Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues.

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy. Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell-based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system.

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821-1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

A chemokine network of T cell exhaustion and metabolic reprogramming in renal cell carcinoma.

Renal cell carcinoma (RCC) is frequently infiltrated by immune cells, a process which is governed by chemokines. CD8+ T cells in the RCC tumor microenvironment (TME) may be exhausted which most likely influence therapy response and survival. The aim of this study was to evaluate chemokine-driven T cell recruitment, T cell exhaustion in the RCC TME, as well as metabolic processes leading to their functional anergy in RCC. Eight publicly available bulk RCC transcriptome collectives (n=1819) and a single cell RNAseq dataset (n=12) were analyzed. Immunodeconvolution, semi-supervised clustering, gene set variation analysis and Monte Carlo-based modeling of metabolic reaction activity were employed. Among 28 chemokine genes available, CXCL9/10/11/CXCR3, CXCL13/CXCR5 and XCL1/XCR1 mRNA expression were significantly increased in RCC compared to normal kidney tissue and also strongly associated with tumor-infiltrating effector memory and central memory CD8+ T cells in all investigated collectives. M1 TAMs, T cells, NK cells as well as tumor cells were identified as the major sources of these chemokines, whereas T cells, B cells and dendritic cells were found to predominantly express the cognate receptors. The cluster of RCCs characterized by high chemokine expression and high CD8+ T cell infiltration displayed a strong activation of IFN/JAK/STAT signaling with elevated expression of multiple T cell exhaustion-associated transcripts. Chemokinehigh RCCs were characterized by metabolic reprogramming, in particular by downregulated OXPHOS and increased IDO1-mediated tryptophan degradation. None of the investigated chemokine genes was significantly associated with survival or response to immunotherapy. We propose a chemokine network that mediates CD8+ T cell recruitment and identify T cell exhaustion, altered energy metabolism and high IDO1 activity as key mechanisms of their suppression. Concomitant targeting of exhaustion pathways and metabolism may pose an effective approach to RCC therapy.

CXCR3 Expression Is Associated with Advanced Tumor Stage and Grade Influencing Survival after Surgery of Localised Renal Cell Carcinoma.

BACKGROUND: Surgery is the standard treatment in localized renal cell carcinoma (RCC). Pembrolizumab is now approved for adjuvant therapy in high-risk patients. However, inhomogeneity of studies gives ambiguity which patient benefit most from adjuvant therapy. A high infiltration of CD8+ T cells is known to be linked with poor prognosis in RCC. CXCR3 is a key player of CD8+ T cell differentiation and infiltration. We aimed to evaluate CXCR3 as a potential marker for predicting recurrence. METHODS: CXCR3 and immune cell subsets (CD4, CD8, CD68 and FoXP3) were measured on RCC samples by multiplex immunofluorescence (mIF) staining. Cellular localization of CXCR3 was evaluated using single-cell RNA analysis on a publicly available dataset. RESULTS: Tumor samples of 42 RCC patients were analyzed, from which 59.5% were classified as clear-cell RCC and of which 20 had recurrence. Single-cell RNA analysis revealed that CXCR3 was predominantly expressed in intratumoral T cells and dendritic cells. CXCR3 expression was higher in advanced tumors stages (p = 0.0044) and grade (p = 0.0518), correlating significantly with a higher CD8+ T cell expression (p < 0.001). Patients with CXCR3high RCCs had also a significant shorter RFS compared to CXCR3low (median: 78 vs. 147 months, p = 0.0213). In addition, also tumor stage pT3/4 (p < 0.0001) as well as grade G3/4 (p = 0.0008) negatively influenced RFS. CONCLUSION: CXCR3high cell density was associated with high T cell infiltration and advanced tumor stage, worsening RFS in surgically resected RCC patients. Beside its prognostic value, CXCR3 might be a predictive biomarker to guide therapy decision for adjuvant therapy in localized RCC.

High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is characterized by molecular heterogeneity with diverse immune cell infiltration patterns, which has been linked to therapy sensitivity and resistance. However, full understanding of how immune cell phenotypes vary across different patient subgroups is lacking. Here, we dissect the NSCLC tumor microenvironment at high resolution by integrating 1,283,972 single cells from 556 samples and 318 patients across 29 datasets, including our dataset capturing cells with low mRNA content. We stratify patients into immune-deserted, B cell, T cell, and myeloid cell subtypes. Using bulk samples with genomic and clinical information, we identify cellular components associated with tumor histology and genotypes. We then focus on the analysis of tissue-resident neutrophils (TRNs) and uncover distinct subpopulations that acquire new functional properties in the tissue microenvironment, providing evidence for the plasticity of TRNs. Finally, we show that a TRN-derived gene signature is associated with anti-programmed cell death ligand 1 (PD-L1) treatment failure.

Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets.

PURPOSE: Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels. EXPERIMENTAL DESIGN: The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4. RESULTS: High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability-high/mismatch repair-deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC. CONCLUSIONS: High intratumoral CXCR4 mRNA expression is linked to a T cell- and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

The Biology of Classic Hairy Cell Leukemia.

Classic hairy cell leukemia (HCL) is a rare mature B-cell malignancy associated with pancytopenia and infectious complications due to progressive infiltration of the bone marrow and spleen. Despite tremendous therapeutic advances achieved with the implementation of purine analogues such as cladribine into clinical practice, the culprit biologic alterations driving this fascinating hematologic disease have long stayed concealed. Nearly 10 years ago, BRAF V600E was finally identified as a key activating mutation detectable in almost all HCL patients and throughout the entire course of the disease. However, additional oncogenic biologic features seem mandatory to enable HCL transformation, an open issue still under active investigation. This review summarizes the current understanding of key pathogenic mechanisms implicated in HCL and discusses major hurdles to overcome in the context of other BRAF-mutated malignancies.

Lynch Syndrome: Its Impact on Urothelial Carcinoma.

Lynch syndrome, known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal-dominant familial cancer syndrome with an increased risk for urothelial cancer (UC). Mismatch repair (MMR) deficiency, due to pathogenic variants in MLH1, MSH2, MSH6, and PMS2, and microsatellite instability, are known for development of Lynch syndrome (LS) associated carcinogenesis. UC is the third most common cancer type in LS-associated tumors. The diversity of germline variants in the affected MMR genes and their following subsequent function loss might be responsible for the variation in cancer risk, suggesting an increased risk of developing UC in MSH2 mutation carriers. In this review, we will focus on LS-associated UC of the upper urinary tract (UUT) and bladder, their germline profiles, and outcomes compared to sporadic UC, the impact of genetic testing, as well as urological follow-up strategies in LS. In addition, we present a case of metastatic LS-associated UC of the UUT and bladder, achieving complete response during checkpoint inhibition since more than 2 years.

Clonal dynamics in a composite chronic lymphocytic leukemia and hairy cell leukemia-variant.

Composite lymphoma is the rare simultaneous manifestation of two distinct lymphomas. Chronic lymphocytic leukemia (CLL) has a propensity for occurring in composite lymphomas, a phenomenon that remains to be elucidated. We applied cytogenetics, droplet digital polymerase chain reaction, and massively parallel sequencing to analyze longitudinally a patient with CLL, who 3 years later showed transformation to a hairy cell leukemia-variant (HCL-V). Outgrowth of the IGHV4-34-positive HCL-V clone at the expense of the initially dominant CLL clone with trisomy 12 and MED12 mutation started before CLL-guided treatment and was accompanied by a TP53 mutation, which was already detectable at diagnosis of CLL. Furthermore, deep sequencing of IGH showed a composite lymphoma with presence of both disease components at all analyzed timepoints (down to a minor clone: major clone ratio of ~1:1000). Overall, our analyses showed a disease course that resembled clonal dynamics reported for malignancies with intratumoral heterogeneity and illustrate the utility of deep sequencing of IGH to detect distinct clonal populations at diagnosis, monitor clonal response to therapy, and possibly improve clinical outcomes.

The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis.

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1-6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

Amplification of 7p12 Is Associated with Pathologic Nonresponse to Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Cancer.

Pathologic downstaging (pDS) to neoadjuvant chemotherapy (NAC) is one of the most important predictors of survival in muscle-invasive bladder cancer (MIBC). The use of NAC is limited as pDS is only achieved in 30% to 40% of cases and predictive biomarkers are still lacking. We performed a comprehensive immunomolecular biomarker analysis to characterize the role of immune cells and inhibitory checkpoints, genome-wide frequencies of copy number alterations, mutational signatures in whole exome, and tumor mutational burden in predicting NAC response. Our retrospective study included 23 primary MIBC patients who underwent NAC, followed by radical cystectomy. pDS to NAC was a significant prognostic factor for better recurrence-free survival (P < 0.001), with a median time to recurrence of 41.2 versus 5.5 months in nonresponders. DNA damage repair alterations were noticed in 38.1% (n = 8), confirming a positive correlation with high tumor mutational burden (P = 0.007). Chromosomal 7p12 amplification, including the genes HUS1, EGFR, ABCA13, and IKZF1, predicted nonresponse in patients with a sensitivity, a negative predictive value, and a specificity of 71.4%, 87.5%, and 100%, respectively. Total count of CD3+ T cells/mm2 tumor was a significant predictor of NAC response. In conclusion, 7p12 amplification may predict nonresponse to NAC and worse survival in MIBC. Multicenter, prospective trials with sufficient statistical power may further fortify these findings.

