RREB1-MKL2 fusion in a spindle cell sinonasal sarcoma: biphenotypic sinonasal sarcoma or ectomesenchymal chondromyxoid tumor in an unusual site?

Biphenotypic sinonasal sarcoma (BSNS) is a rare, low grade spindle cell sarcoma, recently recognized in the WHO classification of head and neck tumors, which is characterized by a dual myogenic and neural differentiation and recurrent gene fusions, often involving PAX3-MAML3, and less commonly PAX3 fusions with other partners such as NCOA1, NCOA2, or WWTR1. Yet, in about 4% of tumors no gene rearrangements are identified. Herein, we describe a RREB1-MKL2 fusion in a BSNS lesion occurring in a 73-year-old female patient with a right maxillo-ethmoidal angle lesion. The polypoid, moderately cellular tumor with infiltrative submucosal growth was composed of fascicles of relatively bland spindle cells embedded in a loose collagenous matrix. The tumor cells showed moderate amounts of eosinophilic cytoplasm with indistinct borders and uniform, pale, ovoid to slender nuclei. The slowly proliferating neoplastic cells co-expressed smooth muscle actin and S100, and showed focal nuclear positivity for ß-catenin, while lacking staining for cytokeratins, desmin, myogenin, caldesmon, glial fibrillary acid protein, and SOX-10. Molecular analysis by targeted RNA-based next-generation sequencing identified an in-frame fusion between exon 8 of RREB1 and exon 11 of MKL2, a genetic event that was reported to be a molecular hallmark of ectomesenchymal chondromyxoid tumor. Gene rearrangements in both genes were independently verified by fluorescence in situ hybridization (FISH). To evaluate its recurrent potential an additional group of 15 fusion negative BSNS were tested for abnormalities in RREB1 and MKL2 genes by FISH, but no additional positive cases were identified.

S1-Guideline: Microscopically controlled surgery.

Microscopically controlled surgery (MCS) comprises various methods allowing histologically proven complete resection of malignant tumors while at the same time sparing the tumor-free tissue in the immediate vicinity as much as possible. All procedures subsumed under MCS have in common the marking of the excised tissue for topographical orientation, which provides an assignment of remaining tumor remnants. Indications for MCS are malignant skin tumors in problem localizations as well as aggressive subtypes of skin tumors. Established indications for MCS include basal cell carcinoma, cutaneous squamous cell carcinoma, Bowen's disease as well as Bowen's carcinoma, dermatofibrosarcoma protuberans, melanoma in chronically light-damaged skin as well as acral lentiginous melanoma and Merkel cell carcinoma. For other tumors such as extramammary Paget's disease and various cutaneous sarcomas, evidence exists that MCS has demonstrated benefits, such as local recurrence rates. In addition, MCS is indicated when it is foreseeable that a complex closure technique is required and complete resection of the tumor must be assured. Various methods of MCS have been described, including 3D histology, horizontal method and Mohs surgery. A close cooperation of qualified surgeons and (dermato)pathologists as well as laboratory staff is essential for the successful application of MCS.

Effective Initial Treatment of Diffuse Pulmonary Lymphangiomatosis with Sirolimus and Propranolol: A Case Report.

Diffuse pulmonary lymphangiomatosis (DPL), an exceptionally rare disease, mainly occurs in children and young adults of both sexes. Even though DPL is considered to be a benign disease, its prognosis is relatively poor. Because of its rarity, little guidance on diagnosis and treatment is available, which makes working with patients with DPL challenging for clinicians. We present here a case of a young man with DPL in whom treatment with sirolimus and propranolol rapidly achieved positive radiological and clinical effects.

Rationale and design of the German-speaking myeloma multicenter group (GMMG) trial HD6: a randomized phase III trial on the effect of elotuzumab in VRD induction/consolidation and lenalidomide maintenance in patients with newly diagnosed myeloma.

