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Familial inheritance in non-medullary thyroid cancer (NMTC) is an area that has yet to be adequately explored. Despite evidence suggesting strong familial clustering of non-syndromic NMTC, known variants still account for a very small percentage of the genetic burden. In a recent whole genome sequencing (WGS) study of five families with several NMTCs, we shortlisted promising variants with the help of our in-house developed Familial Cancer Variant Prioritization Pipeline (FCVPPv2). Here, we report potentially disease-causing variants in checkpoint kinase 2 (CHEK2), Ewing sarcoma breakpoint region 1 (EWSR1) and T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) in one family. Performing WGS on three cases, one probable case and one healthy individual in a family with familial NMTC left us with 112254 variants with a minor allele frequency of less than 0.1%, which was reduced by pedigree-based filtering to 6368. Application of the pipeline led to the prioritization of seven coding and nine non-coding variants from this family. The variant identified in CHEK2, a known tumor suppressor gene involved in DNA damage-induced DNA repair, cell cycle arrest, and apoptosis, has been previously identified as a germline variant in breast and prostate cancer and has been functionally validated by Roeb et al. in a yeast-based assay to have an intermediate effect on protein function. We thus hypothesized that this family may harbor additional disease-causing variants in other functionally related genes. We evaluated two further variants in EWSR1 and TIAM1 with promising in silico results and reported interaction in the DNA-damage repair pathway. Hence, we propose a polygenic mode of inheritance in this family. As familial NMTC is considered to be more aggressive than its sporadic counterpart, it is important to identify such susceptibility genes and their associated pathways. In this way, the advancement of personalized medicine in NMTC patients can be fostered. We also wish to reopen the discussion on monogenic vs polygenic inheritance in NMTC and instigate further development in this area of research.

Multiple myeloma (MM) is a malignancy of plasma cells, characterized by the presence of monoclonal immunoglobulin, known as M protein1. MM is preceded by monoclonal gammopathy of undetermined significance (MGUS) which is also a precursor of immunoglobulin light chain (AL) amyloidosis. Previous studies have reported a 2- to 4-fold increased risk of MGUS or MM in first-degree relatives of MM or MGUS patients, suggesting the existence of inherited susceptibility. For many years, high-risk germline predisposing genes have been lacking for MM. However, recent sequencing efforts have proposed a few novel candidates, most notably loss-offunction (LoF) variants in the tumor suppressor gene DIS3 and in the histone demethylase gene KDM1A, and others as recently reviewed in detail in Pertesi et al. In addition to the suspected rare, high-penetrance variants, genome-wide association studies have identified over 20 common, low-penetrance variants associated with the risk of MM; these were estimated to account for about 15% of the familial MM risk.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancers (CRC) and the remaining genetic burden may be due to rare high- to moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer-predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants: a coding variant in APC downregulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5' untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additionalAPCDD1variants in a cohort of 1705 familial CRC patients and no furtherHDAC5variants. Proliferation assays indicated an insignificant proliferative impact for theAPCDD1variant. Luciferase reporter assays using theHDAC5variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by theHDAC5variant showed a significant impact of TCF4 on promoter activity of mutatedHDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5'UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