Spliced XBP1 Levels Determine Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitor Bortezomib Independent of the Unfolded Protein Response Mediator GRP78.

Background: Mechanisms mediating resistance against the proteasome inhibition by bortezomib (BTZ) in multiple myeloma (MM) cells are still unclear. We analyzed the activation of the unfolded protein response (UPR), induction of prosurvival, and apoptotic pathways after proteasome inhibition in BTZ-sensitive and -resistant cells. Thereafter, these findings from tissue culture were proofed on MM cells of BTZ-sensitive and BTZ-refractory patients. Methods: Proteasomal and ABC transporter activities were measured in sensitive and resistant cell lines by the use of the respective substrates. TP53 gene loss and mutations were determined by cytogenetics and targeted NGS. UPR pathways, proteasome subunit levels and protein secretion were studied by Western Blot analysis, and apoptosis was determined by flow cytometry. MM cell lines were stably transfected with inducible GRP78 expression to study unfolded protein expression. Transient knock-down of GRP78 was done by RNA interference. Splicing of XBP1 and expression of GRP78 was studied by real-time PCR in CD138-enriched MM primary cells of BTZ-refractory and -sensitive patients. Results: BTZ-sensitive cells displayed lower basal proteasomal activities. Similar activities of all three major ABC transporter proteins were detected in BTZ-sensitive and resistant cells. Sensitive cells showed deficiencies in triggering canonical prosurvival UPR provoked by endoplasmic reticulum (ER) stress induction. BTZ treatment did not increase unfolded protein levels or induced GRP78-mediated UPR. BTZ-resistant cells and BTZ-refractory patients exhibited lower sXBP1 levels. Apoptosis of BTZ-sensitive cells was correlating with induction of p53 and NOXA. Tumor cytogenetics and NGS analysis revealed more frequent TP53 deletions and mutations in BTZ-refractory MM patients. Conclusions: We identified low sXBP1 levels and TP53 abnormalities as factors correlating with bortezomib resistance in MM. Therefore, determination of sXBP1 levels and TP53 status prior to BTZ treatment in MM may be beneficial to predict BTZ resistance.

The Plasma Levels of the Angiogenic Cytokine Endocan Are Elevated in Patients with Multiple Myeloma.

BACKGROUND/AIM: Monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma often precede multiple myeloma (MM). The identification of biomarkers predicting progression to MM might facilitate an earlier diagnosis of MM. Our study assessed the diagnostic value of plasma levels of endocan, a 50-kDa soluble dermatan sulfate proteoglycan produced and secreted by endothelial cells, hitherto unknown in MM, in patients with plasma cell dyscrasia. MATERIALS AND METHODS: Endocan levels were determined in 96 peripheral blood plasma samples by sandwich enzyme-linked immunosorbent assay (ELISA) in healthy controls (n=12), in patients with MGUS (n=17), and in patients newly diagnosed with (n=42) or relapsed/refractory (n=25) MM. RESULTS: Median endocan concentration increased from MGUS (315.00 pg/ml) and healthy controls (316.19 pg/ml) to newly-diagnosed MM (371.82 pg/ml; p=0.027). The low endocan levels (median=246.20 pg/ml) in patients with relapsed/refractory MM were similar to those in healthy controls and patients with MGUS. A cut-off value of >220 pg/ml endocan in peripheral blood discriminated patients newly diagnosed with MM from those with MGUS (area under the curve(AUC)=0.66, 95% confidence interval(CI)=0.55-0.81). CONCLUSION: The plasma levels of endocan can non-invasively differentiate patients newly diagnosed with MM from those with MGUS and should therefore be evaluated prospectively as a potential diagnostic marker.

FOXF1 Mediates Endothelial Progenitor Functions and Regulates Vascular Sprouting.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

Expression and release of glucose-regulated protein-78 (GRP78) in multiple myeloma.