BACKGROUND: Despite major advances in therapy, multiple myeloma is still an incurable malignancy in the majority of patients. To increase survival, deeper remissions (i.e. CR) translating into longer PFS need to be achieved. Incorporation of new drugs (i.e. bortezomib and lenalidomide) as induction and maintenance treatment in an intensified treatment concept, including high dose melphalan (200 mg/m2), has resulted in increased CR rates, and is considered the standard of care for younger patients. Elotuzumab in combination with lenalidomide and dexamethasone has given better results as lenalidomide and dexamethasone alone in a phase III trial. The GMMG-HD6 trial will be the first phase III trial investigating the role of elotuzumab in combination with bortezomib, lenalidomide and dexamethasone (VRD) induction/consolidation and lenalidomide maintenance within a high dose concept. METHODS: GMMG-HD6 is a randomized, open, multicenter phase III trial. The planned recruitment number is 564 NDMM patients. All patients will receive 4 VRD cycles as induction and undergo peripheral blood stem cell mobilization and harvesting. Thereafter they will be treated with high dose melphalan therapy plus autologous stem cell transplantation followed by 2 cycles of VRD consolidation and lenalidomide maintenance. Patients in arm B1 + B2 will additionally receive elotuzumab in the induction phase, whereas patients in A2 + B2 will be treated with elotuzumab added to consolidation and maintenance. The primary endpoint of the trial is PFS. Secondary objectives and endpoints are OS, CR rates after induction therapy comparing the two arms VRD (A1 + A2) vs VRD + elotuzumab (B1 + B2), CR rates after consolidation treatment, best response to treatment during the study, time to progression (TTP), duration of response (DOR), toxicity and quality of life. RESULTS: Since this is the publication of a study protocol of an ongoing study, no results can be presented. DISCUSSION: This phase III trial is designed to evaluate whether the addition of elotuzumab to an intensified treatment concept with high dose melphalan chemotherapy plus autologous stem cell transplantation and induction, consolidation and maintenance treatment with bortezomib and lenalidomide is able to improve PFS compared to the same concept without elotuzumab. TRIAL REGISTRATION: NCT02495922 on June 24th, 2015.

Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes.

In this study, targeted sequencing to screen 50 multidrug refractory multiple myeloma (rMM) patients was performed by using the Multiple Myeloma Mutation Panel. Patients were pretreated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), and 88%, 78%, and 68% were refractory to an IMiD, a PI, or both, respectively. The majority of patients had progressive (82%) or refractory (78%) disease immediately before sampling, with 43% being IMiD refractory and 46% being PI refractory in the most recent line of therapy. Compared with newly diagnosed MM, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS, and/or BRAF (72%), as well as TP53 (26%), CRBN (12%), and CRBN pathway genes (10%) was observed. Longitudinal analyses performed in 3 patients with CRBN mutations at time of IMiD resistance confirmed that these mutations were undetectable at earlier, IMiD-sensitive time points. Furthermore, the functional introduction of these mutations in MM cells conferred lenalidomide resistance in vitro. These data indicate a differential genetic landscape in rMM associated with drug response.

BRAF inhibition in hairy cell leukemia with low-dose vemurafenib.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.

Influence of Regioselectively Sulfated Cellulose on in Vitro Vascularization of Biomimetic Bone Matrices.

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined.

Recurrent CDKN1B (p27) mutations in hairy cell leukemia.

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.

Expression of CDKN1C in the bone marrow of patients with myelodysplastic syndrome and secondary acute myeloid leukemia is associated with poor survival after conventional chemotherapy.

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34(+) cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin-dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p < 0.001). In multivariable analysis, higher CDKN1C expression was associated with worse OS (p = 0.02). On the BM level, a total of 84 (38%) patients showed CDKN1C protein expression before start of treatment. Patient, disease and treatment characteristics did not differ between CDKN1C-positive and -negative patients. Positive CDKN1C BM status was associated with shorter OS in multivariable analysis (HR 1.54, p = 0.04). There was an interaction between CDKN1C BM status and subsequent treatment with negative impact on OS being most pronounced in patients receiving conventional cytotoxic chemotherapy (n = 83, 2-year OS 30% versus 58%, p = 0.002). In conclusion, low-proliferative phenotype and CDKN1C expression were associated with shorter OS. CDKN1C protein expression in the BM of newly diagnosed, treatment-naïve MDS and secondary AML patients was identified as a prognostic factor for poor survival in patients treated with antiproliferative chemotherapy.

Aggressive primary cutaneous B-cell lymphomas show increased Angiopoietin-2-induced angiogenesis.

