Predictive factors of response to vismodegib: a French study of 61 patients with multiple or locally advanced basal cell carcinoma

Background: Vismodegib is indicated for the treatment of advanced or metastatic basal cell carcinoma (BCC). The predictive factors of response to vismodegib have so far been poorly described. Objectives: The primary objective was to determine the profile of patients responding to vismodegib and the duration of response. Secondary objectives were to assess whether there is a correlation between the duration of treatment and the risk of relapse, and to define factors associated with relapse. Materials & Methods: We included 61 patients with locally advanced BCC (laBCC) or multiple BCC, treated with vismodegib (150 mg per day), from July 2011 to November 2015, in the Oncodermatology Department of Nantes University Hospital in France. Tumour response was assessed using Response Evaluation Criteria in Solid Tumours version 1.1. Results: Thirty-nine patients had advanced BCC (64%) and 22 patients multiple BCC (36%), including 10 patients with Gorlin syndrome. No factor predicted response to vismodegib. The median progression-free survival (PFS) was 69.5 months for the total population. In multivariate analysis, multiple BCC was the only factor associated with an increased risk of relapse (HR: 13.80 [CI95%, 1.93-98.64, p < 0.01]). Treatment duration decreased the risk of relapse (HR 0.95 [CI95%, 0.90-0.99, p = 0.0467]). Among the 20 patients who experienced relapse during follow-up, 15 (75%) were re-treated with vismodegib, with a response rate of 66%. Conclusion: Although we were unable to establish predictive factors for the response to vismodegib, we demonstrate for the first time that increased treatment duration correlates with a decreased risk of relapse.

Circulating Tumor DNA Early Kinetics Predict Response of Metastatic Melanoma to Anti-PD1 Immunotherapy: Validation Study.

The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti-PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%; 1-year OS = 13%; n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%; n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%; 1-year OS = 81%; n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.

Circulating Tumour DNA Is an Independent Prognostic Biomarker for Survival in Metastatic BRAF or NRAS-Mutated Melanoma Patients.

Circulating tumour DNA (ctDNA) can be used to identify gene alterations. The purpose of this study was to determine whether the detection of ctDNA, based on the identification of BRAF and NRAS mutations before systemic treatment initiation, was associated with the prognosis of metastatic melanoma. In total, 68 BRAF or NRAS-mutated stage IV or unresectable stage III metastatic cutaneous melanoma patients were included and tested for the presence of BRAF and NRAS mutations in circulating DNA before treatment initiation, using the Cobas BRAF/NRAS Mutation Test (Roche). The expected mutation was detected in the plasma of 34/68 patients (50% sensitivity). ctDNA detection was associated with AJCC stage, along with the number and nature of metastases. ctDNA was less frequently detected in NRAS-mutated than in BRAF-mutated melanoma (36% and 66%, respectively). At initiation of first-line treatment, ctDNA detection was associated with poor prognosis in Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis (log-rank: p = 0.002 and p < 0.0001, respectively). In multivariate analysis, ctDNA detection was an independent factor of poor prognosis in OS, after adjustment for AJCC stage, number and nature of metastases and gender (HR = 4.384; 95% CI: (1.308; 14.699); p = 0.017).

Tumor infiltrating lymphocytes as adjuvant treatment in stage III melanoma patients with only one invaded lymph node after complete resection: results from a multicentre, randomized clinical phase III trial.

BACKGROUND: Adoptive tumor-infiltrating lymphocytes (TIL) therapy and interleukin-2 (IL-2) have been investigated in melanoma. AIM: To confirm previously observed preventive effects of TIL + IL2 in a subgroup of patients with relapsing metastatic stage III melanoma. METHODOLOGY: Open-label, randomized two-group, multicenter five-year trial in adult stage III melanoma patients with only one invaded lymph node after complete resection. Patients received TIL + IL2 or abstention. TIL + IL2 was administered within 8 weeks after lymph node resection and 4 weeks after. Disease-free survival was assessed every 2 months up to month 18, every 3 months up to month 36 and every 4 months up to 5 years. A once-a-year follow-up was scheduled beyond the five-year follow-up. Safety was assessed throughout the trial. RESULTS: Overall, 49 patients accounted for the modified intent-to-treat and 47 for the PP. Slightly more male than female patients participated; mean age was 57.7 ± 11.4 years in the TIL + IL2 group and 53.5 ± 13.0 years in the abstention group. After 5 years of follow-up, 11/26 patients in the TIL + IL2 group and 13/23 in the abstention group had relapsed. There was no statistical difference between the groups (HR: 0.63 CI 95% [0.28-1.41], p = 0.258), nine patients in the TIL + IL2 and 11 in the abstention group died with no significant difference between the two groups (HR: 0.65 CI95% [0.27 - 1.59], p = 0.34). Safety was good. CONCLUSION: We did not confirm results of a previous trial. However, ulceration of the primary melanoma may be considered predictive of the efficacy of TIL in melanoma in adjuvant setting, in a manner similar to interferon α.

