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.

Pulmonary Langerhans cell histiocytosis - an update on pathogenesis and treatment.

PURPOSE OF REVIEW: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare diffuse cystic lung disease that affects young to middle-aged smoking adults of both genders. The identification of molecular alterations in the canonical mitogen-activated protein kinase (MAPK) signalling pathway in most specific lesions has demonstrated the clonal/neoplastic nature of PLCH. We will summarize the progress made in the understanding of the pathogenesis of adult PLCH, and briefly highlight the recent findings useful for the management of the patients. RECENT FINDINGS: The MAPK pathway is constantly activated in PLCH lesions. Apart from the BRAFV600E mutation, other driver somatic genomic alterations in this pathway (mainly MAP2K1  mutations/deletions and BRAF deletions) have been identified in the lesions, paving the way for targeted treatment. Smoking appears to promote the recruitment of MAPK-activated circulating myeloid precursors to the lung. The long-term survival of PLCH is more favourable with a 10-year survival >90%. Lung cancer and chronic respiratory failure are the main causes of death. Few patients develop severe pulmonary complications within the 5 years after diagnosis, justifying a close longitudinal follow-up of the patients. SUMMARY: PLCH is a MAPK driven neoplasia with inflammatory properties. The place of targeted therapies in severe forms of PLCH warrants further evaluation.

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.

Paraneoplastic pemphigus uncovers distinct clinical and biological phenotypes of western unicentric Castleman disease.

Unicentric Castleman disease (UCD) is a lymphoproliferative disease of unknown cause. Paraneoplastic pemphigus (PNP) is a major complication shown to be associated with a poor prognosis, with particular severity in patients with bronchiolitis obliterans (BO). This study describes the clinical and biological characteristics of UCD-PNP patients in a large Western cohort. A total of 148 patients diagnosed with UCD were identified, including 14 patients with a defined PNP. PNP was significantly associated with myasthenia gravis (MG) and FDC sarcoma during follow-up (FDCS). PNP was also significantly associated with reduced survival. These data, together with a multivariate analysis by principal components, led to the identification of UCD-PNP as a group at risk of MG, FDCS and death. PDGFRB sequencing performed on UCD lesions from six patients found the gain-of-function p.N666S variant in two. Interestingly, both patients had hyaline-vascular UCD subtype, were in the UCD-PNP subgroup and had FDCS. Sera from 25 UCD-PNP patients and 6 PNP patients without UCD were tested for PNP-associated autoantibodies. Sera from UCD-PNP patients had a strong reactivity against the N-terminal domain of recombinant periplakin (rPPL, 82%) and showed reactivity against at least two domains of rPPL. These features were not found in patients with UCD alone or in the PNP group without UCD. These data indicate that UCD-PNP patients belong to a subgroup sharing strong clinical and biological identity that might help to decipher the different dynamics of UCD natural history.

Recurrent PAK2 rearrangements in poroma with folliculo-sebaceous differentiation.

AIMS: Poroma is a benign adnexal neoplasm with differentiation towards the upper portion of the sweat gland apparatus. In 2019, Sekine et al. demonstrated recurrent YAP1::MAML2 and YAP1::NUTM1 fusion in poroma and porocarcinoma. Follicular, sebaceous and/or apocrine differentiation has been reported in rare cases of poroma and whether these tumours constitute a variant of poroma or represent a distinctive tumour is a matter to debate. Herein we describe the clinical, immunophenotypic, and molecular features of 13 cases of poroma with folliculo-sebaceous differentiation. METHODS AND RESULTS: Most of the tumours were located on the head and neck region (n = 7), and on the thigh (n = 3). All presented were adults with a slight male predilection. The median tumour size was 10 mm (range: 4-25). Microscopically, lesions displayed features of poroma with nodules of monotonous basophilic cells associated with a second population of larger eosinophilic cells. In all cases, ducts and scattered sebocytes were identified. Infundibular cysts were present in 10 cases. In two cases high mitotic activity was noted, and in three cases cytologic atypia and areas of necrosis were identified. Whole transcriptome RNA sequencing demonstrated in-frame fusion transcripts involving RNF13::PAK2 (n = 4), EPHB3::PAK2 (n = 2), DLG1::PAK2 (n = 2), LRIG1::PAK2 (n = 1), ATP1B3::PAK2 (n = 1), TM9SF4::PAK2 (n = 1), and CTNNA1::PAK2 (n = 1). Moreover, fluorescence in situ hybridisation (FISH) analysis revealed PAK2 rearrangement in an additional case. No YAP1::MAML2 or YAP1::NUTM1 fusion was detected. CONCLUSION: Recurrent fusions involving the PAK2 gene in all analysed poroma with folliculo-sebaceous differentiation in this study confirms that this neoplasm represents a separate tumour entity distinct from YAP1::MAML2 or YAP1::NUTM1 rearranged poromas.

