Porocarcinomas with PAK1/2/3 fusions: a series of 12 cases.

AIMS: Porocarcinoma is a malignant sweat gland tumour differentiated toward the upper part of the sweat duct and may arise from the transformation of a preexisting benign poroma. In 2019, Sekine et al. demonstrated the presence of YAP1::MAML2 and YAP1::NUTM1 fusions in most poromas and porocarcinomas. Recently, our group identified PAK2-fusions in a subset of benign poromas. Herein we report a series of 12 porocarcinoma cases harbouring PAK1/2/3 fusions. METHODS AND RESULTS: Five patients were male and the median age was 79 years (ranges: 59-95). Tumours were located on the trunk (n = 7), on the thigh (n = 3), neck (n = 1), or groin area (n = 1). Four patients developed distant metastases. Microscopically, seven cases harboured a benign poroma component and a malignant invasive part. Ductal formations were observed in all, while infundibular/horn cysts and cells with vacuolated cytoplasm were detected in seven and six tumours, respectively. In three cases, the invasive component consisted of a proliferation of elongated cells, some of which formed pseudovascular spaces, whereas the others harboured a predominant solid or trabecular growth pattern. Immunohistochemical staining for CEA and EMA confirmed the presence of ducts. Focal androgen receptor expression was detected in three specimens. Whole RNA sequencing evidenced LAMTOR1::PAK1 (n = 2), ZDHHC5::PAK1 (n = 2), DLG1::PAK2, CTDSP1::PAK1, CTNND1::PAK1, SSR1::PAK3, CTNNA1::PAK2, RNF13::PAK2, ROBO1::PAK2, and CD47::PAK2. Activating mutation of HRAS (G13V, n = 3, G13R, n = 1, Q61L, n = 2) was present in six cases. CONCLUSION: Our study suggests that PAK1/2/3 fusions is the oncogenic driver of a subset of porocarcinomas lacking YAP1 rearrangement.

Inactivation of kindlin-3 increases human melanoma aggressiveness through the collagen-activated tyrosine kinase receptor DDR1.

The role of the focal adhesion protein kindlin-3 as a tumor suppressor and its interaction mechanisms with extracellular matrix constitute a major field of investigation to better decipher tumor progression. Besides the well-described role of kindlin-3 in integrin activation, evidence regarding modulatory functions between melanoma cells and tumor microenvironment are lacking and data are needed to understand mechanisms driven by kindlin-3 inactivation. Here, we show that kindlin-3 inactivation through knockdown or somatic mutations increases BRAFV600mut melanoma cells oncogenic properties via collagen-related signaling by decreasing cell adhesion and enhancing proliferation and migration in vitro, and by promoting tumor growth in mice. Mechanistic analysis reveals that kindlin-3 interacts with the collagen-activated tyrosine kinase receptor DDR1 (Discoidin domain receptor 1) modulating its expression and its interaction with ß1-integrin. Kindlin-3 knockdown or mutational inactivation disrupt DDR1/ß1-integrin complex in vitro and in vivo and its loss improves the anti-proliferative effect of DDR1 inhibition. In agreement, kindlin-3 downregulation is associated with DDR1 over-expression in situ and linked to worse melanoma prognosis. Our study reveals a unique mechanism of action of kindlin-3 in the regulation of tumorigenesis mediated by the collagen-activated tyrosine kinase receptor DDR1 thus paving the way for innovative therapeutic targeting approaches in melanoma.

Combined PDE4+MEK inhibition shows antiproliferative effects in NRASQ61 mutated melanoma preclinical models.

Upregulation of phosphodiesterase type 4 (PDE4) has been associated with worse prognosis in several cancers. In melanomas harboring NRAS mutations, PDE4 upregulation has been shown to trigger a switch in signaling from BRAF to RAF1 which leads to mitogen-activated protein kinase pathway activation. Previous in vitro evidence showed that PDE4 inhibition induced death in NRASQ61mut melanoma cells and such a strategy may thus be a relevant therapeutic option in those cases with no molecular targeted therapies approved to date. In this study, we generated patient-derived xenografts (PDX) from two NRASQ61mut melanoma lesions. We performed ex vivo histoculture drug response assays and in vivo experiments. A significant ex vivo inhibition of proliferation with the combination of roflumilast+cobimetinib was observed compared to dimethyl sulfoxide control in both models (51 and 67%). This antiproliferative effect was confirmed in vivo for PDX-1 with a 56% inhibition of tumor growth. To decipher molecular mechanisms underlying this effect, we performed transcriptomic analyses and revealed a decrease in MKI67, RAF1 and CCND1 expression under bitherapy. Our findings strengthen the therapeutic interest of PDE4 inhibitors and support further experiments to evaluate this approach in metastatic melanoma.

