Molecularly matched targeted therapy: a promising approach for refractory metastatic melanoma.

BACKGROUND: Only a fraction of patients with metastatic melanoma derive durable benefit from approved treatments. The clinical impact of personalized medicine strategies for melanoma, apart from BRAF, NRAS, or CKIT targeting, has rarely been reported. MATERIALS AND METHODS: By means of the Group of Cutaneous Oncology of the French Society of Dermatology, we retrospectively included all patients with advanced melanoma aged 18 years and older for whom molecular testing identified one or more actionable molecular alterations and who accordingly received molecularly matched therapy. We excluded patients with only BRAF, NRAS, or CKIT alterations and patients who received molecularly matched therapy for less than 15 days. RESULTS: We included 26 patients with a median follow-up of 8 months (1-54), a median age of 63 years (24-89), and a sex ratio of 2.7. These patients had been heavily pretreated, and 64% had elevated LDH levels. The disease control rate was 38%, with 4 cases of partial response (overall response rate: 15%) and 6 of stable disease for at least 6 months. The median duration of treatment was 3.1 months (0.9-13.5). Among patients with disease control, the median duration of control was 6.6 months (2.6-13.5) and 3 cases were ongoing at the end of the study. Patients with controlled disease had GNA11, MAP2K1, FYCO1-RAF1, HRAS, ATM, CCND1, MDM2/CDK4, and CDKN2A/NRAS alterations. CONCLUSIONS: High-throughput sequencing followed by matched targeted therapy is a promising approach for patients with advanced melanoma refractory to approved treatments.

Combined Reverse-Transcriptase Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Analyses to Assign Unclassified BCL2-/BCL6- Nonrearranged Small B-Cell Lymphoid Neoplasms as Follicular or Nodal Marginal Zone Lymphoma.

Distinguishing between follicular lymphoma (FL) and nodal marginal zone lymphoma (NMZL) can be difficult when morphologic and phenotypic features are unusual and characteristic cytogenetic rearrangements are absent. We evaluated the diagnostic contribution of ancillary techniques-including fluorescence in situ hybridization (FISH)-detected 1p36 deletion; reverse-transcriptase, multiplex, ligation-dependent probe amplification (RT-MLPA); and next-generation sequencing (NGS)-for tumors that remain unclassified according to standard criteria. After review, 50 CD5-negative small B-cell lymphoid neoplasms without BCL2 and BCL6 FISH rearrangements were diagnosed as FLs (n = 27), NMZLs (n = 5), or unclassified (n = 18) based on the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. FISH helped identify the 1p36 deletion in 3 FLs and 1 unclassified tumor. Most classified FLs had an RT-MLPA germinal center B-cell (GCB) signature (93%) or were noncontributive (7%). Classified NMZLs had an RT-MLPA activated B-cell signature (20%), had an unassigned signature (40%), or were noncontributive (40%). Among unclassified tumors, the RT-MLPA GCB signature was associated with mutations most commonly found in FLs (CREBBP, EZH2, STAT6, and/or TNFRSF14) (90%). An RT-MLPA-detected GCB signature and/or NGS-detected gene mutations were considered as FL identifiers for 13 tumors. An activated B-cell signature or NOTCH2 mutation supported NMZL diagnosis in 3 tumors. Combining the RT-MLPA and NGS findings successfully discriminated 89% of unclassified tumors in favor of one or the other diagnosis. NGS-detected mutations may be of therapeutic interest. Herein, we detected 3 EZH2 and 8 CREBBP mutations that might be eligible for targeted therapies.

A High Programmed Cell Death Protein 1 Hormone Receptor Score on Skin Biopsy is Associated with Sézary Syndrome Diagnosis: A Study of 91 Patients with Erythroderma.