INTRODUCTION: Multiple myeloma (MM) is a plasma cell neoplasm that is mostly incurable due to acquired resistance during the treatment course. Thus, we evaluated expression and release of glucose-regulated protein 78 kDa (GRP78/BiP), an endoplasmic reticulum (ER) based pro-survival chaperone involved in immunoglobulin folding and unfolded protein responses. RESULTS: GRP78 protein expression in the ER and on the cell surface did not significantly differ between MGUS, NDMM and RRMM patients although there was a trend to higher surface expression in RRMM. In bone marrow plasma, the amount of released GRP78 protein was not significantly increased between MGUS-, NDMM- and RRMM patients. MM cells of the three cell lines release GRP78 as full-length protein under apoptotic, but not under acidotic or ER-stress conditions. In necrosis, only proteolytic fragments of GRP78 were detected in supernatants of MM cells. MATERIALS AND METHODS: GRP78 protein expression and plasma levels were quantified in bone marrow aspirates of patients with monoclonal gammopathy of undetermined significance (MGUS, n = 29), newly diagnosed MM (NDMM, n = 29) and with relapsed/refractory MM (RRMM, n = 15) by immunohistochemistry and sandwich ELISA. The human MM cell lines U266, NCI-H929 and OPM-2 were used for functional GRP78 release- and processing studies after induction of acidosis, ER stress, apoptosis and necrosis. CONCLUSIONS: Ectopic expression of GRP78 on cell membrane or its release in the microenvironment is not a suitable marker to distinguish MGUS from NDMM and RRMM.

High levels of FLT3-ligand in bone marrow and peripheral blood of patients with advanced multiple myeloma.

INTRODUCTION: Multiple myeloma (MM) is still incurable due to resistance against various therapies. Thus, the identification of biomarkers predicting progression is urgently needed. Here, we evaluated four biomarkers in bone marrow and peripheral blood of MM patients for their prognostic significance. MATERIALS & METHODS: Bone marrow- and peripheral blood plasma levels of FLT3-L, soluble TIE2, endostatin, and osteoactivin were determined in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 14/n = 4), patients with newly diagnosed MM (NDMM, n = 42/n = 31) and patients with relapsed/refractory MM (RRMM, n = 27/n = 16) by sandwich ELISA. RESULTS: Median FLT3-L expression increased from MGUS (58.77 pg/ml in bone marrow; 80.40 pg/ml in peripheral blood) to NDMM (63.15 pg/ml in bone marrow; 85.05 pg/ml in peripheral blood) and was maximal in RRMM (122 pg/ml in bone marrow; 160.47 pg/ml in peripheral blood; NDMM vs. RRMM p<0.001). A cut-off value of FLT3-L >92 pg/ml in bone marrow and >121 pg/ml in peripheral blood was associated with relapse or refractoriness in MM patients. FLT3-L was found to be a high predictive marker for discrimination between NDMM and RRMM as well in bone marrow as in peripheral blood (AUC 0.75 in bone marrow; vs 0.84 in peripheral blood). CONCLUSION: High levels of FLT3-L in bone marrow and peripheral blood of MM patients identify patients with progressive disease and are associated with relapse or refractoriness in MM patients. FLT3-L could be useful as a marker to identify RRMM patients and should be evaluated as target for future therapies.

CCR10/CCL27 crosstalk contributes to failure of proteasome-inhibitors in multiple myeloma.

The bone marrow microenvironment plays a decisive role in multiple myeloma progression and drug resistance. Chemokines are soluble mediators of cell migration, proliferation and survival and essentially modulate tumor progression and drug resistance. Here we investigated bone marrow-derived chemokines of naive and therapy-refractory myeloma patients and discovered that high levels of the chemokine CCL27, known so far for its role in skin inflammatory processes, correlated with worse overall survival of the patients. In addition, chemokine levels were significantly higher in samples from patients who became refractory to bortezomib at first line treatment compared to resistance at later treatment lines.In vitro as well as in an in vivo model we could show that CCL27 triggers bortezomib-resistance of myeloma cells. This effect was strictly dependent on the expression of the respective receptor, CCR10, on stroma cells and involved the modulation of IL-10 expression, activation of myeloma survival pathways, and modulation of proteasomal activity. Drug resistance could be totally reversed by blocking CCR10 by siRNA as well as blocking IL-10 and its receptor.From our data we suggest that blocking the CCR10/CCL27/IL-10 myeloma-stroma crosstalk is a novel therapeutic target that could be especially relevant in early refractory myeloma patients.