TERT Expression Induces Resistance to BRAF and MEK Inhibitors in BRAF-Mutated Melanoma In Vitro.

Because BRAF-mutated melanomas are addicted to the Mitogen Activated Protein Kinase (MAPK) pathway they show a high response rate to BRAF and MEK inhibitors. However, the clinical responses to these inhibitors are often short-lived with the rapid onset of resistance to treatment. Deciphering the molecular mechanisms driving resistance has been the subject of intense research. Recent in vitro and clinical data have suggested a link between expression of telomerase and resistance to targeted therapy in melanoma. TERT promoter mutations are the main mechanism for the continuous upregulation of telomerase in melanoma and co-occur frequently with BRAF alterations. To understand how TERT promoter mutations could be associated with resistance to targeted therapy in melanoma, we conducted translational and in vitro studies. In a cohort of V600E-BRAF-mutated melanoma patients, we showed that the TERT promoter mutation status and TERT expression tended to be associated with response to BRAF and MEK inhibitors. We demonstrated that TERT overexpression in BRAF-mutated melanoma cells reduced sensitivity to BRAF and MEK independently of TERT's telomer maintenance activity. Interestingly, inhibition of TERT reduced growth of BRAF-mutated melanoma including resistant cells. TERT expression in melanoma can therefore be a new biomarker for resistance to MAPK inhibitors as well as a novel therapeutic target.

Microsecretory adenocarcinoma of the skin, a novel type of sweat gland carcinoma: Report of three additional cases.

Microsecretory adenocarcinoma (MSA) is a newly described salivary gland tumor harboring a characteristic balanced chromosomal translocation resulting in MEF2C::SS18 gene fusion. Six primary cutaneous MSA cases have been recently described. We report three additional cases confirming the relevance of this recently identified entity of primary cutaneous adnexal tumor. Three patients aged 53-, 64- and 78-year-old were retrospectively diagnosed with MSA of the skin (MSAS) as consultation cases of the CARADERM (CAncers RAres DERMatologiques) national network. The clinical presentation was an indolent nodule on the upper extremities. There was no history of salivary gland tumor. Histopathologically, the tumors presented as dermal nodular proliferation with slightly infiltrative borders, composed of cribriform and microcystic structures with abundant myxoid intraluminal secretion embedded in a fibromyxoid stroma. They diffusely expressed cytokeratin 8 and SOX10, focally p63 and heterogeneously smooth muscle actin. All tumors harbored the MEF2C::SS18 gene fusion. A complete surgical excision was performed. No local recurrence or distant metastases were observed so far (follow-up: 17, 38, and 45 months). MSAS is the cutaneous homologue of MSA of the salivary gland, a low-grade adnexal neoplasm whose prognosis seems to be excellent once the complete removal of the tumor is assured.

Clinicopathologic and molecular characterization of melanomas mutated for CTNNB1 and MAPK.

Wnt/ß-catenin signaling plays crucial roles in melanocyte biology and may be implicated in melanoma progression. In this study, we retrospectively examined a real-life cohort of melanomas mutated for ß-catenin (CTNNB1), in association or not with a MAPK mutation (of BRAF or NRAS), and analyzed their clinical, histopathological, and molecular characteristics. Our results indicate that, regardless of the presence of a concurrent MAPK mutation, CTNNB1mut cutaneous primary melanomas display more proliferative hallmarks (increased Breslow thickness, mitotic index, and ulceration) than their CTNNB1 wild-type counterparts. Accordingly, they often progress to the metastatic stage. Furthermore, concurrent CTNNB1 and MAPK mutations do not necessarily confer a deep penetrating nevi phenotype. Altogether, this study provides evidence that CTNNB1 mutations in melanomas are associated with specific clinical and pathological features.

Macrophage-derived CXCL9 and CXCL11, T-cell skin homing, and disease control in mogamulizumab-treated CTCL patients.