Quantitative monitoring of circulating tumor DNA predicts response of cutaneous metastatic melanoma to anti-PD1 immunotherapy.

Immunotherapies have changed the medical management of metastatic melanoma. However, the early detection of patients who do not respond to these treatments is a key issue. We evaluated the quantitative monitoring of circulating tumor DNA (ctDNA) as an early predictor of response to anti-PD1. Patients treated with anti-PD1 for metastatic mutated melanoma were selected. The somatic alteration detected on the tumor tissue was quantified on plasma DNA by digital PCR (dPCR) at treatment initiation, after 2 and 4 weeks of treatment, and then every 4 weeks until progression. The absence of biological response (defined as a significant decrease in the amount of ctDNA relative to the baseline level) after 2 weeks of treatment was associated with a lack of clinical benefit under anti-PD1. In the presence of a biological response at week 2, detection of subsequent biological progression (significant increase in the amount of ctDNA relative to its nadir) was 100% predictive of progressive disease, on average 75 days prior to radiological detection. Patients with a persistent biological response beyond week 16 did not experience any progressive disease and exhibited sustained responses. In conclusion, we show that quantitative monitoring of ctDNA, using criteria accounting for dPCR measurement imprecision, allows the early and specific detection of patients who do not respond to anti-PD1 therapy.

Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes in Advanced Melanoma Patients.

Immunotherapy for melanoma includes adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TILs). This monocenter retrospective study was undertaken to evaluate the efficacy and safety of this treatment of patients with advanced melanoma. All advanced melanoma patients treated with TILs using the same TIL expansion methodology and same treatment interleukin-2 (IL-2) regimen between 2009 and 2012 were included. After sterile intralesional excision of a cutaneous or subcutaneous metastasis, TILs were produced according to a previously described method and then infused into the patient who also received a complementary subcutaneous IL-2 regimen. Nine women and 1 man were treated for unresectable stage IIIC (n = 4) or IV (n = 6) melanoma. All but 1 patient with unresectable stage III melanoma (1st line) had received at least 2 previous treatments, including anti-CTLA-4 antibody for 4. The number of TILs infused ranged from 0.23 × 109 to 22.9 × 109. Regarding safety, no serious adverse effect was reported. Therapeutic responses included a complete remission, a partial remission, 2 stabilizations, and 6 progressions. Among these 4 patients with clinical benefit, 1 is still alive with 9 years of follow-up and 1 died from another cause after 8 years of follow-up. Notably, patients treated with high percentages of CD4 + CD25 + CD127lowFoxp3+ T cells among their TILs had significantly shorter OS. The therapeutic effect of combining TILs with new immunotherapies needs further investigation.

BRAF mutations might be more common than supposed in vulvar melanomas.

Data on BRAF, NRAS and KIT mutations are scarce in patients with vulvo-vaginal melanomas and are associated with important therapeutic issues. We investigated their prevalence in a cohort of patients with female lower genital tract melanomas between 2003 and 2017. Of the 22 patients, 5 (22.7%) harboured a BRAF mutation, which was much higher than the rate of 5% reported in the literature. One patient, who was tested negative on the primary melanoma, had a NRAS mutation in a cutaneous metastasis. Our data provide a rationale for prospective and repeated mutations testing in female lower genital tract melanomas.

Emergence of High-Avidity Melan-A-Specific Clonotypes as a Reflection of Anti-PD-1 Clinical Efficacy.

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy. Cancer Res; 77(24); 7083-93. ©2017 AACR.

Circulating tumour DNA: analytical aspects and clinical applications for metastatic melanoma patients.

