Subungual melanoma: molecular analysis of 31 cases from early stage to invasive melanoma.

AIMS: The distinction between the benign subungual melanocytic lesions and an early lesion of subungual melanoma (SUM) remains a diagnostic challenge. We evaluated the routine diagnostic utility of array Comparative Genomic Hybridization (aCGH) to detect whole-genome copy number variations (CNV) as well as targeted next-generation sequencing (NGS) in SUM. METHODS AND RESULTS: This retrospective study included 20 cases of in situ SUM and 11 cases of invasive SUM. Analysis by aCGH detected common oncogene amplifications in all but one case of invasive SUM (n = 10) and in all cases of in situ SUM with a melanocyte count (MC) >45/mm (n = 4 true positive) and the average number of CNV was 8.5. Thirteen remaining cases of in situ SUM gave false negative results (n = 13), owing to a lack of sufficient melanocytes to analyse (median MC of 35.35; range: 10.16-39.5). Molecular analysis failed in four cases (three in situ SUM and one invasive SUM) due to insufficient amounts of DNA. Across the whole cohort, the sensitivity of aCGH was 52%, but when adjusting the cutoff to MC >45/mm, the sensitivity was 93%. Targeted NGS was less informative than aCGH analyses in our series of SUM. CONCLUSION: To distinguish malignant from benign lesions, especially in situ SUM versus atypical lentiginous melanocytic proliferations, aCGH analysis should be performed when the MC is above 45 melanocytes per linear millimetre. This pangenomic method can detect oncogene amplifications, as well as a number of CNV >3, which strongly support the diagnosis of malignancy.

Molecular relapse after first-line intensive therapy in patients with CBF or NPM1-mutated acute myeloid leukemia - a FILO study.

Patients with Core-Binding Factor (CBF) and NPM1-mutated acute myeloid leukemia (AML) can be monitored by quantitative PCR after having achieved first complete remission (CR) to detect morphologic relapse and drive preemptive therapy. How to best manage these patients is unknown. We retrospectively analyzed 303 patients with CBF and NPM1-mutated AML, aged 18-60 years, without allogeneic hematopoietic cell transplantation (HCT) in first CR, with molecular monitoring after first-line intensive therapy. Among these patients, 153 (51%) never relapsed, 95 (31%) had molecular relapse (53 received preemptive therapy and 42 progressed to morphologic relapse at salvage therapy), and 55 (18%) had upfront morphologic relapse. Patients who received preemptive therapy had higher OS than those who received salvage therapy after having progressed from molecular to morphologic relapse and those with upfront morphologic relapse (three-year OS: 78% vs. 51% vs. 51%, respectively, P = 0.01). Preemptive therapy included upfront allogeneic HCT (n = 19), intensive chemotherapy (n = 21), and non-intensive therapy (n = 13; three-year OS: 92% vs. 79% vs. 58%, respectively, P = 0.09). Although not definitive due to the non-randomized allocation of patients to different treatment strategies at relapse, our study suggests that molecular monitoring should be considered during follow-up to start preemptive therapy before overt morphologic relapse.

A 78-year-old woman with diffuse white matter infiltration and predominant involvement of bilateral temporo-parieto-occipital regions.

Diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (H3/IDH-wt-pHGG) is a newly defined entity amongst brain tumours, primarily reported in children. It is a rare, ill-defined type of tumour and the only method to diagnose it is DNA methylation profiling. The case we report here carries new knowledge about this tumour which may, in fact, occur in elderly patients, be devoid of evocative genomic abnormalities reported in children and harbour a misleading mutation.

Poor prognosis of SRSF2 gene mutations in patients treated with VEN-AZA for newly diagnosed acute myeloid leukemia.

