Max Schlaak

Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

BackgroundThe approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.MethodsSamples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).ResultsThere was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.ConclusionTherefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Mast cells play a protumorigenic role in primary cutaneous lymphoma

Primary cutaneous lymphomas (PCLs) are clonal T- or B-cell neoplasms, which originate in the skin. In recent years, mast cells were described as regulators of the tumor microenvironment in different human malignancies. Here, we investigated the role of mast cells in the tumor microenvironment of PCL. We found significantly increased numbers of mast cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL). Mast cell infiltration was particularly prominent in the periphery, at lymphoma rims. Interestingly, CTCL and CBCL patients with a progressive course showed higher mast cell counts than stable patients, and mast cell numbers in different stages of CTCL correlated positively with disease progression. In addition, mast cell numbers positively correlated with microvessel density. Incubating primary CTCL cells with mast cell supernatant, we observed enhanced proliferation and production of cytokines. In line with our in vitro experiments, in a mouse model of cutaneous lymphoma, tumor growth in mast cell-deficient transgenic mice was significantly decreased. Taken together, these experiments show that mast cells play a protumorigenic role in CTCL and CBCL. Our data provide a rationale for exploiting tumor-associated mast cells as a prognostic marker and therapeutic target in PCL.

Local Tumor Treatment in Combination with Systemic Ipilimumab Immunotherapy Prolongs Overall Survival in Patients with Advanced Malignant Melanoma.

Too few patients benefit from immune checkpoint inhibition alone. However, patients with melanoma receiving systemic anti-CTLA-4 plus localized treatments had significantly prolonged overall survival. In a multivariate analysis, adding local treatment was an independent factor for improved survival. Immune checkpoint inhibition with ipilimumab has revolutionized cancer immunotherapy and significantly improved outcomes of patients with advanced malignant melanoma. Local peripheral treatments (LPT), such as radiotherapy or electrochemotherapy, have been shown to modulate systemic immune responses, and preliminary data have raised the hypothesis that the combination of LPT with systemic immune checkpoint blockade might be beneficial. Clinical data from 127 consecutively treated melanoma patients at four cancer centers in Germany and Switzerland were analyzed. Patients received either ipilimumab (n = 82) or ipilimumab and additional LPT (n = 45) if indicated for local tumor control. The addition of LPT to ipilimumab significantly prolonged overall survival (OS; median OS 93 vs. 42 weeks, unadjusted HR, 0.46; P = 0.0028). Adverse immune-related events were not increased by the combination treatment, and LPT-induced local toxicities were in most cases mild. In a multivariable Cox regression analysis, we show that the effect of added LPT on OS remained statistically significant after adjusting for BRAF status, tumor stage, tumor burden, and central nervous system metastases (adjusted HR, 0.56; 95% confidence interval, 0.31–1.01, P = 0.05). Our data suggest that the addition of LPT to ipilimumab is safe and effective in patients with metastatic melanoma irrespective of clinical disease characteristics and known risk factors. Induction of antitumor immune responses is most likely the underlying mechanism and warrants prospective validation. Cancer Immunol Res; 4(9); 744–54. ©2016 AACR.

Allogeneic stem cell transplantation versus conventional therapy for advanced primary cutaneous T-cell lymphoma