Highlights • That Sphingolipid derivatives are promising drug candidates for the management of novel COVID-19 disease. • C-1P based tailoring of Th1 effector immunity for the eradication of infection is a translationally viable approach and deserves immediate attention. • That C-1P would promote the killing of infected cells and resolve infection in moderate to severely infected cases. • Ceramide derivatives can be exploited as drug candidates for controlling SARS-CoV-2 against novel COVID-19 disease., Sphingolipids are potent bioactive agents involved in the pathogenesis of various respiratory bacterial infections. To date, several sphingolipid derivatives are known, but S1P (Sphingosine-1-phosphate) and Ceramide are the best-studied sphingolipid derivatives in the context of human diseases. These are membrane-bound lipids that influence host-pathogen interactions. Based on these features, we believe that sphingolipids might control SARS-CoV-2 infection in the host. SARS-CoV-2 utilizes the ACE-II receptor (Angiotensin-converting enzyme II receptor) on epithelial cells for its entry and replication. Activation of the ACE-II receptor is indirectly associated with the activation of S1P Receptor 1 signaling which is associated with IL-6 driven fibrosis. This is expected to promote pathological responses during SARS-CoV-2 infection in COVID-19 cases. Given this, mitigating S1P signaling by application of either S1P Lyase (SPL) or S1P analog (Fingolimod / FTY720) seems to be potential approach for controlling these pathological outcomes. However, due to the immunosuppressive nature of FTY720, it can modulate hyper-inflammatory responses and only provide symptomatic relief, which may not be sufficient for controlling the novel COVID-19 infection. Since Th1 effector immune responses are essential for the clearance of infection, we believe that other sphingolipid derivatives like Cermaide-1 Phosphate with antiviral potential and adjuvant immune potential can potentially control SARS-CoV-2 infection in the host by its ability in enhancing autophagy and antigen presentation by DC to promote T cell response which can be helpful in controlling SARS-CoV-2 infection in novel COVID-19 patients.

Colorectal cancer (CRC) is the third most known type of cancer in developing countries and is one of the leading causes of deaths around the world. The current treatment therapies for CRC are increasing nowadays since there are large variations seen among individuals in terms of diagnosis and response to drugs used to manage CRC owing to biomolecular heterogeneity. Around 15% of CRC cases are due to defect in the DNA mismatch repair (MMR) system, featured by microsatellite instability (MSI) characteristics. We highliht the role of DNA mismatch repair genes in CRC, its diagnosis and management including targeted therapies and immune checkpoint blockade therapies for controlling the individual CRC therapy response and treatment.

Background The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. Methods In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. Results Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. Conclusions We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

Background Based on the possible shared mechanisms of chemotherapy-induced peripheral neuropathy (CIPN) for different drugs, we aimed to aggregate results of all previously published genome-wide association studies (GWAS) on CIPN, and to replicate them within a cohort of multiple myeloma (MM) patients. Methods Following a systematic literature search, data for CIPN associated single nucleotide polymorphisms (SNPs) with P-values

Superalloys with burr-free parts are most preferably used in biomedical, aerospace, marine and automotive applications. In order to reduce the global pollution content, industries strive to execute stringent green manufacturing technologies. There is a need to investigate the different available tools in high-speed micro-milling process to achieve desired burr free with good surface finish on super alloys without using traditional coolants. In machining of titanium and its alloys, because of low thermal conductivity and reactivity with tool materials instigate the burr formation on work material and lowers the tool performance. The main objective of this article is to investigate the top burr formation in high-speed micro-end milling of alpha + beta-titanium alloy-grade 23 ELI (Ti-6Al-4V) under dry cutting conditions using Uncoated and physical vapor deposition coated AlTiN, TiAlN tungsten carbide end mills. Machining performance of the three cutting tools was compared. From the comparison of cutting tools for machining titanium alloy-grade 23, it is found that coated TiAlN tools produce less burr formation than coated AlTiN and uncoated tungsten carbide tools.