Dramatic Response After Switching MEK Inhibitors in a Patient With Refractory Mixed Histiocytosis.

We report the case of a patient with progressive multisystem mixed histiocytosis associating Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) involving the bone marrow, whose lesions harbored the MAP2K1 E102-I103del. After initial improvement under the MEK inhibitor trametinib, the treatment was only partially efficient and poorly tolerated. Eventually, although the trough blood level of trametinib at steady state was within expected ranges, the disease progressed to a life-threatening situation, with peritoneal involvement and anasarca. Switching to the MEK inhibitor cobimetinib as a salvage therapy resulted in a dramatic, rapid disease response, and the patient remains disease-free 3 years later with the treatment. The load of the MAP2K1 deletion in peripheral blood was correlated with the disease activity and strongly declined with cobimetinib, although it remained detectable at the last follow-up.

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.

The PI3K/mTOR Pathway Is Targeted by Rare Germline Variants in Patients with Both Melanoma and Renal Cell Carcinoma.

Background: Malignant melanoma and RCC have different embryonic origins, no common lifestyle risk factors but intriguingly share biological properties such as immune regulation and radioresistance. An excess risk of malignant melanoma is observed in RCC patients and vice versa. This bidirectional association is poorly understood, and hypothetic genetic co-susceptibility remains largely unexplored. Results: We hereby provide a clinical and genetic description of a series of 125 cases affected by both malignant melanoma and RCC. Clinical germline mutation testing identified a pathogenic variant in a melanoma and/or RCC predisposing gene in 17/125 cases (13.6%). This included mutually exclusive variants in MITF (p.E318K locus, N = 9 cases), BAP1 (N = 3), CDKN2A (N = 2), FLCN (N = 2), and PTEN (N = 1). A subset of 46 early-onset cases, without underlying germline variation, was whole-exome sequenced. In this series, thirteen genes were significantly enriched in mostly exclusive rare variants predicted to be deleterious, compared to 19,751 controls of similar ancestry. The observed variation mainly consisted of novel or low-frequency variants (<0.01%) within genes displaying strong evolutionary mutational constraints along the PI3K/mTOR pathway, including PIK3CD, NFRKB, EP300, MTOR, and related epigenetic modifier SETD2. The screening of independently processed germline exomes from The Cancer Genome Atlas confirmed an association with melanoma and RCC but not with cancers of established differing etiology such as lung cancers. Conclusions: Our study highlights that an exome-wide case-control enrichment approach may better characterize the rare variant-based missing heritability of multiple primary cancers. In our series, the co-occurrence of malignant melanoma and RCC was associated with germline variation in the PI3K/mTOR signaling cascade, with potential relevance for early diagnostic and clinical management.

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.

Mitogen-activating protein kinase pathway alterations in Langerhans cell histiocytosis.

PURPOSE OF REVIEW: Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the infiltration of involved tissues by specialized dendritic cells. The demonstration of the constant activation of the mitogen-activated protein kinase (MAPK) pathway in LCH lesions has been a breakthrough in the understanding of the pathogenesis of this rare disease. We will summarize the current knowledge on MAPK alterations in LCH and the new therapeutic options indicated by these findings. RECENT FINDINGS: Since the description of the B-Raf proto-oncogene, serine/threonine kinase (BRAF)V600E mutation in LCH lesions, several other molecular alterations affecting the MAPK pathway have been identified in most cases. Based on these driver alterations, LCH cells were shown to be derived from hematopoietic precursors, which yielded the current concept of LCH as a myeloid inflammatory neoplasia. MAPK pathway inhibitors have emerged as an innovative therapy in severe forms of LCH, resulting in virtually no acquired resistance. However, although they are highly effective, their effect is only temporary, as the disease relapses upon discontinuation of the treatment. SUMMARY: LCH is an inflammatory myeloid neoplastic disorder, driven by mutations activating the MAPK pathway. MAPK-targeted treatments represent an important stepforward in the management of patients with severe progressive LCH.

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.

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.