The management of metastatic melanoma has evolved since the onset of treatments with BRAF inhibitors. In order to predict which patients are likely to respond to these treatments, the therapeutic strategy is now conditioned by the search for the activating mutations of the BRAF gene. Tumor genotyping is routinely performed from DNA extracted from tissue or cellular specimens from the primary tumor, metastases, or neoplastic effusions. Due to their invasiveness, these specimens are rarely repeated during the management. In addition, the analysis of the tumor material requires a pretreatment of the sample (formalin fixation, paraffin inclusion, preparation of tissue sections) and may take up to several weeks, making emergency treatment with BRAF inhibitors impossible. Circulating tumor DNA (ctDNA), released by cancer cells in the blood stream, appears as an alternative to tissue sampling. The pre-analytical conditions are now well defined, and several technological approaches can be used to demonstrate the desired molecular alterations. ctDNA is less affected by tumor heterogeneity, can be collected in a minimally invasive manner and analyzed rapidly. Furthermore, ctDNA can be repeatedly analyzed during follow-up, which makes it possible to envisage its use as a specific tumor marker, in order to monitor the response to the treatment and to detect treatment failure.

Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report.

BACKGROUND: Fixed tissues are the standard samples used in routine practice for molecular testing. But sometimes tissues are lacking or difficult to obtain. In these cases, circulating tumor DNA released from tumor cells can be used as an alternative source of tumor DNA. CASE PRESENTATION: We present the case of a 63-year-old Caucasian woman with a metastatic melanoma and a very poor performance status. A plasma sample was tested and the BRAF p.V600E mutation was detected. Based on this result, a treatment combining a BRAF inhibitor and a MEK inhibitor was immediately started. This patient achieved a complete response. In addition, by repeating the plasma test, we could obtain a precise kinetic of release of mutated BRAF DNA in plasma. CONCLUSIONS: We report here for the first time the efficient treatment of a metastatic melanoma patient on the basis of circulating tumor DNA analysis. This urgent treatment provided a dramatic response in a patient with a very poor initial condition. The kinetic data most likely reflect treatment efficacy.

Impact of the time interval between lymph node recurrence and lymphadenectomy on melanoma patient survival.

Lymphadenectomy is currently the standard treatment for melanoma patients with palpable lymph node (LN) metastasis. There is no recommendation as to the time when surgery should be performed in France. The aim of this retrospective study was to assess the impact of the time interval preceding lymphadenectomy on patient outcomes. Patients who underwent lymphadenectomy for LN macrometastasis (Stage IIIB/C AJCC) between 2005 and 2012 were included. Both the time interval between the first suspicion of LN recurrence (physical examination and imaging) and lymphadenectomy and the time interval between the multidisciplinary team meeting and lymphadenectomy were recorded. The impact of these time intervals on patient relapse-free survival (RFS) and overall survival (OS) were analysed. The regression optimized (ROP) model was used to identify ghost factors for the Cox model. A total of 154 patients were included. The median time interval between the multidisciplinary team meeting and lymphadenectomy was 22 days (IQR: 6 to 66). The median time interval between the first suspicion of LN recurrence and lymphadenectomy was 59 days (IQR: 15 to 676). Taking into account the effect identified by the regression optimized (ROP) model, these times were associated with an increased risk of recurrence and mortality (p<0.001 and p = 0.01, respectively). Our study demonstrates that increasing the time interval preceding lymphadenectomy significantly reduces patient RFS and OS.

Histopathological aspects of cutaneous erythematous-papular eruptions induced by immune checkpoint inhibitors for the treatment of metastatic melanoma.