Simple Summary Radiomimetic drugs induce extensive genotoxic insults to their target cells. Irreparable DNA damage leaves cells with the choice between a program leading to cell death or senescence, but not DNA repair. Among the challenges of an advanced stage of small cell lung carcinoma (SCLC), the resistance to radiomimetic drugs is the most prominent one. In SCLC, the initial chemotherapeutic treatment primes cell to modify their DNA repair and cell cycle regulatory systems, using alternative but highly efficient forms of DNA repair and auxiliary factors. This modulated system now bypasses several regulatory controls. Thus, at this stage, cells become resistant to any beneficial effects of chemotherapeutic drugs. In the present study, we observed that variant-V of the receptor for advanced glycation end-products (RAGE) is abundantly expressed in advancing and metastasizing SCLC. Therefore, it may serve as a potential target for specific therapeutic interventions directed to SCLC. Abstract Small cell lung carcinoma (SCLC) is a highly aggressive malignancy with a very high mortality rate. A prominent part of this is because these carcinomas are refractory to chemotherapies, such as etoposide or cisplatin, making effective treatment almost impossible. Here, we report that elevated expression of the RAGE variant-V in SCLC promotes homology-directed DNA DSBs repair when challenged with anti-cancer drugs. This variant exclusively localizes to the nucleus, interacts with members of the double-strand break (DSB) repair machinery and thus promotes the recruitment of DSBs repair factors at the site of damage. Increased expression of this variant thus, promotes timely DNA repair. Congruently, the tumor cells expressing high levels of variant-V can tolerate chemotherapeutic drug treatment better than the RAGE depleted cells. Our findings reveal a yet undisclosed role of the RAGE variant-V in the homology-directed DNA repair. This variant thus can be a potential target to be considered for future therapeutic approaches in advanced SSLC.

Introduction: The risk of developing Multiple Myeloma (MM) is 2-4 fold higher in first-degree relatives of patients with MM compared to the general population, suggesting genetic predisposition to this cancer. Indeed, recent genome-wide association studies have identified common risk alleles that predispose for MM. Yet, the impact of these variants on MM risk is too low to explain familial aggregation of MM. High-impact alleles have been identified for other cancers such as ovarian and breast cancer (BRCA1,-2) and melanoma (CDKN2A) but the search for such alleles in MM is still in its infancy. In order to identify high-impact alleles in MM we have performed whole genome/exon sequencing (WGS/WES) in members of MM high risk families. Methods: We included 21 families with multiple cases of MM/MGUS. Whole genome/exome sequencing was performed on a total of 46 affected and 20 unaffected family members. Filtering and prioritization of the variants were performed in accordance with the criteria of our in-house familial cancer variant prioritization pipeline version 2 (FCVPPv2). Loss-of-function variants were further screened using MutPred-LOF, Translate tool and IntOGen/c-BioPortal in order to discriminate pathogenic and neutral variants, to translate a nucleotide sequence to a protein sequence and to visualize the domain affected by the variant and the portion of the protein lost after the newly formed stop codon. Variants were analyzed for predicted effects on splicing by using Human Splicing Finder. Results: We found a total of 148 potentially pathogenic variants, 109 non-synonymous and 39 LOF, in 18 out of 21 MM families. Among our genes, many affect protein metabolism, immune system, and other have known links to carcinogenesis. Additionally, some of them are known to interact with key signaling pathways in MM, including PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, JAK/STAT, NF-κB, Wnt/β-catenin, and RANK/RANKL/OPG, showing congruency with previously reported literature. Interestingly, we also found different missense variants in the same two genes in two unrelated families. Conclusions: We have identified potentially pathogenic gene variants in 85% of MM/MGUS families. Our results can offer a useful reference to gene finding efforts by others in order to improve screening, early diagnosis and personalized therapy of individuals at risk of developing MM. Disclosures Durie: Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Goldschmidt:Merck Sharp and Dohme (MSD): Research Funding; Molecular Partners: Research Funding; Incyte: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

To circumvent the problem of haphazard movement of trucks in the Kandla port area, it becomes important to plan the parking terminal to satisfy the desired demand for parking of all goods-carrying vehicles under the influence of the port. This will result in time and cost savings for all the associated transport agencies. In lieu of that the vehicle may charge an entry fee to meet out the construction and maintenance cost. The study is taken up to access the financial feasibility of the project for the concession period of 10 years. Hence, a financial analysis is carried out to determine the viability of the idea. The objective of such a study was to ensure that a project is worth investment or not. The financial evaluation of the project is carried out considering three growth scenarios: normal, optimistic, and pessimistic with three levels of occupancy (100, 80, and 70%). Three patterns of parking charges are also considered for providing useful tool to the policy makers for implementing the project.

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight into the biological basis of MM.

