Significant Genetic Alterations Research Articles

Size distribution of cell-free DNA in oncology.

Tumor-specific, circulating cell-free DNA (cfDNA) in liquid biopsy test is a novel promising biomarker in the advancement of cancer management, including early diagnosis, screening, prognosis, identification of actionable targets, and serial tumor monitoring. The specific size pattern of DNA fragments derived from cancer cells is observed to differ from that of cfDNA fragments shed by non-cancer cells. Research into the physiological and biological properties of cfDNA reveals the molecular signature carried by each cfDNA fragments, which can reflect their tissue origins, as well as the mutational profiles with significant genetic alterations. Understanding the fragmentation and size distribution of cfDNA might be a valuable hotspot in liquid biopsy research, with the potential to drive innovation in oncology.

Next-generation sequencing in thyroid cancers: do targetable alterations lead to a therapeutic advantage?: A multicenter experience.

Radioiodine-refractory thyroid cancers (IRTCs) are uncommon and have a poor prognosis. Treatment options for radioiodine-refractory and anaplastic tumors (ATCs) are limited. Although the genomic landscape of thyroid cancer has been studied, there is little evidence on whether next-generation sequencing (NGS) findings translate to tumor control.We analyzed all patients with IRTC and ATC who underwent commercially available NGS in 3 cancer centers.Twenty-two patients were identified, 16 patients with IRTCs and 6 patients with ATCs. Eighteen (82%) had targetable findings in NGS, nine patients were treated accordingly. Median progression-free survival for targeted treatment was 50 months [95% confidence interval (CI95%) 9.8–66.6] and2 months (CI95% 0.2–16.5) for IRTC and ATC, respectively. Of 4 patients who achieved durable responses of 7 to 50 months, 2 are ongoing. The estimated median OS of IRTC receiving targeted treatment was not reached (CI95% 89.7–111.4 months) and was 77.8 months (CI95% 52.5–114.6) for patients treated conventionally (P = .3).NGS may detect clinically significant genetic alterations and benefit patients with advanced thyroid cancers.

What Every Neuropathologist Needs to Know: Practical Aspects and Pitfalls in Molecular Diagnosis of Brain Tumors.

Molecular testing has become part of the routine diagnostic workup of brain tumors after the implementation of integrated histomolecular diagnoses in the 2016 WHO classification update. It is important for every neuropathologist to be aware of practical preanalytical, analytical, and postanalytical factors that impact the performance and interpretation of molecular tests. Prior to testing, optimizing tumor purity and tumor amount increases the ability of the molecular test to detect the genetic alteration of interest. Recognizing basic molecular testing platform analytical characteristics allows selection of the optimal platform for each clinicopathological scenario. Finally, postanalytical considerations to properly interpret molecular test results include understanding the clinical significance of the detected genetic alteration, recognizing that detected clinically significant genetic alterations are occasionally germline constitutional rather than somatic tumor-specific, and being cognizant that recommended and commonly used genetic nomenclature may differ. Potential pitfalls in brain tumor molecular diagnosis are also discussed.

Effect of An 84-bp Deletion of the Receptor-Binding Domain on the ACE2 Binding Affinity of the SARS-CoV-2 Spike Protein: An In Silico Analysis.

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the Spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the RBD that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the ACE2 receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understand key elements of Spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.

Plasma biopsy by Tag-sequencing: an acceptable alternative to tumor tissue profiling in non-small-cell lung cancer.

Tag-sequencing is amodified next-generation sequencing (NGS) approach wherein targeted regions are tagged with unique molecular identifiers enabling error-free detection ofrare genomic alterations. We aimed to perform this high- fidelity sequencing to identify actionable variants from theplasma oflung cancer patients. Targeted sequencing was performed from plasma-derived cell-free nucleic acid in twenty-one advanced, treatment naïve, non-small-cell lung cancer (NSCLC) patients. Clinically significant genetic alterations were compared with matched tumor NGS profile for each patient (patient-level), and separately for each alteration (variant-level). Cross-platform validation was done for EGFR and KRAS mutations (real-time PCR) and ALK1 rearrangement (immunohistochemistry). Forty-seven alterations (26 in plasma and 21 in tumor tissue) were detected in 19/21 tested cases. Overall-concordance between thetwo assays was 94.87% (κ of0.71, 95% CI: 0.54-0.89). Patient-level and genic-concordance was 57.1% (12/21 cases) and 67.86%, respectively. Almost perfect agreement was reached for detecting actionable EGFR mutations and ALK1 rearrangement (κ of0.89 and κ of1, respectively), which was confirmed by single-gene testing. Substantial agreement between theassays makes Tag-sequencing aviable option for identifying multibiomarkers from theplasma ofadvanced NSCLC patients in special circumstances where tissue has depleted/tumor is inaccessible/high risk ofbiopsy due to existing comorbidities.