BACKGROUND: Immune checkpoint blockade therapy (ICBT) for the treatment of melanoma has led to an important improvement of overall survival in advanced stage patients. However, secondary cutaneous maculopapular eruptions (CMPEs) are frequent and remain poorly characterized. METHODS: We performed a retrospective analysis of melanoma patients from our institution who developed CMPEs during ICBT. Clinical information was retrieved, and histopathological and immunohistochemical characterization was performed by two pathologists. For comparison, a group of biopsies from CMPE caused by anti-v-raf murine sarcoma viral oncogene homolog B1 (BRAF) therapy was analyzed. RESULTS: Eleven patients met the inclusion criteria. On clinical grounds, CMPE developed mainly on early onset of immunotherapy and were of low grade. Typical lesions included erythematous papules and macules affecting the trunk and/or extremities with associated pruritus. The histopathological patterns consisted of a superficial perivascular lymphocytic dermatitis (SPLD) with eosinophils followed by a granulomatous dermatitis. Other patterns included lichenoid, spongiotic, and a case of Grover's disease. The inflammatory infiltrate consisted of T lymphocytes (CD3+ ) with a predominance of CD4+ over CD8+ cells; isolated Foxp3+ cells were invariably present, and PD-1 was not expressed. Biopsies from CMPE caused by anti-BRAF therapy showed an SPLD and a similar lymphocytic immunophenotype. CONCLUSIONS: Our study showed the clinical features of a group of melanoma patients with CMPE for ICBT and emphasized the wide spectrum of histological findings as well as their immunohistochemical profile. Differential diagnosis can be difficult with CMPE provoked by other therapies as was seen in our comparison group of anti-BRAF-induced eruptions.

Clinical, Genetic and Innate Immunity Characteristics of Melanoma in Organ Transplant Recipients.

The aims of this study were to determine the clinical and histological characteristics of melanoma in transplant recipients, the mutation profile (BRAF, NRAS and c-KIT genes), and the immune tolerance of the tumour microenvironment by immunohistochemical study of the expression of indoleamine 2,3-dioxygenase (IDO), PD1, PD-L1, CD8 and FoxP3. The study population comprised patients who had undergone a renal transplant in Nantes University Hospital who developed post-transplantation melanoma. Twenty cases of melanoma out of 4,663 transplant recipients were studied. The results differed from the usual data with respect to melanoma site: 40% were located on the face and were of the malignant lentigo type. The mutation profile was concordant with that of the immunocompetent population. IDO was expressed in all the sections tested, while CD8, FoxP3, PD1 and PD-L1 were poorly expressed. This reflected a highly immunodepressed tumour environment, raising the question of the inductive role of IDO on tumour immune tolerance in patients presenting with long-term immunodepression.

A new protoparvovirus in human fecal samples and cutaneous T cell lymphomas (mycosis fungoides).

We genetically characterized seven nearly complete genomes in the protoparvovirus genus from the feces of children with diarrhea. The viruses, provisionally named cutaviruses (CutaV), varied by 1-6% nucleotides and shared ~76% and ~82% amino acid identity with the NS1 and VP1 of human bufaviruses, their closest relatives. Using PCR, cutavirus DNA was found in 1.6% (4/245) and 1% (1/100) of diarrhea samples from Brazil and Botswana respectively. In silico analysis of pre-existing metagenomics datasets then revealed closely related parvovirus genomes in skin biopsies from patients with epidermotropic cutaneous T-cell lymphoma (CTCL or mycosis fungoides). PCR of skin biopsies yielded cutavirus DNA in 4/17 CTCL, 0/10 skin carcinoma, and 0/21 normal or noncancerous skin biopsies. In situ hybridization of CTCL skin biopsies detected viral genome within rare individual cells in regions of neoplastic infiltrations. The influence of cutavirus infection on human enteric functions and possible oncolytic role in CTCL progression remain to be determined.

Immunohistochemical markers of advanced basal cell carcinoma: CD56 is associated with a lack of response to vismodegib.

Vismodegib is an effective treatment for advanced basal cell carcinoma (BCC), but primary resistance to vismodegib remains to be elucidated. Alternative approaches are warranted to help selecting patients most likely to be responsive to treatment. The identification of immunohistochemical markers may support this perspective, as well as better understanding of resistance mechanisms. To determine the level of expression of CD56, PDGF-R, CD117, MMP9, TIMP3, and CXCR4 in advanced BCC, and explore whether expression levels are associated with non-response to vismodegib. A cross-sectional study was conducted. Immunohistochemical markers were selected based on their roles in tumour proliferation and/or migration in skin tumours. Tissue samples included pretreatment advanced BCC samples from patients treated with vismodegib, with an available response after six months of treatment. Regression optimised models were used to build hypotheses regarding a possible association between expression levels and non-response to vismodegib, which was then tested by logistic regression. Twenty-three patients were included. The percentage of samples expressing markers ranged from 43.5% (CD117) to 91.3% (CXCR4). CD56 expression was significantly associated with an increased risk of non-response to vismodegib (OR = 5.5; CI 95%: 3.4-29.8; p = 0.0488); a similar association was suggested for CXCR4 (p = 0.066), but not identified for other markers. These results provide a better understanding of the expression of immunohistochemical markers in advanced BCC. Further detailed analysis of CD56 expression may provide insights into guiding further investigation of the correlation between this marker and non-response to vismodegib.