Relapse of T-cell acute lymphoblastic leukemia (T-ALL) has a dismal prognosis, with only 20% of afflicted children surviving.[1][1] Children with relapsed T-ALL are commonly treated within high-risk arms of second-line treatment protocols that include mandatory hematopoietic stem cell

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that accounts for approximately 15% of pediatric acute lymphoblastic leukemias. A variety of genetic events affecting cellular processes such as the cell cycle, differentiation and survival have been identified in

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The NOTCH1 signaling pathway is essential for hematopoiesis and a critical regulatory step for T-cell proliferation and maturation. The E3 ubiquitin ligase FBXW7 controls NOTCH1 protein stability. Mutations in NOTCH1/FBXW7 activate NOTCH signaling and are of prognostic significance in patients with T-cell acute lymphoblastic leukemia (T-ALL). In this study we analyzed NOTCH1 and FBXW7 mutations in 50 South Indian T-ALL patients treated by a modified ALL BFM 95 regimen. The hot spot exons (HD-N, HD-C, TAD, and PEST) of NOTCH1 and exons 9 of the 10 of FBXW7 were polymerase chain reaction amplified and sequenced. In total, 20 of the 50 (40%) T-ALL patients revealed heterozygous mutations in the NOTCH1 domains, and a predominance of missense mutations in HD-N (70%) and PEST (15%) domains. FBXW7 mutations were detected in 5 of the 50 (10%) T-ALL patients. T-ALL patients with NOTCH1/FBXW7 mutations expressed higher protein level of NOTCH1 compared with patients without NOTCH1/FBXW7 mutations. Six of the mutations detected in NOTCH1 were not reported previously. When tested in a Dual Luciferase Renilla reporter assay some of these conferred increased NOTCH activity, suggesting that these are activating mutations. Importantly, 13 of the 20 (65%) NOTCH1/FBXW7-mutated T-ALL patients showed a good prednisone response (P=0.01) and a better clinical outcome compared with NOTCH1/FBXW7 nonmutated patients (P=0.03). These data suggest that NOTCH1/FBXW7 mutations are present in T-ALL patients from Southern India and may be useful biomarkers to predict prognosis in T-ALL.

Super alloys are widely used in many applications such as aerospace, automotive, biomedical, marine, mining and oil industries. In high-speed micro end milling (HSMEM) of super alloys, the proper selection of cutting tool is necessary to achieve good surface quality without burrs. Surface roughness characterisation is crucial on the work materials to achieve the desired production rate in limited time and at low cost. In this research work, surface roughness characterisation was investigated in order to opt for the better possible tool for machining Near Alpha Titanium Alloy Grade-12 under feasible conditions. The influence of machining parameters i.e. cutting speed, feed and depth of cut on surface roughness was investigated in the present work. Experiments were conducted under dry cutting conditions using Uncoated, PVD Coated TiAlN & AlTiN tungsten carbide tools. It was observed that machining with uncoated tungsten carbide tools generates less surface roughness on near alpha titanium alloy.