Abstract IA06: Molecular classification of diffuse large B-cell lymphoma

Abstract Key PointsSequencing-based approaches are readily used for molecular classifications of diffuse large B-cell lymphomaGenetically distinct diffuse large B-cell lymphoma provides insights into unique lymphomagenesis, prognosis prediction, and combination of targeted treatmentsGenetic classifiers identify novel vulnerabilities and inform clinical trial designs Heterogeneity of Diffuse Large B-Cell Lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell non-Hodgkin lymphoma and is thought to arise from antigen-exposed B cells. Although the majority of patients with DLBCL are curable with combination immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), a substantial fraction of patients develop recurrent or progressive disease that is often fatal. The clinical heterogeneity of these tumors prompted the development of various classification schemes and included molecular classifiers that i.) improved the accuracy of diagnosis, ii.) identified relevant molecular subtypes with distinct biology, iii.) developed prognostic models for relevant clinical endpoints, and iv.) stratified patients for disease management. The heterogeneity of DLBCL is partially captured in clinically and/or transcriptionally defined subtypes that provide insights into disease pathogenesis and candidate treatment targets. However, patient stratification for treatment-based transcriptional subtypes, including the activated B-cell type (ABC) and the germinal center B-cell type (GCB), has been largely unsuccessful thus far and suggested that additional heterogeneity had not been fully captured. Technical advantages paved the way for next-generation sequencing (NGS)-based techniques to be included in clinical and/or low-throughput-based molecular classifiers. These technologies allowed detection and prioritization of all genetic alteration types—recurrent mutations, somatic copy number alterations (SCNAs), and structural variants (SVs) —followed by an integration and assessment of their temporal ordering and associated transcriptional profiles. Genetic Heterogeneity of DLBCL. Recently, two independent comprehensive genomic efforts at base-pair resolution of hundreds of patients diagnosed with primary DLBCL have captured the complex intrinsic genetic heterogeneity of these lymphomas and have shed light on the genomic architecture of DLBCL. Integration of significant genetic alterations composed of recurrent somatic mutations, SCNAs and SVs, resulted in the discovery of at least 5 genetically distinct DLBCL subsets that predicted outcome to state-of-the-art frontline treatment, suggesting new insights into the lymphomagenesis of DLBCLs and rational combination treatments. These 5 subsets included: 1) a high-risk ABC DLBCLs with near-uniform BCL2 copy gain, frequent activating MYD88 and CD79B mutations, and extranodal tropism (C5 DLBCLs); 2) favorable-risk ABC-DLBCLs with genetic features of an extrafollicular, possibly marginal zone origin (C1 DLBCLs); 3) poor-risk GCB-DLBCLs with BCL2 SVs, inactivating mutations, and/or copy loss of PTEN and alterations of epigenetic enzymes (C3 DLBCLs); 4) a good-risk GCB-DLBCLs with distinct alterations in BCR/PI3K, JAK/STAT, and BRAF pathway components and multiple histones (C4 DLBCLs); and 5) an ABC/GCB-independent group of tumors with biallelic inactivation of TP53, 9p21.3/CDKN2A, and associated genomic instability (C2 DLBCLs). The biology of these 5 DLBCL subsets was largely confirmed by an independent non-overlapping large-scale study that followed a completely different analytical approach and discovered similar groups with shared pathogenetic mechanisms. In particular, C1 DLBCLs were similar to BN2 (BCL6/NOTCH2), C3 DLBCLs were similar to EZB (EZH2/BCL2), and C5 DLBCLs were similar to MCD (MYD88/CD79B). In subsequent work, the biology of the remaining DLBCL subtypes (C2 and C4 DLBLCs) was recently independently validated by two other groups (C4 ~ ST2; C2 ~ A53 | C4 ~ SGK1; C2 ~ TP53). These studies underscore that DLBCL is a genetically heterogenous disease with at least 5-7 different molecular subtypes. At the same time, they also highlight that despite having several hundreds of sequenced tumors, we still do not capture the full spectrum of DLBCL subsets, and for low-frequency subtypes in particular larger sample sizes are needed. More work is needed, to link genetically defined subtypes to actionable vulnerabilities, and eventually build molecular classifiers that link genetics to dependencies with the hope of using them for precision medical trials. Citation Format: Bjoern Chapuy. Molecular classification of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA06.

Molecular and clinicopathologic features of gliomas harboring NTRK fusions

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) genes are detected in ≤2% of gliomas and can promote gliomagenesis. The remarkable therapeutic efficacy of TRK inhibitors, which are among the first Food and Drug Administration-approved targeted therapies for NTRK-fused gliomas, has generated significant clinical interest in characterizing these tumors. In this multi-institutional retrospective study of 42 gliomas with NTRK fusions, next generation DNA sequencing (n = 41), next generation RNA sequencing (n = 1), RNA-sequencing fusion panel (n = 16), methylation profile analysis (n = 18), and histologic evaluation (n = 42) were performed. All infantile NTRK-fused gliomas (n = 7) had high-grade histology and, with one exception, no other significant genetic alterations. Pediatric NTRK-fused gliomas (n = 13) typically involved NTRK2, ranged from low- to high-histologic grade, and demonstrated histologic overlap with desmoplastic infantile ganglioglioma, pilocytic astrocytoma, ganglioglioma, and glioblastoma, among other entities, but they rarely matched with high confidence to known methylation class families or with each other; alterations involving ATRX, PTEN, and CDKN2A/2B were present in a subset of cases. Adult NTRK-fused gliomas (n = 22) typically involved NTRK1 and had predominantly high-grade histology; genetic alterations involving IDH1, ATRX, TP53, PTEN, TERT promoter, RB1, CDKN2A/2B, NF1, and polysomy 7 were common. Unsupervised principal component analysis of methylation profiles demonstrated no obvious grouping by histologic grade, NTRK gene involved, or age group. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, MAPK, and other pathways. In summary, the study highlights the clinical, histologic, and molecular heterogeneity of NTRK-fused gliomas, particularly when stratified by age group.