Primary cutaneous large B-cell lymphomas, leg type (PCLBCL/LT) are primary cutaneous B-cell lymphoma (PCBCL) with an intermediate prognosis. Therefore, antracycline-based polychemotherapy combined with rituximab has been recommended as first-line treatment. Yet, despite this regimen, the 5-year survival rate remains 50-66% only. Angiogenesis, the formation of a vascular network, is essential for the pathogenesis of nodal lymphomas. So far, no study has analysed angiogenesis and its key factors in PCLBCL/LT. The present study was aimed at characterizing angiogenesis in PCLBCL/LT to identify the angiogenic molecules as potential therapeutic targets. The intra-tumoral microvessel density (MVD) was assessed by immunohistochemical studies of CD20 and CD31. The MVD was higher in PCLBCL/LT compared with indolent PCBCL. Analyses of open-source microarray data showed correlation between the angiogenic molecule angiopoietin-2 (Ang-2) and pan-endothelial cell markers. ELISA studies determined a shift between Ang-2 and Ang-1 towards Ang-2 in the peripheral blood of PCLBCL/LT patients. Immunofluorescence costainings against the Ang receptor Tie2/angiogenic integrins/CD34 revealed that the vasculature in both aggressive and indolent PCBCL tumors harbours an endothelial cell subpopulation with reduced expression of Tie2. In contrast, the alternative Ang-2 binding partners, angiogenic integrins, are strongly expressed in PCBCL. In line with these findings, downstream targets of Ang-2-integrin signalling, that is phosphorylation of focal adhesion kinase at Tyr397, and sprouting angiogenesis are enhanced in PCLBCL/LT. Our data present Ang-2 as a promising therapeutic target and anti-angiogenic therapy as a new line in treatment of PCLBCL/LT as a hitherto intractable disease.

Expression of Mucin-1 in multiple myeloma and its precursors: correlation with glycosylation and subcellular localization.

AIMS: Recent reports suggest a possible role for extracellular (MUC1N) and transmembrane (MUC1C) subunits of Mucin 1 (MUC1) in the pathogenesis of multiple myeloma (MM). Nuclear translocation of MUC1C is involved in activation of various oncogenic signalling pathways and both MUC1 subunits are potential therapeutic targets. We aimed at performing a comprehensive expression analysis of the MUC1 subunits in plasma cell dyscrasias. METHODS AND RESULTS: Immunohistochemistry with monoclonal antibodies against the MUC1N subunit (EMA and 5E10) tumour-associated glycoforms of MUC1N (5E5) and the MUC1C subunit were applied to a series of biopsies from normal controls (n = 10) and plasma cell dyscrasias (n = 121). Clonal plasma cells showed reduced MUC1N expression, and the 5E5 MUC1N epitope was expressed only in neoplastic plasma cells. Nuclear localization of MUC1C was equally frequent in all disease stages and did not differ from the control cases. Loss of both MUC1 subunits in MM (n = 12) was associated with significantly shorter overall survival and was more frequent in pretreated MM samples. CONCLUSIONS: Our findings indicate that aberrant glycosylation of MUC1 is an early event in the pathogenesis of MM. In contrast, MUC1C nuclear localization is not likely to be a driver of tumour progression.

Endothelial vulnerability and endothelial damage are associated with risk of graft-versus-host disease and response to steroid treatment.

There is accumulating evidence indicating that endothelial factors are involved in the pathogenesis of GVHD. We have recently shown that steroid-refractory, but not sensitive, GVHD is characterized by higher pretransplantation serum levels of angiopoetin-2 (ANG2), a hormone mediating endothelial vulnerability. To evaluate whether endothelial vulnerability is a risk factor for GVHD per se or becomes important only when noticeable GVHD is established, we measured ANG2 along with additional serum markers of endothelial stress, including soluble thrombomodulin (sTM), IL-8 (CXCL8), and hepatocyte growth factor (HGF), in patients with no, low-grade, or severe GVHD. Patients with refractory GVHD exhibited elevated serum levels of ANG2, sTM, HGF, and IL-8 posttransplantation compared with patients with sensitive GVHD and patients without GVHD. Pretransplantation ANG2 was the only growth factor correlated with the risk of refractoriness and mortality, and then only within the subset of patients who developed grade III-IV GVHD. In contrast, ANG2 was not predictive of GVHD or nonrelapse mortality (NRM) in patients with no GVHD or low-grade GVHD. These findings provide evidence that endothelial function plays an important role in the pathogenesis of steroid refractoriness in ongoing GVHD; however, endothelial vulnerability does not predict incidence of GVHD.

The heat shock transcription factor 1 as a potential new therapeutic target in multiple myeloma.