Relapsed T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology as it is often resistant to treatment and fatal. In the search for genes that define critical steps of relapse and can serve as prognostic markers we compared 67 relapses (REL) and 147 samples collected at initial diagnosis (INI) by targeted sequencing of 313 leukemia-related genes. In addition to the analysis of single nucleotide variants (SNV) and small insertions and deletions (InDels), we made use of the available coverage data for profiling of copy number alterations (CNA). Of the 147 INI patients, 31 were treated according to the ALL-BFM 2000 and 116 according to the AIEOP-BFM ALL 2009 protocol. All REL patients were recruited from the ALL-REZ BFM 2002 trial. We analyzed bone marrow DNA by targeted capture of 313 genes (5964 exons) using the Haloplex Target Enrichment Kit (Agilent). We used Varscan to detect both SNV and InDels. In the absence of available remission samples, we subtracted known SNPs (dbSNP, 1000 gp) and variants present in at least one of 20 non-leukemic samples that we sequenced in parallel to the patients' samples. Only mutations with an AF >10% were considered. Copy number analysis based on read-depth data was validated by MLPA analysis of 14 genes showing a sensitivity of 99% and by low coverage WGS. In total, we have SNV data on all 147 INI and 67 REL patients and CNA data on 144 and 58 patients, respectively. Altogether, we identified relapse specific genetic events in 32 of 67 RELs. Our results confirm that NT5C2 mutations are highly enriched in relapse (REL: 17/67 vs. INI: 1/147; p=0.0001). Although activation of NT5C2 was associated with the occurrence of early first relapse (p=0.02), it did not correlate with induction failure and therefore has no prognostic impact. Similarly, amplifications of chromosome 17 q11.2-24.3, a region that contains genes of the STAT and ABCA families were significantly more frequent in REL than in INI (REL 7/58 vs. INI 3/144, p=0.0068), but also had no prognostic implications. By contrast, TP53 mutations were highly predictive of a second event: all 8 patients who carried a total of 9 TP53 mutations and deletions died within 9 months after first relapse (p=0.002), whereas 17 of the other 58 (29%) survived. Inactivation of TP53 was significantly correlated with higher mutation rates in other genes compared to those with wild-type TP53 (ttest= p In conclusion, targeted sequencing of relapsed T-ALL identified a molecular signature predicting an exquisitely poor outcome in 50% of relapses, who failed salvage treatment. This group of patients does not benefit from current treatment strategies thus identifying a subgroup with a dire clinical need for experimental therapy. Notably, approx. 25% REL patients who failed induction carried RAS and IL7R mutations. These patients may be considered for personalized treatment with either MEK- or JAK/STAT-inhibitors. Another 4 patients carry TP53 missense mutations and may benefit from treatment with p53 refolding compounds. Disclosures No relevant conflicts of interest to declare.

Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75(th) percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P

Despite improvements in treatment results for pediatric T-cell acute lymphoblastic leukemia, approximately 20% of patients relapse with dismal prognosis. PTEN inactivation and NOTCH1 activation are known frequent leukemogenic events but their effect on outcome is still controversial. We analyzed the effect of PTEN inactivation and its interaction with NOTCH1 activation on treatment response and long-term outcome in 301 ALL-BFM treated children with T-cell acute lymphoblastic leukemia. We identified PTEN mutations in 52 of 301 (17.3%) of patients. In univariate analyses this was significantly associated with increased resistance to induction chemotherapy and a trend towards poor long-term outcome. By contrast, patients with inactivating PTEN and activating NOTCH1 mutations showed marked sensitivity to induction treatment and excellent long-term outcome, which was similar to patients with NOTCH1 mutations only, and more favorable than in patients with PTEN mutations only. Notably, in the subgroup of patients with a prednisone- and minimal residual disease (MRD)-response based medium risk profile, PTEN-mutations without co-existing NOTCH1-mutations represented an MRD-independent highly significant high-risk biomarker. Mutations of PTEN highly significantly indicate a poor prognosis in T-ALL patients who have been stratified to the medium risk group of the BFM-protocol. This effect is clinically neutralized by NOTCH1 mutations. Although these results have not yet been explained by an obvious molecular mechanism, they contribute to the development of new molecularly defined stratification algorithms. Furthermore, these data have unexpected potential implications for the development of NOTCH1 inhibitors in the treatment of T-cell acute lymphoblastic leukemia in general, and in those with a combination of PTEN and NOTCH1 mutations in particular.

According to the GLOBOCAN statistics, cervical cancer is one of the leading causes of death among women worldwide. It is found to be gradually increasing in the younger population, specifically in the developing countries. We analyzed the protein-protein interaction networks of the uterine cervix cells for the normal and disease states. It was found that the disease network was less random than the normal one, providing an insight into the change in complexity of the underlying network in disease state. The study also portrayed that, the disease state has faster signal processing as the diameter of the underlying network was very close to its corresponding random control. This may be a reason for the normal cells to change into malignant state. Further, the analysis revealed VEGFA and IL-6 proteins as the distinctly high degree nodes in the disease network, which are known to manifest a major contribution in promoting cervical cancer. Our analysis, being time proficient and cost effective, provides a direction for developing novel drugs, therapeutic targets and biomarkers by identifying specific interaction patterns, that have structural importance.