Erythroderma is challenging to diagnose. The aim of this single-centre retrospective study was to identify factors that can be used to improve the diagnosis of erythroderma. Among 91 patients with erythroderma, 21 were diagnosed with eczema, 17 with psoriasis, 20 with drug-induced erythroderma, 13 with erythrodermic mycosis fungoides and 20 with Sézary syndrome. Nail alterations, ear involvement, and severe scaling were significantly associated with psoriasis (p = 0.044). Fever and hypereosinophilia were associated with drug-induced erythroderma. Expression of programmed cell death protein 1 was observed in all skin biopsies. However, with Sézary syndrome, programmed cell death protein 1 expression was significantly higher than with other aetiologies. A programmed cell death protein 1 hormone receptor score (H-score) >50 was associated with Sézary syndrome (p < 0.001, sensitivity 75%, specificity 92%) as well as CXCL13 expression (p < 0.044). CD7 loss was more frequent with erythrodermic mycosis fungoides and Sézary syndrome (p = 0.022). This study reports the importance of programmed cell death protein 1 expression for the differential diagnosis of Sézary syndrome and other aetiologies, including erythrodermic mycosis fungoides.

Integrative diagnosis of primary cutaneous large B-cell lymphomas supports the relevance of cell of origin profiling.

Primary cutaneous large B-cell lymphomas (PCLBCL) represent a diagnostic challenge because they are classified as PCLBCL, leg type (PCLBCL, LT) or primary cutaneous follicle centre lymphoma, large cell (PCFCL, LC), which differ by prognosis and therapeutic requirement. Unclassified cases with discordant clinical presentations, morphologies, and immunophenotypes may be classified into the not otherwise specified (PCLBCL, NOS) category based on ancillary molecular analyses. Cell-of-origin profiling as germinal centre (GC) type or non-GC type by immunohistochemistry is not considered reproducible because of variable CD10 expression. In a series of 55 PCLBCL cases with > 80% large cells, we reported 21 PCFCL, LC cases as GC-type and 27 PCLBCL, LT as non-GC-type; 7 cases were considered PCLBCL, NOS. Here, we demonstrate the accuracy of molecular profiling of PCLBCL as GC or non-GC type using a reverse transcriptase multiplex ligation assay (RT-MLPA). RT-MLPA classified the seven PCLBCL, NOS cases in accordance with their mutational profile. An integrative principal component analysis confirmed the main criteria and the relevance of genomic profiling of PCFCL, LC as GC-derived, and PCLBCL, LT as non-GC-derived. Both the cell-of-origin classification of PCLBCL and the integrative analysis identified two clinically relevant subgroups according to overall survival, which may help to standardize PCLBCL diagnosis and patient management.

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas.

The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

Xenograft and cell culture models of Sézary syndrome reveal cell of origin diversity and subclonal heterogeneity.

Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.

The Need for a Consensus Next-generation Sequencing Panel for Mature Lymphoid Malignancies.

Supplemental Digital Content is available in the text.

Mutations of the B-Cell Receptor Pathway Confer Chemoresistance in Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

Clinical, histological and molecular predictors of metastatic melanoma responses to anti-PD-1 immunotherapy.

BACKGROUND: Prescribing anti-programmed death-1 (PD-1) immunotherapy for advanced melanoma is currently not restricted by any biomarker assessment. Determination of programmed death-ligand-1 (PD-L1)-expression status is technically challenging and is not mandatory, because negative tumours also achieve therapeutic responses. However, reproducible biomarkers predictive of a response to anti-PD-1 therapy could contribute to improving therapeutic decision-making. METHODS: This retrospective study on 70 metastatic melanoma patients was undertaken to evaluate the relationships between clinical, histological, immunohistochemical and/or molecular criteria, and the 6-month objective response rate. RESULTS: Better objective response rates were associated with metachronous metastases (P = 0.04), PD-L1 tumour- and/or immune-cell status (P = 0.01), CD163+ histiocytes at advancing edges (P = 0.009) of primary melanomas and NRAS mutation (P = 0.019). Moreover, CD163+ histiocytes at advancing edges (P = 0.04) were associated with longer progression-free survival (PFS), and metachronous metastases with longer overall survival (P = 0.02) and PFS (P = 0.049). CONCLUSIONS: Combining these reproducible biomarkers could help improve therapeutic decision-making for patients with progressive disease.

Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.

In nodal diffuse large B-cell lymphoma, the search for double-hit with MYC and BCL2 and/or BCL6 rearrangements or for dual expression of BCL2 and MYC defines subgroups of patients with altered prognosis that has not been evaluated in primary cutaneous large B-cell lymphoma. Our objectives were to assess the double-hit and dual expressor status in a cohort of 44 patients with primary cutaneous large B-cell lymphoma according to the histological subtype and to evaluate their prognosis relevance. The 44 cases defined by the presence of more than 80% of large B-cells in the dermis corresponded to 21 primary cutaneous follicle centre lymphoma with large cell morphology and 23 primary cutaneous diffuse large B-cell lymphoma, leg type. Thirty-one cases (70%) expressed BCL2 and 29 (66%) expressed MYC. Dual expressor profile was observed in 25 cases (57%) of either subtypes (n = 6 or n = 19, respectively). Only one primary cutaneous follicle centre lymphoma, large-cell case had a double-hit status (2%). Specific survival was significantly worse in primary cutaneous diffuse large B-cell lymphoma, leg type than in primary cutaneous follicle centre lymphoma, large cell (p = 0.021) and for the dual expressor primary cutaneous large B-cell lymphoma group (p = 0.030). Both overall survival and specific survival were worse for patients belonging to the dual expressor primary cutaneous diffuse large B-cell lymphoma, leg type subgroup (p = 0.001 and p = 0.046, respectively). Expression of either MYC and/or BCL2 negatively impacted overall survival (p = 0.017 and p = 0.018 respectively). As the differential diagnosis between primary cutaneous follicle centre lymphoma, large cell and primary cutaneous diffuse large B-cell lymphoma, leg type has a major impact on prognosis, dual-expression of BCL2 and MYC may represent a new diagnostic criterion for primary cutaneous diffuse large B-cell lymphoma, leg type subtype and further identifies patients with impaired survival. Finally, the double-hit assessment does not appear clinically relevant in primary cutaneous large B-cell lymphoma.

A Single-Arm Phase II Trial of Lenalidomide in Relapsing or Refractory Primary Cutaneous Large B-Cell Lymphoma, Leg Type.

Although the combination of rituximab and polychemotherapy has improved prognosis of primary cutaneous diffuse large B-cell lymphoma, leg type, the advanced age of patients limits therapeutic options in relapsing/refractory cases. A multicenter, single-arm, phase II trial was conducted to assess the benefits and safety of lenalidomide in refractory/relapsing primary cutaneous diffuse large B-cell lymphoma, leg type. The primary endpoint was the 6-month overall response rate. Secondary endpoints were 12-month overall response rate, overall and specific survival, duration of response, progression-free survival, safety, and identification of prognostic factors. Among the 19 patients included, the 6-month overall response rate was 26.3% (90% confidence interval [CI] = 11-47.6), including four complete responses and one partial response. At 12 months, there were still two complete responses and one partial response. Median progression-free survival was 4 months. Median overall and specific survivals were 19.4 and 23.8 months, respectively. Reduced doses tended to be associated with higher 6-month overall response rate and progression-free survival. Absence of the MYD88L265P mutation was associated with a higher overall response under treatment (80.0% vs. 33.3%; P = 0.05). The most common grade 3 adverse events were hematologic. Two grade 5 adverse events occurred (sepsis and pulmonary embolism). Lenalidomide at reduced doses may allow prolonged responses in a few patients and represents a therapeutic option in relapsing/refractory primary cutaneous diffuse large B-cell lymphoma, leg type.

Assessment of BRAFV600E mutation in pulmonary Langerhans cell histiocytosis in tissue biopsies and bronchoalveolar lavages by droplet digital polymerase chain reaction.