The heat shock transcription factor 1 (HSF1) has recently been reported to promote malignant transformation and growth. Here we provide experimental evidence for a role of HSF1 in the pathogenesis of multiple myeloma (MM). Immunohistochemical analyses revealed that HSF1 was overexpressed in half of the investigated MM samples, including virtually all cases with extramedullary manifestations or anaplastic morphology. HSF1 function was inhibited either by siRNA-mediated knockdown or pharmacologically through treatment with triptolide. Both approaches caused depletion of HSF1, lowered the constitutively high expression of a multitude of protective HSPs (such as HSP90, HSP70, HSP40 and HSP27), induced apoptosis in human MM cells in vitro, and strongly reduced MM tumour growth in vivo. Furthermore, we observed that treatment-induced upregulation of HSPs after proteasome or HSP90 inhibition was critically dependent on HSF1. Importantly, the apoptotic effects of the HSP90 inhibitor NVP-AUY922 or the proteasome inhibitor bortezomib were strongly enhanced in combination with triptolide, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Collectively, our data indicate that inhibition of HSF1 affects multiple protective HSPs and might therefore represent a therapeutic strategy - in particular in combination with proteasome or HSP90 inhibitors.

The PI3K/Akt signaling pathway regulates the expression of Hsp70, which critically contributes to Hsp90-chaperone function and tumor cell survival in multiple myeloma.

Despite therapeutic advances multiple myeloma remains largely incurable, and novel therapeutic concepts are needed. The Hsp90-chaperone is a reasonable therapeutic target, because it maintains oncogenic signaling of multiple deregulated pathways. However, in contrast to promising preclinical results, only limited clinical efficacy has been achieved through pharmacological Hsp90 inhibition. Because Hsp70 has been described to interact functionally with the Hsp90-complex, we analyzed the suitability of Hsp72 and Hsp73 as potential additional target sites. Expression of Hsp72 and Hsp73 in myeloma cells was analyzed by immunohistochemical staining and western blotting. Short interfering RNA-mediated knockdown or pharmacological inhibition of Hsp72 and Hsp73 was performed to evaluate the role of these proteins in myeloma cell survival and for Hsp90-chaperone function. Furthermore, the role of PI3K-dependent signaling in constitutive and inducible Hsp70 expression was investigated using short interfering RNA-mediated and pharmacological PI3K inhibition. Hsp72 and Hsp73 were frequently overexpressed in multiple myeloma. Knockdown of Hsp72 and/or Hsp73 or treatment with VER-155008 induced apoptosis of myeloma cells. Hsp72/Hsp73 inhibition decreased protein levels of Hsp90-chaperone clients affecting multiple oncogenic signaling pathways, and acted synergistically with the Hsp90 inhibitor NVP-AUY922 in the induction of death of myeloma cells. Inhibition of the PI3K/Akt/GSK3ß pathway with short interfering RNA or PI103 decreased expression of the heat shock transcription factor 1 and down-regulated constitutive and inducible Hsp70 expression. Treatment of myeloma cells with a combination of NVP-AUY922 and PI103 resulted in additive to synergistic cytotoxicity. In conclusion, Hsp72 and Hsp73 sustain Hsp90-chaperone function and critically contribute to the survival of myeloma cells. Translation of Hsp70 inhibition into the clinic is therefore highly desirable. Treatment with PI3K inhibitors might represent an alternative therapeutic strategy to target Hsp70.

Intravoxel incoherent motion imaging for assessment of bone marrow infiltration of monoclonal plasma cell diseases.

Modern imaging techniques have demonstrated that monoclonal plasma cell diseases infiltrate the bone marrow in a diffuse, focal, mixed pattern. While focal lesions can be easily counted and measured, the diffuse lesions of the infiltration are hard to assess. We therefore investigated 31 patients with monoclonal plasma cell diseases of all stages with intravoxel incoherent motion imaging of the same region of the pelvis from where afterwards a biopsy was obtained. We found a significant correlation between plasma cell percentage in bone marrow histology and the imaging parameters "apparent diffusion coefficient" and the diffusion coefficient D. Furthermore, those parameters correlated with other factors of disease activity, e.g., monoclonal protein, hemoglobin, and immunoparesis. In summary, we found that the non-invasively acquired imaging parameters correlated with the degree of plasma cell infiltration in the bone marrow.

Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease.

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.