Novel treatment approaches are needed for patients with acute lymphoblastic leukemia (ALL) who respond poorly to current therapies. The genotypic diversity identified recently by next generation sequencing technologies within ALL calls for individualized novel strategies. However, functional correlations of oncogenic lesions with drug response profiles are ill defined for ALL. We have established an imaging-based cell viability analysis platform to generate drug response profiles for primary patient-derived ALL samples co-cultured with mesenchymal stroma cells, expecting to derive functional information directly from individual patient samples. Such response profiles may mirror perturbations in relevant cellular programs that could be exploited therapeutically. Our pipeline integrates high-content screening, newly developed bioinformatics tools and biochemical approaches. We screened a library of 65 compounds for activity in 37 precursor B-ALL and 23 T-ALL samples including refractory cases. Cross-sample comparisons revealed that cells from relapsed refractory patients showed a more resistant phenotype for most of the drugs. While only a few agents including genotoxic drugs showed activity across all samples, we detected selective activity of given drugs that distinguish patient sample groups. MLL-rearranged and TCF3-HLF-positive ALL samples as well as a subgroup of T-ALL cases were highly sensitive to the BCL-2 specific BH3-mimetic ABT-199 suggesting BCL-2 dependency for these cases. Multiparametric analyses of in vitro responses predicted in vivo activity of ABT-199 in xenografted mice. Moreover, we could identify synergistic activity of ABT-199 with clinical and preclinical compounds, such as topotecan or epigenetic modifiers. Our drug response profiling pipeline contributes to the identification of distinct vulnerabilities in leukemia and may facilitate the selection of candidate drugs for further development into clinical application. Citation Format: Viktoras Frismantas, Anna Rinaldi, Maria Pamela Dobay, Salome Higi, Sabrina Eugster, Blerim Marovca, Peter Horvath, Mauzro Delorenzi, Joachim Kunz, Obul R. Bandapalli, Gunnar Cario, Martin Stanulla, Andreas E. Kulozik, Martina Muckenthaler, Cornelia Eckert, Thomas Radimerski, Jean-Pierre Bourquin, Beat C. Bornhauser. Drug response profiling to inform individualized treatment approaches in high risk leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2015-493

Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms “extracellular matrix,” “cell communication,” “response to biotic stimulus,” “structural molecule activity” and “cell growth,” indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale. © 2005 Wiley-Liss, Inc.

IL7R-activating mutations identified in B-ALL and T-ALL patient leukemic cells facilitate cytokine-independent growth., Interleukin-7 receptor α (IL7R) is required for normal lymphoid development. Loss-of-function mutations in this gene cause autosomal recessive severe combined immune deficiency. Here, we describe somatic gain-of-function mutations in IL7R in pediatric B and T acute lymphoblastic leukemias. The mutations cause either a serine-to-cysteine substitution at amino acid 185 in the extracellular domain (4 patients) or in-frame insertions and deletions in the transmembrane domain (35 patients). In B cell precursor leukemias, the mutations were associated with the aberrant expression of cytokine receptor-like factor 2 (CRLF2), and the mutant IL-7R proteins formed a functional receptor with CRLF2 for thymic stromal lymphopoietin (TSLP). Biochemical and functional assays reveal that these IL7R mutations are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells. A cysteine, included in all but three of the mutated IL-7R alleles, is essential for the constitutive activation of the receptor. This is the first demonstration of gain-of-function mutations of IL7R. Our current and recent observations of mutations in IL7R and CRLF2, respectively suggest that the addition of cysteine to the juxtamembranous domains is a general mechanism for mutational activation of type I cytokine receptors in leukemia.