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF V600E and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAFV600E detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS). The presence of BRAFV600E mutation was assessed by at least two concordant techniques to further evaluate specificity and sensitivity of each method. The BRAF V600E mutation prevalence was detected in 18 out of 41 cases by ddPCR, 10 out of 36 cases by HRM PCR, and 16 out of 31 cases by NGS. BRAFV600E immunohistochemistry sensitivity was 94%, and specificity was 79%. HRM PCR sensitivity was only 59%, and specificity was 100%. NGS sensitivity and specificity were 100% for interpretable cases (n = 31), but in 11 cases, this technique was non-contributive. The analysis of BAL samples by ddPCR revealed a BRAFV600E mutation both in tissue and in BAL samples in one patient, a wild-type status both in tissue and in BAL samples in two patients, and a wild-type BRAF status in BAL and a BRAFV600E mutation in tissue samples in four patients. The study supports the usefulness of ddPCR for BRAF status assessment in either tissue or BAL samples to increase the accuracy of PLCH diagnosis.

TP53 alterations in primary and secondary Sézary syndrome: A diagnostic tool for the assessment of malignancy in patients with erythroderma.

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene.

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

Challenging a dogma: co-mutations exist in MAPK pathway genes in colorectal cancer.

Sequencing of genes encoding mitogen-activated protein kinase (MAPK) pathway proteins in colorectal cancer (CRC) has established as dogma that of the genes in a pathway only a single one is ever mutated. We searched for cases with a mutation in more than one MAPK pathway gene (co-mutations). Tumor tissue samples of all patients presenting with CRC, and referred between 01/01/2008 and 01/06/2015 to three French cancer centers for determination of mutation status of RAS/RAF+/-PIK3CA, were retrospectively screened for co-mutations using Sanger sequencing or next-generation sequencing. We found that of 1791 colorectal patients with mutations in the MAPK pathway, 20 had a co-mutation, 8 of KRAS/NRAS, and some even with a third mutation. More than half of the mutations were in codons 12 and 13. We also found 3 cases with a co-mutation of NRAS/BRAF and 9 with a co-mutation of KRAS/BRAF. In 2 patients with a co-mutation of KRAS/NRAS, the co-mutation existed in the primary as well as in a metastasis, which suggests that co-mutations occur early during carcinogenesis and are maintained when a tumor disseminates. We conclude that co-mutations exist in the MAPK genes but with low frequency and as yet with unknown outcome implications.

Spleen Histologic Appearance in Common Variable Immunodeficiency: Analysis of 17 Cases.

Histologic and phenotypic analyses of splenectomy samples from 17 patients with common variable immunodeficiency (CVID) showed the following nonspecific, evocative, white-pulp lesions: white-pulp hyperplasia (WPH) with reactive follicles, giant follicles (GFs), marginal zone hyperplasia, periarteriolar T-zone hyperplasia (PATH) and/or granulomas, which enabled us to discern 2 groups: the first (n=6) composed of WPH with reactive follicles without granulomas, and the second (n=9) characterized by the presence of granulomas with or without WPH. All specimens were Epstein-Barr virus negative by in situ hybridization. Molecular analyses revealed a polyclonal immunoglobulin heavy chain gene (IGH) rearrangement (n=12). WPH-only patients were mostly female individuals and younger at CVID onset, diagnosis, and splenectomy, but their interval between the first symptom and diagnosis was longer; they had more associated infectious events, autoimmune disease, pulmonary complications, and liver regenerative nodular hyperplasia; their IgG, IgA, and IgM concentrations were also higher. Granuloma-group patients were mostly male individuals; were older at CVID onset, diagnosis, and splenectomy; had disseminated granulomatous disease, but infectious events, autoimmune disease, pulmonary complications, and liver regenerative nodular hyperplasia were less common; their immunoglobulin concentrations were lower. Histologic comparisons between the WPH-only and granuloma groups showed more intense WPH and more intense marginal zone hyperplasia and fewer GFs in the former versus more developed PATH and more common GFs in the latter. The results of this novel comparative study of the histologic patterns of 17 CVID patients' evocative splenic lesions suggested different biological and clinical profiles.