Prevalence of calreticulin exon 9 indel mutations in vascular risk patients.

INTRODUCTION: Acquired JAK2 as well as calreticulin (CALR) mutations are involved in the development of Philadelphia-negative myeloproliferative neoplasms (MPN). We previously showed that the JAK2 V617F mutation could also been found in coronary patients and in patients with peripheral arterial disease (PAD). However, prevalence of CALR mutations is unknown in vascular risk patients and its evaluation subject of the present study. MATERIALS AND METHODS: We determined the prevalence of CALR exon 9 indel mutations in a cohort of 1052 angiographied coronary patients, including 141 patients with PAD, and, additionally, 86 patients with PAD, but without coronary angiography. CALR mutation analysis was performed using PCR fragment analysis and JAK2 V617F mutation analysis with allele-specific real-time PCR. RESULTS: From included 1138 patients, 18 individuals were tested positive for the JAK2 V617F mutation. CALR exon 9 mutations were not detected in any of our patients. CONCLUSIONS: We conclude that CALR exon 9 mutations are infrequent in vascular risk patients. Routine testing for the presence of CALR mutations is not recommended for unselected patients with vascular disease. Future studies are warranted to define a putative pathologic role of CALR mutations in patients without MPN.

Predominant expression of truncated EpCAM is associated with a more aggressive phenotype and predicts poor overall survival in colorectal cancer.

Regulated intramembrane proteolysis (RIP) has been shown to be an important mechanism for oncogenic activation of EpCAM through nuclear translocation of the intracellular domain EpICD. Recently, we identified two different membranous EpCAM variants namely EpCAM(MF) (full-length) and EpCAM(MT) (truncated) to be expressed in the majority of human epithelial tumors. The aim of our study was to evaluate the potential role of these two protein variants as additional prognostic biomarkers in colorectal cancer. In most studies only one antibody targeting the extracellular domain of EpCAM (EpEX) has been used, whereas in the present study additionally an antibody which detects the intracellular domain (EpICD) was applied to discriminate between different EpCAM variants. Using immunohistochemistry, we analyzed the expression of EpCAM(MF) and EpCAM(MT) variants in 640 patients with colorectal cancer and determined their correlations with other prognostic factors and clinical outcome. A statistically significant association was observed for EpCAM(MT) with advanced tumor stage (p < 0.001), histological grade (p = 0.01), vascular (p < 0.001) and marginal (p = 0.002) invasion. Survival analysis demonstrated reduced overall survival (p < 0.004) in patients with tumors expressing the EpCAM(MT) phenotype when compared to patients with tumors expressing the EpCAM(MF) variant. In conclusion, this study for the first time indicates that expression of EpCAM(MT) is associated with a more aggressive phenotype and predicts poor survival in patients with colorectal cancer.

Biomarkers of evasive resistance predict disease progression in cancer patients treated with antiangiogenic therapies.

Numerous antiangiogenic agents are approved for the treatment of oncological diseases. However, almost all patients develop evasive resistance mechanisms against antiangiogenic therapies. Currently no predictive biomarker for therapy resistance or response has been established. Therefore, the aim of our study was to identify biomarkers predicting the development of therapy resistance in patients with hepatocellular cancer (n = 11), renal cell cancer (n = 7) and non-small cell lung cancer (n = 2). Thereby we measured levels of angiogenic growth factors, tumor perfusion, circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP) and tumor endothelial markers (TEM) in patients during the course of therapy with antiangiogenic agents, and correlated them with the time to antiangiogenic progression (aTTP). Importantly, at disease progression, we observed an increase of proangiogenic factors, upregulation of CEC/CEP levels and downregulation of TEMs, such as Robo4 and endothelial cell-specific chemotaxis regulator (ECSCR), reflecting the formation of torturous tumor vessels. Increased TEM expression levels tended to correlate with prolonged aTTP (ECSCR high = 275 days vs. ECSCR low = 92.5 days; p = 0.07 and for Robo4 high = 387 days vs. Robo4 low = 90.0 days; p = 0.08). This indicates that loss of vascular stabilization factors aggravates the development of antiangiogenic resistance. Thus, our observations confirm that CEP/CEC populations, proangiogenic cytokines and TEMs contribute to evasive resistance in antiangiogenic treated patients. Higher TEM expression during disease progression may have clinical and pathophysiological implications, however, validation of our results is warranted for further biomarker development.