Cutaneous T-cell lymphomas (CTCLs) are rare malignancies involving primarily the skin. Responses to treatment are usually short-lived in advanced CTCL. The determinants of long-term CTCL control are unclear. Mogamulizumab, an anti-human CCR4 antibody that acts by antibody-dependent cell cytotoxicity against CCR4+ CTCL tumor cells and peripheral memory blood regulatory T cells, has been associated with long-lasting remissions and immune adverse events. Here, we reported skin rashes in 32% of 44 patients with CTCL treated with mogamulizumab, associated with significantly higher overall survival (hazard ratio, 0.16; 0.04-0.73; P = .01). Rash occurred in patients with Sézary syndrome and was associated with longer time to progression. These rashes were characterized by a CD163+ granulomatous and/or CD8+ lichenoid skin infiltrate. High-throughput sequencing analysis of T-cell receptor ß genes in skin and blood flow cytometry confirmed the depletion of CTCL tumor cells, as well as the recruitment of new reactive T-cell clones in skin at the time of skin rash. CXCL9 and CXCL11, two macrophage-derived chemokines that recruit CXCR3+ T cells to skin, were overexpressed in skin rashes. A higher frequency of TIGIT+ and PD1+ exhausted reactive blood T cells was observed at baseline in patients with rash, and this frequency decreased with mogamulizumab treatment. These data are consistent with mogamulizumab-induced long-term immune CTCL control by activation of the macrophage and T-cell responses in patients with rash.

Pharmacogenomics in solid cancers and hematologic malignancies: Improving personalized drug prescription.

The discovery of molecular alterations involved in oncogenesis is evolving rapidly and has led to the development of new innovative targeted therapies in oncology. High-throughput sequencing techniques help to identify genomic targets and to provide predictive molecular biomarkers of response to guide alternative therapeutic strategies. Besides the emergence of these theranostic markers for the new targeted treatments, pharmacogenetic markers (corresponding to genetic variants existing in the constitutional DNA, i.e., the host genome) can help to optimize the use of chemotherapy. In this review, we present the current clinical applications of constitutional PG and the recent concepts and advances in pharmacogenomics, a rapidly evolving field that focuses on various molecular alterations identified on constitutional or somatic (tumor) genome.

Phase I-II Open-Label Multicenter Study of Palbociclib + Vemurafenib in BRAF V600MUT Metastatic Melanoma Patients: Uncovering CHEK2 as a Major Response Mechanism.

PURPOSE: In BRAF V600MUT metastatic melanoma, cyclin D-CDK4/6-INK4-Rb pathway alterations are involved in resistance to MAPK inhibitors, suggesting a clinical benefit of cyclin-dependent kinase 4 (CDK4) inhibitors. In this phase I-II study, we aimed to establish the MTD of palbociclib when added to vemurafenib. PATIENTS AND METHODS: Patients with BRAF V600E/KMUT metastatic melanoma harboring CDKN2A loss and RB1 expression were included and stratified into two groups according to previous BRAF inhibitor treatment (no:strata 1; yes:strata 2). Treatment comprised palbociclib once daily for 14 days followed by a 7-day break + continuous dosing of vemurafenib. The primary endpoint was the occurrence of dose-limiting toxicity (DLT), and the secondary endpoints included the best response, survival, pharmacokinetics, and tumor molecular profiling. RESULTS: Eighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (N = 16; 88.9%), high lactate dehydrogenase (N = 9; 50.0%), and median number of previous treatments of 2. One and 5 patients experienced DLT in strata 1 and 2, respectively, defining the MTD at palbociclib 25 mg and vemurafenib 960 mg in strata 2. No significant evidence for drug-drug interactions was highlighted. The median progression-free survival was 2.8 months, and 5 (27.8%) patients showed a clinical response. The baseline differential mRNA expression analysis and in vitro data revealed the role of CHEK2 in the response to palbociclib. CONCLUSIONS: Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

Custom pyrosequencing assay to detect short BRAF deletions in Langerhans cell histiocytic lesions.

