression and highlighted a prognostic role of miR-17 expression in LKB1WT patients, which was further confirmed by The Cancer Genome Atlas data analysis. CONCLUSIONS: We identified miR-17 as a mediator of LKB1 expression in NSCLC tumors. This study proposes a miR-17 expression score potentially exploitable to discriminate LKB1WT patients with NSCLC with impaired LKB1 expression and poor outcome, eligible for energy-stress-based treatments.

Molecular Signatures for Combined Targeted Treatments in Diffuse Malignant Peritoneal Mesothelioma.

Background-There are currently no effective therapies for diffuse malignant peritoneal mesothelioma (DMPM) patients with disease recurrence. In this study, we investigated the biology of DMPM by analyzing the EGFR family, Axl, and MET, in order to assess the presence of cross-talk between these receptors, suggesting the effectiveness of combined targeted treatments in DMPM. Method-We analyzed a series of 22 naïve epithelioid DMPM samples from a single institute, two of which showed higher-grade malignancy ("progressed"). EGFR, HER2, HER3, Axl, and MET activation and expression were investigated by biochemical analysis, real-time PCR immunofluorescence, immunohistochemistry, next-generation sequencing, miRNA, and mRNA in situ hybridization. Results-In most DMPMs, a strong EGFR activation was associated with HER2, HER3, Axl, and MET co-activation, mediated mainly by receptor heterodimerization and autocrine-paracrine loops induced by the expression of their cognate ligands. Axl expression was downregulated by miRNA34a. Mutations in MET Sema domain were exclusively found in two "progressed" DMPMs, and the combined Axl and MET inhibition reduced cellular motility in a DMPM cell line obtained from a "progressed" DMPM. Conclusion-The results indicate that the coordinated activity of multiple cross-talks between RTKs is directly involved in the biology of DMPM, suggesting the combined inhibition of PIK3 and mTOR as an effective strategy that may be easily implemented in clinical practice, and indicating that the combined inhibition of EGFR/HER2 and HER3 and of Axl and MET deserves further investigation.

Identification of potentially druggable molecular alterations in skin adnexal malignancies.

Skin adnexal cancers (SAC) are a heterogeneous group of rare malignancies with histological differentiation towards epithelial adnexa, which lack effective systemic treatments. The aim of this work is to identify any potentially druggable genomic alterations for possible targeted therapies. Cases of primary or recurrent/metastatic (RM) SAC between 2002 and 2014 were identified by searching the institutional cancer registration database. Histological sections of all referral cases were reviewed by a dedicated pathologist to confirm diagnosis. Immunohistochemistry was performed to assess the expression of androgen receptors (AR) and human epidermal growth factor receptor type 2 (HER2). Targeted next-generation sequencing (T-NGS) was performed to identify targetable mutations (panel of 50 genes analyzed by Cancer Hotspot Panel, Ion-Torrent Personal Genome Machine). Mutational analysis of the PTCH1 gene not present in the T-NGS panel was assessed by Sanger sequencing. A total of 45 cases with available histological samples were identified (35 primary, 10 RM). The most frequent histological type was porocarcinoma (n = 12). Globally, 14 cases (31%) were AR+ (6/10 RM, 60%; 8/35 primary, 23%). HER2 was shown as 2+ in eight of 42 (19%) cases (2/9 RM, 22%; 6/33 primary, 18%). DNA was adequate for T-NGS analysis in 25 cases. In the majority of cases (17 cases, 68%) at least one mutation in oncogenes or tumor suppressor genes was found: the most frequent ones involved TP.53 (13 cases, 76% of mutated SAC) and PIK3CA (three cases, 18%). The rate of PTCH1 mutation was 30%. These findings support the use of molecular screening in patients with advanced SAC.

Pulmonary adenocarcinoma with mucin production modulates phenotype according to common genetic traits: a reappraisal of mucinous adenocarcinoma and colloid adenocarcinoma.