Mutations in spliceosome genes (SRSF2, SF3B1, U2AF1, ZRSR2) correlate with inferior outcomes in patients treated with intensive chemotherapy for Acute Myeloid Leukemia. However, their prognostic impact in patients treated with less intensive protocols is not well known. This study aimed to evaluate the impact of Spliceosome mutations in patients treated with Venetoclax and Azacitidine for newly diagnosed AML. 117 patients treated in 3 different hospitals were included in the analysis. 34 harbored a mutation in at least one of the spliceosome genes (splice-mut cohort). K/NRAS mutations were more frequent in the splice-mut cohort (47% vs 19%, p=0.0022). Response rates did not differ between splice-mut and splice-wt cohorts. With a median follow-up of 15 months, splice mutations were associated with a lower 18-month LFS (p=0.0045). When analyzing splice mutations separately, we found SRSF2 mutations to be associated with poorer outcomes (p=0.034 and p=0.037 for OS and LFS respectively). This negative prognostic impact remained true in our multivariate analysis. We believe this finding should warrant further studies aimed at overcoming this negative impact.

Comparison of clinical outcomes of several risk stratification tools in newly diagnosed AML patients: A real-world evidence in our current therapeutic era.

BACKGROUND OF THE STUDY: AML classification tools have been developed to stratify the risk at AML diagnosis. There is a need to evaluate these tools in the current therapeutic era. COHORT CHARACTERISTICS: In this retrospective study, we compared five classifiers: ELN 2017, ELN 2022, ALFA classifier, Papaemmanuil et al. classifier, and Lindsley et al. classifier, in a real-life cohort of 281 patients newly diagnosed for AML in Nice University Hospital. In our cohort median age was 68 years old, sex ratio was M/F 56%/44%, performance status was lower than 2 in 73.1% of patients, AML subtype was "De novo" in 71.5%, "secondary" in 22.4%, and "therapy-related" in 6.0% of patients. Intensive chemotherapy was used in 53.0% of patients, and non-intensive chemotherapy in 40.6% of patients. Molecular analysis was available in a large majority of patients and the main mutations found were NPM1 (22.7%), DNMT3A (17.4%), TP53 (13.1%), TET2 (12.4%), and FLT3-ITD (12.4%). RESULTS: In our findings, the comparison of overall survival between the three prognostic groups in the global cohort was statistically significant in all classifiers: ELN 2017 p < 0.0001, ELN 2022 p < 0.0001, ALFA classifier p < 0.0001, Papaemmanuil classifier p < 0.0001, Lindsley classifier p = 0.001. ELN 2017, ELN 2022, ALFA classifier, Papaemmanuil classifier, and Lindsley classifier were calculated respectively in 99%, 99%, 89%, 90%, and 89% of patients. CONCLUSIONS: Using Akaike's information criteria (AIC) to compare all five classifiers, ELN 2022 is the best classifier into younger and older patients and for prognosis.

Vanished MDM2 amplification in multiple recurrences of an irradiated poorly differentiated sarcoma with amplified TRIO::TERT fusion gene.

Among sarcomas, MDM2 amplification is usually a molecular hallmark of well-differentiated liposarcoma and dedifferentiated liposarcoma (DDLPS) and occasionally a secondary genetic anomaly in other sarcomas. Histological evaluation and FISH analysis showing MDM2 amplification led to the diagnosis of DDLPS for a tumor located on the left arm of a 71-year-old patient. The patient was treated by adjuvant radiotherapy (RT) but the tumor recurred soon after. Array-comparative genomic hybridization and targeted RNA/DNA sequencing of the primary tumor and of four recurrences were done. Strikingly, the MDM2 amplification observed in the primary tumor had vanished in the recurrences. In contrast, other rearrangements, such as amplification of the genes TRIO and TERT as well as TRIO::TERT fusion were detected retrospectively in the primary tumor and in all the recurrences. The transitory nature of the MDM2 amplification raised the hypothesis that RT was active on cells that contained MDM2 amplification but not on other tumor cells with only the TERT and TRIO alterations. In contrast to MDM2 amplification, the TRIO::TERT amplified fusion was stable over time. The detection of this fusion was crucial in the analysis of the diagnostically challenging last tumor; it allowed to determine that it was a fourth recurrence, instead of a new independent tumor. It also suggested the diagnosis undifferentiated pleomorphic sarcoma rather than DDLPS. The TRIO::TERT fusion is not well explored. The current study shows that its role in sarcomas, with or without MDM2 amplification, should be more extensively researched.