Abstract 740 Despite recent advances in treatment results of pediatric T-ALL, approximately 20% of the patients relapse and then have a dismal prognosis. Therefore, we aimed to identify molecular biomarkers that help in stratifying patients according to risk early in the course of treatment. We have analysed the clinical interaction of PTEN and NOTCH1 mutations in a cohort of 301 children with T-ALL who were treated on ALL-BFM protocols. We found heterozygous exon 7 PTEN mutations in 50 patients (17%). PTEN mutations occurred in 19% of male and 9% of female patients (p=0.05) and were more common in leukemias without (21%) than in those with (12%) activating NOTCH1 mutations (p=0.04). Patients with PTEN mutations showed a poor prednisone response (PPR) significantly more frequently than those patients without PTEN mutations (58% vs. 37%; p= 0.007; odds ratio 2.4; 95%CI = 1.3–4.4, p=0.006). Furthermore, in a multivariate analysis including variables known to be associated with prednisone response (sex, age at diagnosis, presenting WBC count at diagnosis and T-cell immunophenotype), the negative effect of PTEN mutations retained its significant effect (odds ratio =2.6, 95% CI= 1.3–5.2, p=0.005). On day 33, only 4% of the patients with PTEN mutations showed a favorable MRD response as compared to 29% of PTEN non-mutated patients (p=0.001). On day 78, the MRD response remained unfavorable significantly more frequently than in patients without a PTEN mutation, although this difference was less pronounced (57% vs. 39%; p = 0.05). Logistic regression analysis showed that patients with PTEN mutations carried a 9.2 fold higher risk of not achieving a favorable MRD level on day 33 (95% CI 2.2–3.9, p=0.003) and a 2.1 fold higher risk for an unfavorable MRD on day 78 (95% CI 1.1–1.4, p=0.02). In a multivariate analysis with known variables to be associated with treatment response (sex, age at diagnosis, presenting WBC count at diagnosis and T-cell immunophenotype), the negative effect of PTEN mutation retained its significant effect on day 33 (Odds ratio= 11, 95% CI= 2.5–48.5, p= 0.001) and on day 78 (Odds ratio =2, 95% CI= 1–4.1, p=0.05). These differences between PTEN-mutated and PTEN-non-mutated patients in early treatment response were maintained as a trend towards an inferior pEFS of 0.82 vs. 0.70 (p=0.07). We next tested how PTEN mutations clinically interact with NOTCH1 mutations. The comparison of early treatment response showed that leukemias with NOTCH1 and PTEN mutations had a similar rate of PPR and unfavorable MRD day 33 responses as those leukemias with PTEN mutations only (61% vs. 56%, p=0.8; 94% vs. 96 %, p=1), whereas patients with NOTCH1 mutations only exhibited the known low rate of PPR and unfavorable day 33 MRD response (27%, p=0.006 and 64%, p=0.008). Leukemias with neither mutation showed intermediate rates of PPR (48%) and unfavorable day 33 MRD response (79%). These data indicate that the unfavorable effect of PTEN mutations dominates over the favorable effect of NOTCH1 activation during early induction. By contrast, the analysis of MRD responses on day 78 and of long term survival showed that the favorable effect of NOTCH1 neutralizes the unfavorable effect of PTEN. The groups with both, PTEN and NOTCH1 mutations or NOTCH1 mutations only show a similar pEFS of 0.88 and 0.87, respectively, while the group with PTEN mutations only shows a significantly inferior pEFS of 0.61. These results indicate that PTEN mutations represent unfavorable biomarkers in BFM-treated pediatric T-ALL patients when not combined with NOTCH1 mutations and that NOTCH1 activation neutralizes this unfavorable effect at the end of induction and in long- term outcome. Activating NOTCH1 mutations thus remain the dominant favorable risk marker in BFM-treated children with T-ALL. Disclosures: No relevant conflicts of interest to declare.