BACKGROUNDnPrimary cutaneous T-cell lymphomas (CTCL) belong to the group of non-Hodgkin lymphomas and usually run an indolent course. However, some patients progress to advanced tumour or leukaemic stages. To date, there is no cure for those cases. In the last few years, several publications reported durable responses in some patients following allogeneic stem cell transplantation (alloSCT). This is an update of a Cochrane review first published in 2011 and updated in 2013.nnnOBJECTIVESnTo compare the efficacy and safety of conventional therapies with allogeneic stem cell transplantation in patients with advanced primary cutaneous T-cell lymphomas.nnnSEARCH METHODSnWe searched the Cochrane Central Register of Controlled Trials (CENTRAL, Issue 1), MEDLINE (1950 to January 2013), Internet-databases of ongoing trials, conference proceedings of the American Society of Clinical Oncology (ASCO, 2009 to July 2013) and the American Society of Hematology (ASH, 2009 to July 2013). We also contacted members of the European Organisation for Research and Treatment of Cancer (EORTC) Cutaneous Lymphoma Task Force to check for ongoing study activities. We handsearched citations from identified trials and relevant review articles. In addition, we handsearched randomised controlled trials from the European Group for Blood and Marrow Transplantation (EBMT) and International Conference on Cutaneous T-cell Lymphoma, ASCO and ASH up to July 2013.nnnSELECTION CRITERIAnTrials eligible for inclusion were genetically randomised controlled trials (RCTs) comparing alloSCT plus conditioning therapy (regardless of agents) with conventional therapy as treatment for advanced CTCL.nnnDATA COLLECTION AND ANALYSISnTwo review authors would have extracted data from eligible studies and assessed their quality. The primary outcome measure was overall survival; secondary outcomes were time to progression, response rate, treatment-related mortality, adverse events and quality of life.nnnMAIN RESULTSnWe did not identify any randomised controlled trials from the updated search in January 2013. In 2011, we found 2077 citations but none were relevant genetically or non-genetically randomised controlled trials. All 41 studies that were thought to be potentially suitable were excluded after full text screening for being non-randomised, not including CTCL or being review articles.nnnAUTHORS CONCLUSIONSnWe planned to report evidence from genetically or non-genetically randomised controlled trials comparing conventional therapy and allogeneic stem cell transplantation. However, we did not identify any randomised controlled trials addressing this question. Nevertheless, prospective genetically randomised controlled trials need to be initiated to evaluate the precise role of alloSCT in advanced CTCL.

Induction of durable responses by oral etoposide monochemotherapy in patients with metastatic Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive, rare tumour of the skin. For advanced cases with distant organ metastases several different regimens of chemotherapeutics have been described. Disease specific 5-year survival rates for these patients are approximately 11%. In this case series we report our experience with orally administered etoposide (100xa0mg at days 1 to 10 in a 31 day cycle) in 4 patients. We treated two male and two female patients with a median age of 68.5 years. In our four treated patients the disease control rate (complete remission, partial remission, stable disease) was 100%. Three out of four patients reached complete remission. Promisingly, two of our patients had long lasting, durable responses which, until now, have lasted for 16 and 36 months, respectively. The mean follow up time after start of therapy was 14.25 months (range 1-36 months). Etoposide treatment was generally well tolerated, the most common side effect was neutropenia, in one case CTC grade 3. In conclusion, orally administered etoposide in metastatic Merkel cell carcinoma was highly effective and well tolerated.

Assessment of clinical parameters associated with mutational status in metastatic malignant melanoma: a single-centre investigation of 141 patients.

Backgroundu2002 Inhibitors of the mutated, constitutively activated BRAF protein have shown efficacy in the treatment of metastatic melanoma in clinical trials. Mutation analysis especially of the BRAF, NRAS and KIT genes is essential to identify patients suitable for targeted therapies and has been introduced into routine patient care.

Electrochemotherapy in advanced skin tumors and cutaneous metastases – a retrospective multicenter analysis

Once classic treatments such as chemotherapy or radiation therapy have been exhausted, only few therapeutic options remain for extensive skin tumors or cutaneous metastases. In such cases, electrochemotherapy may be considered as alternative therapy.

Brentuximab vedotin in CD30+ cutaneous lymphoma: How do we treat, how shall we treat? A review of the literature