BRCA1/2 Pathogenic Variants Are Not Common in Merkel Cell Carcinoma: Comprehensive Molecular Study of 30 Cases and Meta-Analysis of the Literature.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine cancer. Management of advanced MCC is mainly based on immune-checkpoint inhibitors. The high failure rate warrants an investigation of new therapeutic targets. The recent identification of BRCA1 or BRCA2 (BRCA1/2) mutations in some MCC raises the issue of the use of poly-(ADP-Ribose)-polymerase inhibitors in selected advanced cases. The main objective of our study is to determine the accurate frequency of BRCA1/2 pathogenic variants. We studied a series of 30 MCC and performed a meta-analysis of BRCA1/2 variants of published cases in the literature. In our series, we detected only one BRCA2 pathogenic variant. The low frequency of BRCA1/2 pathogenic variants in our series of MCC (3%) was confirmed by the meta-analysis of BRCA1/2 variants in the literature. Among the 915 MCC from 13 published series studied for molecular alterations of BRCA1/2, only 12 BRCA1/2 pathogenic mutations were identified (1-2% of MCC), whereas many other BRCA1/2 variants were variants of unknown significance or benign. BRCA1/2 pathogenic variants are uncommon in MCC. However, in BRCA-mutated MCC, poly-(ADP-Ribose)-polymerase inhibitors might be a valuable therapeutic option requiring validation by clinical trials.

Onychomatricoma: a clinicopathological, immunohistochemical, and molecular study of 10 cases highlighting recurrent RB1 deletion and the potential diagnostic value of LEF-1.

AIMS: Onychomatricoma (OM), an uncommon benign fibroepithelial neoplasm of the nail unit, is sometimes diagnostically challenging for clinicians and pathologists. OM consistently expresses CD34, but no specific immunohistohemical markers or recurrent genetic alterations have been identified to date. Recent studies have suggested that Wnt signalling is a key molecular characteristic of OM. METHODS AND RESULTS: Ten cases were analysed: four classical OM including two with pleomorphic cells; two superficial acral fibromyxoma-like variants of OM; three micropapilliferum variants of OM including one with pleomorphic cells; and one proliferating variant of OM. Immunohistochemically, the spindle cells were positive with CD34 (n = 10) and CD99 (n = 1), with focal reactivity for CD10 (n = 5). The epithelial component of the tumours expressed immunopositivity for LEF-1. Using array comparative genomic hybridization (aCGH), we demonstrated that all OM, including its variants that were tested (n = 8), harboured a few copy number alterations with losses of whole or part of chromosome 13 including the RB1 gene (n = 8) and chromosome 16 (n = 6). CONCLUSION: We report a recurrent loss of RB1 (13q) as a possible driver molecular event in OM indicating a relationship between OM and other lesions of the spectrum of the so-called '13q/RB1' family of tumours. We did not identify a role for the Wnt/beta-catenin signalling pathway, as has been proposed in a recent study. LEF-1 could be a potential sensitive and specific marker of OM and should be used in the differential diagnosis between OM, superficial acral fibromyxoma, and the CD34-positive fibrosing family of tumours.

Pembrolizumab combined with low-dose cyclophosphamide and intra-tumoral injection of the toll-like receptor 4 agonist G100 in patients with advanced pretreated soft tissue sarcoma: results from the PEMBROSARC basket study.

Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors with limited therapeutic options in the advanced setting. Immune checkpoint inhibitors have been shown to have significant clinical activity in inflamed STS which are characterized by the presence of tertiary lymphoid structures (TLS). New strategies are needed to sensitize TLS-negative STS to immunotherapy. Engagement of the toll-Like Receptor 4 (TLR4) signal pathway contributes to the development of a favorable tumor microenvironment in solid tumors. G100 is a highly potent toll-like receptor 4 (TLR4) agonist. We hypothesized that intra-tumoral G100 would induce a robust local and potentially systemic anti-tumor immune response in the microenvironment of TLS-negative sarcoma, leading to improved response to PD1 inhibition. Twenty metastatic STS patients who had a superficial injectable lesion were treated with 50 mg of cyclophosphamide (CP) orally twice daily (1 week on and 1 week off), 200 mg of pembrolizumab intravenously on day 8 of a planned 21-day cycle and G100 20 µg one weekly intra-tumoral injection for at least 6 weeks and for a maximum of 12 weeks (1st injection one week before CP administration, ie. Day -7). Biopsies and blood were collected pre and post treatment. Of the 17 patients assessable for efficacy analysis, 2 were progression-free at 6 months, and the 6-month non-progression rate was 11.8% (95% CI: 1.5-36.4), indicating that the first endpoint of the study was not reached. In 8 patients, there was an increase in T-cell infiltration into tumor after treatment. The ratio CD8/Fox-P3 + CD4 on treatment decreased in 11 cases out of 14 suggesting a predominant induction of Treg. Soluble PDL1 levels at baseline were also with adverse outcome. G100 appears to modulate the tumor microenvironment with significant infiltration of T cells. However, clinical activity in combination with PD1 inhibition was limited and no clear correlation was observed between tumor shrinkage and increased inflammation. TLR4 stimulation might have both antitumor and pro-tumor consequences.Trial registration: This study was registered with ClinicalTrial.gov, number NCT02406781.

AGAP3: A novel BRAF fusion partner in pediatric pancreatic-type acinar cell carcinoma.

Most available molecular data on pancreatic acinar cell carcinoma (PACC) are provided by studies of adult cases. BRAF, RAF1, or RET rearrangements have been described in approximately 30% of cases. To the best of our knowledge, only seven cases with molecular data have been reported in pediatric PACC. We report here the comprehensive study of a pancreatic-type ACC from a 6-year-old patient. We detected an AGAP3::BRAF fusion. This result showing a BRAF rearrangement demonstrates a molecular link between adult and pediatric PACC. Moreover, it identifies AGAP3, a gene located at 7q36.1 that encodes a major component of the N-methyl-d-aspartate (NMDA) receptor signaling complex, as a partner gene of BRAF. The variability of BRAF partners is consistent with a driver role of BRAF alterations in PACC. The identification of such alterations is noteworthy for considering the use of MEK inhibitors in metastatic cases. We did not detect associated genomic instability. The better outcome of pediatric cases might be related to their stable genomic background.

CLEC12B Decreases Melanoma Proliferation by Repressing Signal Transducer and Activator of Transcription 3.

The potential role of CLEC12B, a gene predominantly expressed by skin melanocytes discovered through transcriptomic analysis, in melanoma is unknown. In this study, we show that CLEC12B expression is lower in melanoma and melanoma metastases than in melanocytes and benign melanocytic lesions and that its decrease correlates with poor prognosis. We further show that CLEC12B recruits SHP2 phosphatase through its immunoreceptor tyrosine-based inhibition motif domain, inactivates signal transducer and activator of transcription 1/3/5, increases p53/p21/p27 expression/activity, and modulates melanoma cell proliferation. The growth of human melanoma cells overexpressing CLEC12B in nude mice after subcutaneous injection is significantly decreased compared with that in the vehicle control group and is associated with decreased signal transducer and activator of transcription 3 phosphorylation and increased p53 levels in the tumors. Reducing the level of CLEC12B had the opposite effect. We show that CLEC12B represses the activation of the signal transducer and activator of transcription pathway and negatively regulates the cell cycle, providing a proliferative asset to melanoma cells.

RBM10-TFE3 fusions: A FISH-concealed anomaly in adult renal cell carcinomas displaying a variety of morphological and genomic features: Comprehensive study of six novel cases.