Whether invasive mucinous adenocarcinoma (IMA) and colloid adenocarcinoma (ICA) of the lung represent separate tumour entities, or simply lie within a spectrum of phenotypic variability, is worth investigating. Fifteen ICA, 12 IMA, 9 ALK-rearranged adenocarcinomas (ALKA), 8 non-mucinous KRAS-mutated adenocarcinomas (KRASA) and 9 mucinous breast adenocarcinomas (MBA) were assessed by immunohistochemistry for alveolar (TTF1, cytoplasmic MUC1), intestinal (CDX-2, MUC2), gastric (membrane MUC1, MUC6), bronchial (MUC5AC), mesenchymal (vimentin), neuroendocrine (chromogranin A, synaptophysin), sex steroid hormone-related (oestrogen and progesterone receptors), pan-mucinous (HNF4A) and pan-epithelial (keratin 7) lineage biomarkers and by targeted next generation sequencing (TNGS) for 50 recurrently altered cancer genes. Unsupervised clustering analysis using molecular features identified cluster 1 (IMA and ICA), cluster 2 (ALKA and KRASA) and cluster 3 (MBA) (p < 0.0001). Cluster 1 showed four histology-independent sub-clusters (S1 to S4) pooled by HFN4A and MUC5AC but diversely reacting for TTF1, MUC1, MUC2, MUC6 and CDX2. Sub-cluster S1 predominantly featured intestinal-alveolar, S2 gastrointestinal, S3 gastric and S4 alveolar differentiation. In turn, KRASA and ALKA shared alveolar lineage alongside residual MUC5AC expression, with additional focal CDX2 and diffuse vimentin, respectively. A proximal-to-distal scheme extending from terminal (TB) and respiratory (RB) bronchioles to alveolar cells was devised, where S3 originated from distal TB (cellular mucinous adenocarcinoma), S2 from proximal RB (secreting mucinous adenocarcinoma), S1 from intermediate RB (mucin lake-forming colloid adenocarcinoma), S4 from distal RB (colloid alveolar adenocarcinoma), KRASA from juxta-alveolar RB (KRAS-mutated non-mucinous adenocarcinoma) and ALKA from juxta-bronchial alveolar cells (ALK-translocated adenocarcinoma). TNGS analysis showed KRAS, LKB1, TP53, APC and CDKN2A mutation predominance. In conclusion, IMA and ICA are basket categories, which likely originate from distinct domains of stem/progenitor cells spatially distributed along bronchioles upon common molecular features and genetic alterations.

Thymus neuroendocrine tumors with CTNNB1 gene mutations, disarrayed ß-catenin expression, and dual intra-tumor Ki-67 labeling index compartmentalization challenge the concept of secondary high-grade neuroendocrine tumor: a paradigm shift.

We herein report an uncommon association of intimately admixed atypical carcinoid (AC) and large cell neuroendocrine (NE) carcinoma (LCNEC) of the thymus, occurring in two 20- and 39-year-old Caucasian males. Both tumors were treated by maximal thymectomy. The younger patient presented with a synchronous lesion and died of disease after 9 months, while the other patient was associated with a recurrent ectopic adrenocorticotropic hormone Cushing's syndrome and is alive with disease at the 2-year follow-up. MEN1 syndrome was excluded in either case. Immunohistochemically, disarrayed cytoplasmic and nuclear ß-catenin expression was seen alongside an intra-tumor Ki-67 antigen labeling index (LI) ranging from 2 to 80% in the younger patient's tumor and from 3 to 45% in the other. Both exhibited upregulated cyclin D1 and retinoblastoma, while vimentin was overexpressed in the recurrent LCNEC only. Next-generation sequencing revealed CTNNB1, TP53, and JAK3 mutations in the synchronous tumor and CTNNB1 mutation alone in the metachronous tumor (the latter with the same mutation as the first tumor of 17 years prior). None of the 23 T-NET controls exhibited this hallmarking triple alteration (p = 0.003). These findings suggested that LCNEC components developed from pre-existing CTNNB1-mutated AC upon loss-of-function TP53 and gain-of-function JAK3 mutations in one case and an epithelial-mesenchymal transition upon vimentin overexpression in the other case. Both tumors maintained intact cyclin D1-retinoblastoma machinery. Our report challenges the concept of secondary LCNEC as an entity that develops from pre-existing AC as a result of tumor progression, suggesting a paradigm shift to the current pathogenesis of NET.