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplastic disease driven by activating mutations in the mitogen-activating protein kinase signalling pathway, including the BRAF V600E mutation and BRAF deletions (BRAFdel). Next-generation sequencing and whole exome sequencing (WES) are valuable and powerful approaches for BRAFdel identification, but these techniques are costly and time consuming. Pyrosequencing is an alternative method that has the potential to rapidly and reliably identify gene deletions. We developed a custom pyrosequencing assay to detect the exon-12 BRAFdel in 18 biopsies from adult patients with LCH, which were all genotyped in parallel using Sanger sequencing and WES. A BRAFdel was detected in 7/18 (39%), 6/18 (33%) and 3/18 (17%) LCH lesions using WES, pyrosequencing and Sanger, respectively, with good concordance between the WES and pyrosequencing results (Kappa-coefficient=0.88). Therefore, our pyrosequencing assay is reliable and useful for detecting BRAFdel, particularly in BRAF V600E-negative LCH lesions, for which targeted treatment is indicated.

Premature termination codons in SOD1 causing Amyotrophic Lateral Sclerosis are predicted to escape the nonsense-mediated mRNA decay.

Amyotrophic lateral sclerosis (ALS) is the most common and severe adult-onset motoneuron disease and has currently no effective therapy. Approximately 20% of familial ALS cases are caused by dominantly-inherited mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), which represents one of the most frequent genetic cause of ALS. Despite the overwhelming majority of ALS-causing missense mutations in SOD1, a minority of premature termination codons (PTCs) have been identified. mRNA harboring PTCs are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD), which limits the production of truncated proteins. The rules of NMD surveillance varying with PTC location in mRNA, we analyzed the localization of PTCs in SOD1 mRNA to evaluate whether or not those PTCs can be triggered to degradation by the NMD pathway. Our study shows that all pathogenic PTCs described in SOD1 so far can theoretically escape the NMD, resulting in the production of truncated protein. This finding supports the hypothesis that haploinsufficiency is not an underlying mechanism of SOD1 mutant-associated ALS and suggests that PTCs found in the regions that trigger NMD are not pathogenic. Such a consideration is particularly important since the availability of SOD1 antisense strategies, in view of variant treatment assignment.

A Melanoma-Tailored Next-Generation Sequencing Panel Coupled with a Comprehensive Analysis to Improve Routine Melanoma Genotyping.

BACKGROUND: Tumor molecular deciphering is crucial in clinical management. Pan-cancer next-generation sequencing panels have moved towards exhaustive molecular characterization. However, because of treatment resistance and the growing emergence of pharmacological targets, tumor-specific customized panels are needed to guide therapeutic strategies. OBJECTIVE: The objective of this study was to present such a customized next-generation sequencing panel in melanoma. METHODS: Melanoma patients with somatic molecular profiling performed as part of routine care were included. High-throughput sequencing was performed with a melanoma tailored next-generation sequencing panel of 64 genes involved in molecular classification, prognosis, theranostic, and therapeutic resistance. Single nucleotide variants and copy number variations were screened, and a comprehensive molecular analysis identified clinically relevant alterations. RESULTS: Four hundred and twenty-one melanoma cases were analyzed (before any treatment initiation for 94.8% of patients). After bioinformatic prioritization, we uncovered 561 single nucleotide variants, 164 copy number variations, and four splice-site mutations. At least one alteration was detected in 368 (87.4%) lesions, with BRAF, NRAS, CDKN2A, CCND1, and MET as the most frequently altered genes. Among patients with BRAFV600 mutated melanoma, 44.5% (77 of 173) harbored at least one concurrent alteration driving potential resistance to mitogen-activated protein kinase inhibitors. In patients with RAS hotspot mutated lesions and in patients with neither BRAFV600 nor RAS hotspot mutations, alterations constituting potential pharmacological targets were found in 56.9% (66 of 116) and 47.7% (63 of 132) of cases, respectively. CONCLUSIONS: Our tailored next-generation sequencing assay coupled with a comprehensive analysis may improve therapeutic management in a significant number of patients with melanoma. Updating such a panel and implementing multi-omic approaches will further enhance patients' clinical management.

The key role of oncopharmacology in therapeutic management, from common to rare cancers: A literature review.

The therapeutic management of cancers has undergone considerable changes due to the emergence of genomics tools and tumor molecular deciphering. In this context, a dual pharmacological approach based on pharmacogenomic analyses and therapeutic drug monitoring is now part of the routine care in cancer management for personalized therapies. First, molecular and immune profiling of tumors allows the emergence of new pharmacological targets in common as well as in rare cancers. Second, pharmacogenomic analyses coupled to therapeutic drug monitoring guide the prescription by adjusting regimen and managing drug resistance.