The accurate diagnosis of Xp11-translocation renal cell carcinoma (RCC) in adults is challenging. TFE3 (located on chromosome X) fuses with a partner gene generally located on another chromosome. In rare cases TFE3 may fuse with a neighboring gene: RBM10. Because TFE3 false-positive immunostaining is a common pitfall in many laboratories, demonstration of the chromosomal rearrangement is required in order to ascertain the diagnosis. Fluorescence in situ hybridization (FISH)-that has been considered as the gold standard method-reaches its limits for detecting small Xp11 paracentric inversions. We performed a comprehensive clinical, histological and genomic study of six novel cases of RCC with RBM10-TFE3 fusion. Using FISH, TFE3 rearrangement was equivocal in one case and negative in others. RBM10-TFE3 fusion was discovered using targeted RNA sequencing (RNASeq). As all the previously reported cases (mean age: 50), the six patients were adults (mean age: 42), suggesting an epidemiologic difference between RBM10-TFE3 RCC and tumors harboring some other partner genes, such as ASPSCR1 that rather occur in children. Array-comparative genomic hybridization showed several alterations, notably a gain of 17q in four cases with papillary features and loss of 3p in one case with clear cells. Our study demonstrates that, though rare among adult cases of RCC, RBM10-TFE3 fusion is not exceptional and warrants appropriate molecular detection. Notably, it would be worthy to systemically investigate by RNASeq challenging RCC with type-2 papillary features and 17q gain.

High throughput profiling of undifferentiated pleomorphic sarcomas identifies two main subgroups with distinct immune profile, clinical outcome and sensitivity to targeted therapies.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) is the most frequent, aggressive and less-characterized sarcoma subtype. This study aims to assess UPS molecular characteristics and identify specific therapeutic targets. METHODS: High-throughput technologies encompassing immunohistochemistry, RNA-sequencing, whole exome-sequencing, mass spectrometry, as well as radiomics were used to characterize three independent cohorts of 110, 25 and 41 UPS selected after histological review performed by an expert pathologist. Correlations were made with clinical outcome. Cell lines and xenografts were derived from human samples for functional experiments. FINDINGS: CD8 positive cell density was independently associated with metastatic behavior and prognosis. RNA-sequencing identified two main groups: the group A, enriched in genes involved in development and stemness, including FGFR2, and the group B, strongly enriched in genes involved in immunity. Immune infiltrate patterns on tumor samples were highly predictive of gene expression classification, leading to call the group B 'immune-high' and the group A 'immune-low'. This molecular classification and its prognostic impact were confirmed on an independent cohort of UPS from TCGA. Copy numbers alterations were significantly more frequent in immune-low UPS. Proteomic analysis identified two main proteomic groups that highly correlated with the two main transcriptomic groups. A set of nine radiomic features from conventional MRI sequences provided the basis for a radiomics signature that could select immune-high UPS on their pre-therapeutic imaging. Finally, in vitro and in vivo anti-tumor activity of FGFR inhibitor JNJ-42756493 was selectively shown in cell lines and patient-derived xenograft models derived from immune-low UPS. INTERPRETATION: Two main disease entities of UPS, with distinct immune phenotypes, prognosis, molecular features and MRI textures, as well as differential sensitivity to specific anticancer agents were identified. Immune-high UPS may be the best candidates for immune checkpoint inhibitors, whereas this study provides rational for assessing FGFR inhibition in immune-low UPS. FUNDING: This work was partly founded by a grant from La Ligue.

Novel Therapeutic Insights in Dedifferentiated Liposarcoma: A Role for FGFR and MDM2 Dual Targeting.