Brentuximab vedotin is an antibody–drug conjugate that brings the antimicrotubule agent monomethyl auristatin E into CD30‐expressing cells. Some prior studies demonstrated good efficacy in cutaneous lymphomas. The standard therapeutic scheme is 1·8 mg kg−1 every 3 weeks. The background of this work is the fact that cutaneous lymphoma has a different pathophysiology and a dynamic other than systemic lymphomas. The objectives of this review were to get an overview of the currently used therapeutic regimen, and to check whether dose reduction or modified time intervals could be of benefit in a similar way with less toxicity. Therefore, we conducted a systematic review of the literature indexed in PubMed and the Cochrane Central Register of Controlled Trials up to April 2016. The procedure was based on the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) criteria. The review showed that the currently used therapeutic regimen is 1·8 mg kg−1 every 3 weeks. No publications of dose‐finding studies in CD30+ cutaneous T‐cell lymphoma (CTCL) were found. Two cases of patients, treated with a dose < 1·8 mg kg−1, have been published. Brentuximab vedotin seems to be a powerful treatment option in refractory CD30+ CTCL, and there is a trend that dose reductions, as well as prolonged treatment intervals, work without any loss of response and with fewer side‐effects.

Targeting Tumor-Infiltrating B Cells in Cutaneous T-Cell Lymphoma

Introduction The tumor microenvironment and infiltrating immune cells are important for cancer biology and progression which has been exploited therapeutically in recent years. Whereas infiltrating T cells, macrophages, or natural-killer (NK) cells have been extensively analyzed, recent studies also suggest a role for B cells in cancer biology. Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas of which the most common subtype is mycosis fungoides (MF). At early stages ( IIA; European Organisation for Research and Treatment of Cancer [EORTC]/International Society for Cutaneous Lymphomas [ISCL]), MF usually runs an indolent course with almost normal life expectancy. However, advanced-stage MF ( IIB), the folliculotropic MF subtype (FMF) and also Sézary syndrome (SS) are more aggressive (median survival, 13-48 months). Preclinical data suggest that chronic inflammation promotes CTCL progression with a critical role for macrophages and mast cells. Occasionally, B-cell infiltrations have been reported in CTCL and although a well-established CTCL model is lacking, B-cell deficient mice exhibited significant regressions of EL4-T-cell lymphoma grafts. Therefore, we analyzed diagnostic skin biopsies of 33 consecutively treated CTCL patients for infiltrating B cells (CD20/ CD79a) and reviewed respective clinical data from 1979 to 2011 (Table 1). Psoriasis and eczema tissues served as controls. Staging followed ISCL/EORTC criteria. Lymphoma-tissues containing 50 B-cells/mm of lymphoma-infiltrate were assigned as B-cell positive. The study was approved by the institutional review board (No. 08-144). Immunohistochemistry revealed remarkable differences of CD20 B-cell infiltrates between CTCL subtypes (Fig 1A) and additional flow-cytometric analysis in available frozen samples supported the B-cell nature of CD20 cells (Fig 3A). Eighteen out of 33 CTCL patients (MF [n 25], FMF [n 5], SS [n 3]) had significantly increased median B-cell numbers within the lymphoma infiltrate, whereas controls (psoriasis [n 5], eczema [n 5]) were B-cell negative (Fig 1B and Table 1). Remarkably, all FMF and SS cases showed significantly increased median B-cell numbers compared with classic MF. However, in classic MF 40% of the cases were B-cell positive. Given the fact that FMF and SS represent more aggressive subtypes, we asked whether B-cell infiltrations correspond with the clinical stage and behavior. Comparison of B-cell infiltrates in early ( IIB) versus advanced ( IIB) stages revealed a significant correlation of B-cell positivity with advanced stages. This correlation not only applied to the entire CTCL cohort but also to classic MF cases solely (Fig 1C). Moreover, clinical data analyses revealed a significant correlation of B-cell infiltrates with progression-free survival (PFS). As shown in Figure 1D, patients with B-cell positive lymphoma had a significantly shortened median PFS, ie, 50 months (entire cohort) and 126 months (classic MF only). In contrast, median PFS in B-cell negative cases was not reached at the end of observation (360 months).

Allogeneic stem cell transplantation in patients with aggressive primary cutaneous T‐cell lymphoma – a case series of the ADF working group „cutaneous lymphomas”

Allogeneic stem cell transplantation (alloSCT) is a treatment option for primary cutaneous T‐cell lymphomas that may induce long‐lasting complete remissions. Little information is available on safety and efficacy.