About 15% of colorectal cancer (CRC) patients have first-degree relatives affected by the same malignancy. However, for most families the cause of familial aggregation of CRC is unknown. To identify novel high-to-moderate-penetrance germline variants underlying CRC susceptibility, we performed whole exome sequencing (WES) on four CRC cases and two unaffected members of a Polish family without any mutation in known CRC predisposition genes. After WES, we used our in-house developed Familial Cancer Variant Prioritization Pipeline and identified two novel variants in the solute carrier family 15 member 4 (SLC15A4) gene. The heterozygous missense variant, p. Y444C, was predicted to affect the phylogenetically conserved PTR2/POT domain and to have a deleterious effect on the function of the encoded peptide/histidine transporter. The other variant was located in the upstream region of the same gene (GRCh37.p13, 12_129308531_C_T; 43 bp upstream of transcription start site, ENST00000266771.5) and it was annotated to affect the promoter region of SLC15A4 as well as binding sites of 17 different transcription factors. Our findings of two distinct variants in the same gene may indicate a synergistic up-regulation of SLC15A4 as the underlying genetic cause and implicate this gene for the first time in genetic inheritance of familial CRC.

Background When germline mutations are suspected as causal in cancer, patient DNA may be sequenced to detect variants in relevant genes. If a particular mutation has not been reported in reliable family studies, genetic counselors are facing a dilemma of appropriately informing patients. Many sequencing facilities provide an interpretation of the findings based on the available sequence databases or on prediction tools that are curated from bioinformatics and mechanistic datasets. The counseling dilemma is exacerbated if the pedigree data are not informative but the in silico predictions suggest pathogenicity. Methods We present here a real world example of the c.256G > A CDKN2A variant, which was detected in one melanoma patient where two siblings were diagnosed with melanoma in situ. We investigated a detailed family history of the affected siblings in order to survey probability of the cancer risks within the context to this mutation. Results This c.256G > A CDKN2A variant was detected in one of the brothers and in the melanoma-free mother while the other brother in the family tested negative. The variant had been previously described in one patient from a melanoma family. In the family under investigation, the mother’s 16 first-and second-degree relatives had survived past the median onset age for melanoma and none presented melanoma. We tested the variant using multiple bioinformatic tools that all predicted deleteriousness of the variant. The genetic counseling report to the melanoma patient stated that the CDKN2A variant was ‘likely pathogenic’ and the disease was defined as ‘likely hereditary melanoma’. Conclusions The pedigree data showed at the most a low penetrance variant, which, if taken into consideration, might have altered the provided diagnosis. When dealing with ‘practically’ unknown variants the counselors would be advised to incorporate a detailed family history rather than basing predictions on functionality provided by sequencing facilities.

Background Tissue inhibitors of metalloproteinases (TIMPs) and the corresponding metalloproteinases are integral parts of the protease network and have been shown to be involved in cancer development and metastasis. Paradoxically, for TIMP-1, tumor promoting as well as tumor inhibitory effects have been observed. Methods To address this paradox, we utilized the BALB/c/CT26 mouse model that reliably leads to liver metastasis after splenic tumor cell injection and variegated the type of target cells for therapeutic intervention and the modalities of gene transfer. Since we have observed before that over-expression of TIMP-1 in liver host cells leads to efficient tumor growth inhibition in this model, we now examined whether targeting the tumor cells themselves will have a similar effect. Results In concordance with the earlier results, TIMP-1 over-expression in tumor cells led to a dramatic reduction of tumor growth as well. To evaluate any influence of treatment modality, we further examined whether TIMP-1 knockdown in the same animal model would have the opposite effect on tumor growth than TIMP-1 over-expression. Indeed, TIMP-1 knockdown led to a marked increase in tumor burden. Conclusion These data indicate that in the BALB/c/CT26 model, the modification of TIMP-1 has concordant effects irrespective of the type of target cell or the technique of modulation of TIMP-1 activity, and that TIMP-1 is unequivocally tumor inhibitory in this model.