We aimed to evaluate the therapeutic potential of the pan-FGFR inhibitor erdafitinib to treat dedifferentiated liposarcoma (DDLPS). FGFR expression and their prognostic value were assessed in a series of 694 samples of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). The effect of erdafitinib-alone or in combination with other antagonists-on tumorigenicity was evaluated in vitro and in vivo. We detected overexpression of FGFR1 and/or FGFR4 in a subset of WDLPS and DDLPS and demonstrated correlation of this expression with poor prognosis. Erdafitinib treatment reduced cell viability, inducing apoptosis and strong inhibition of the ERK1/2 pathway. Combining erdafitinib with the MDM2 antagonist RG7388 exerted a synergistic effect on viability, apoptosis, and clonogenicity in one WDLPS and two DDLPS cell lines. Efficacy of this combination was confirmed in vivo on a DDLPS xenograft. Importantly, we report the efficacy of erdafitinib in one patient with refractory DDLPS showing disease stabilization for 12 weeks. We provide evidence that the FGFR pathway has therapeutic potential for a subset of DDLPS and that an FGFR1/FGFR4 expression might constitute a powerful biomarker to select patients for FGFR inhibitor clinical trials. In addition, we show that combining erdafitinib with RG7388 is a promising strategy for patients with DDLPS that deserves further investigation in the clinical setting.

Detection of tetraploidization in chromophobe renal cell carcinoma: Insights and pitfalls.

Chromosomal losses resulting in a marked hypodiploidy are a specificity of chromophobe renal cell carcinoma (ChRCC), the third most frequent type of kidney cancer. Its detection is useful in challenging cases. However some ChRCC, especially the eosinophilic variant, do not exhibit hypodiploidy and deserve to be better explored. Using comparative genomic hybridization (array-CGH) we observed chromosomal gains in five cases of nonmetastatic ChRCC. Our objective was to determine whether these apparent chromosomal gains were instead losses within a near-polyploid genome. We performed a retrospective and prospective molecular study of 26 cases of ChRCC retrieved among 643 renal tumors (2012-2019). All tumors were analyzed using array-CGH (Agilent) and array-CGH (Affymetrix) coupled to single nucleotide polymorphism analysis (array-SNP). In silico manual centralization of the fluorescence ratio, fluorescence in situ hybridization (FISH) and next generation sequencing were made in the five cases suspected of polyploidy. Tetraploidization was observed in 19% of our series of ChRCC. None of the methods used individually could identify both chromosomal losses and tetraploidy. Only the combination of manual recentring of array-CGH and FISH provided relevant results. B-allele frequency results indicated that tetraploidization occurred secondarily to chromosomal losses in four cases while it preceded losses in one case. Tetraploidization is a frequent but underestimated phenomenon in ChRCC that may be overlooked using the individual standard genomic methods. Its potential clinical consequences are not identified yet. Whether the mechanisms that induce chromosomal losses in ChRCC are the same that generate tetraploidization is not known.

MDM2 amplification and fusion gene ss18-ssx in a poorly differentiated synovial sarcoma: A rare but puzzling conjunction.

The detection of specific alterations by genetic analyses has been included in the diagnostic criterions of the World Health Organization's classification of soft tissues tumors since 2013. The presence of a SS18 rearrangement is pathognomonic of synovial sarcoma (SS). MDM2 amplification is strongly correlated to well-differentiated or dedifferentiated liposarcoma (DDLPS) in the context of sarcoma. We identified one case of poorly differentiated sarcoma harboring both SS18-SSX2 fusion and MDM2 amplification. The review of the literature showed high discrepancies, concerning the incidence of MDM2 amplification in SS: from 1.4% up to 40%. Our goal was to precisely determine the specific clinico-pathological features of this case and to estimate the frequency and characteristics of the association of SS18-SSX fusion/MDM2 amplification in sarcomas. We performed a retrospective and prospective study in 96 sarcomas, (56 SS and 40 DDLPS), using FISH and/or array-CGH to detect MDM2 amplification and SS18 rearrangement. None of the 96 cases presented both genetic alterations. Among the SS, only the index case (1/57: 1.7 %) presented the double anomaly. We concluded that MDM2 amplification in SS is a very rare event. The final diagnosis of the index case was a SS with SS18-SSX2 and MDM2 amplification as a secondary alteration. If the detection of MDM2 amplification is performed first in a poorly differentiated sarcoma, that may lead to not search other anomalies such as SS18 rearrangement and therefore to an erroneous diagnosis. This observation emphasizes the strong complementarity between histomorphology, immunohistochemistry and molecular studies in sarcoma diagnosis.