Adoptive T cell therapy combined with intralesional administrations of TG1042 (adenovirus expressing interferon-γ) in metastatic melanoma patients.

Tumor immune escape has recently been shown to be related to the development of an immune tolerance state of the microenvironment. Cytokines activating the immune system such as IFN-γ can be used to reverse the immune escape and thus to potentiate the efficacy of immunotherapy. A clinical study was conducted in 18 stage IIIc/IV melanoma patients treated with tumor-infiltrating lymphocytes (TILs) in combination with intratumoral TG1042 injection (adenovirus expressing IFN-γ). The primary objective was to investigate the safety of treatment. Secondary objectives were to study the clinical response and translational research. The treatment was well tolerated. Among the 13 patients evaluable for tumor response, 38.5% had an overall objective response (OOR = CR + PR) and disease control rate (DCR = CR + PR + S) of 46%. The clinical response of the 37 targeted lesions led to an OOR of 51% and a DCR of 75%. Translational research on predictive markers did not significantly differ between responder and non-responder patients. However, specifically regarding injected lesions, the clinical response correlated with CD3-/CD56+ NK cells which could be activated by TG1042. Further larger studies of this combined immunotherapy are needed to confirm our findings. Intralesional TG1042 combined with antigen-selected TILs should be discussed.

Tight junction proteins in the canine epidermis: a pilot study on their distribution in normal and in high IgE-producing canines.

Epidermal tight junctions (TJ) have been well-described in human medicine and are involved in many skin diseases such as atopic dermatitis (AD). In dogs, there are no data regarding the implication of TJ in skin diseases including canine AD. The aim of this study was to compare the expression and the distribution of ZO-1, occludin and claudin-1 in the epidermis of healthy and atopic dogs. Skin biopsies from 6 high IgE-producing beagles sensitized to house dust mite (atopic group) were used. Skin specimens from nine healthy dogs without skin issues were sampled (healthy group). Immunoperoxydase staining was used to study the staining pattern of zonula occludens-1 (ZO-1), occludin and claudin-1 in the epidermis of healthy and atopic dogs. Positive controls were healthy human skin samples. Labeling patterns were assessed by 2 examiners blinded to the identities of the specimens. Comparisons between groups were performed using an exact Wilcoxon-Mann-Whitney test. The mean total expression score of claudin-1 was lower in atopic dogs as compared to healthy subjects. Occludin and ZO-1 expression remained unchanged within each group. These results suggest a defect in claudin-1 expression in the nonlesional epidermis of atopic dogs.

Les jonctions serrées (JS) épidermiques sont bien décrites en médecine humaine et sont impliquées dans de nombreuses affections cutanées telles que la dermatite atopique (DA). Dans l'espèce canine, il n'existe aucune donnée concernant l'implication des JS dans la DA canine ou dans d'autres affections dermatologiques.Le but de cette étude est de comparer l'expression et la distribution de ZO-1, de l'occludine et de la Claudine-1 dans l'épiderme de chiens atopiques et de chiens sains.Les biopsies cutanées de six chiens sensibilisés dans leur jeune âge aux acariens de poussière et produisant de forts taux d'IgE (groupe atopique) on été utilisées. Des échantillons de peau exempte de lésions cutanées ont été prélevés avant tout challenge allergique. Des échantillons de peau saine provenant de neuf chiens sans problème dermatologique ont été recueillis (groupe sain).Deux examinateurs ont évalué l'immunomarquage, en aveugle. Des comparaisons entre les différents groupes ont été réalisées à l'aide du test statistique de Wilcoxon-Mann-Whitney.L'expression de la claudine-1 était plus faible dans l'épiderme de chiens atopiques par comparaison aux sujets sains. L'expression de ZO-1 et de l'occludine était identique dans chaque groupe.Ces résultats suggèrent un défaut d'expression de la Claudine-1 dans l'épiderme non lésionnel des chiens atopiques.(Traduit par les auteurs).