Non-amplification genetic alterations of HER2 gene in non-small cell lung carcinoma

AimsThe present study investigated the incidence and spectrum of human epidermal growth factor receptor 2 (HER2) mutations, associated clinicopathological characteristics and the co-occurrence of HER2 gene amplification in the HER2...

Next generation sequencing reveals CCNE1 amplification as an independent prognostic factor for triple negative breast cancer (TNBC) patients.

558 Background: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer due to the heterogeneity as well as lack of better therapeutic approach. It remains controversial whether BRCA status is the predictor of survival in TNBC. Besides, both germline and somatic mutation may contribute to the prognosis. This study is to explore the potential predictors and therapeutic targets based on genetic data and clinicopathological parameters. Methods: Seventy-five TNBC patients were enrolled with approximately 2:1 based on BRCA status. Genetic data was analysed by comprehensive genomic profiling 508 key cancer related genes. DAVID was applied to perform pathway enrichment analysis of significant enriched genetic alterations. Cox regression model was applied to evaluate disease-free survival (DFS) and overall survival (OS). Immuno-chemistry (IHC) was used to validate clinically meaningful genetic alteration. Results: In this study, 27 germline mutations were detected, including 26 homologous recombination repair (HRR) pathway gene mutations and 1 mismatch repair gene mutation among them 16 BRCA1 mutations and 5 BRCA2 mutations were found. Germline HRR including BRCA1/2 mutation marginally affected DFS ( p = 0.0624 and 0.15, respectively). We found 480 somatic genetic alterations including 110 copy number variations (CNV). The median value of TMB was determined to be 4.1 Muts/Mb which divided 74 TNBC patients into TMB-low (TMB-l) and TMB-high (TMB-h) group. TMB-l group had inferior DFS to TMB-h ( p = 0.0457). CCNE1 (with 5% frequency) copy number gain was specifically enriched in TMB-l group but mutually exclusive with BRCA1/2 mutation. TNBC with CCNE1 gain displayed worse DFS ( p< 0.0001). Cox multivariate regression analysis indicated CCNE1 gain was an independent risk factors for DFS [HR = 13.48 (95% CI 2.62-69.23), p= 0.002)]. Pathway analysis indicated CCNE1 harmed prognosis through regulation of transcription in G1/S phase. Expression of cyclin E1 was validated by IHC, which would be presented later. Conclusions: Comprehensive genomic profiling disclosed various potential prognostic markers for TNBC by integrating clinical characters. Especially, amplified CCNE1 may be a potential prognostic marker and therapeutic target. [Table: see text]

Establishment of patient derived xenograft model of high-grade mucinous carcinoma peritonei accompanied with signet ring cells and identification of biological characteristics

Objective: To establish the patient derived xenograft (PDX) model of pseudomyxoma peritonei (PMP), and identify the key characteristics of tumor biology of this model, in order to provide a reliable model for studying the pathological mechanisms and new therapeutic strategies of PMP. Methods: PMP tumor tissue was obtained from surgery and cut into pieces after washing. Then tumor pieces were implanted subcutaneously in BAL B/c-nu mice for 6 stable passages. In the 7th passage, tumor tissue was implanted orthotopically into abdomen. Subcutaneous tumor and orthotopic tumor were then homogenized to make tumor cell suspension, implanted into abdomen of 10 BAL B/c-nu mice through midline laparotomy, 100 μl for each. The key experimental parameters including body weight changes in the observation period, experimental peritoneal cancer index (ePCI) score at the autopsy, histopathological and immunohistochemical characteristics, and gene expression profiles by high-throughput whole-genome exon sequencing were detected and recorded. Results: The successful rate of established orthotopic PDX model of human PMP was 100% (10/10). The animals showed smooth body weight increases after tumor inoculation until day 27, then the body weight began to decrease steadily. Widespread tumor dissemination of PMP tumor through the whole abdomen was found by autopsy, including the diaphragm, liver, spleen, stomach, kidney, parietal peritoneum, bowel and mesenterium. Gelatinous ascites was also observed in abdominopelvic cavity. The ePCI score ranged from 5 to 9, with a 8 of median ePCI. Histopathological studies showed peritoneal mucinous carcinomatosis accompanied with signet ring cells (PMCA-S), obvious tumor cell atypia and parenchymal invasion.Immunohistochemistry showed the expressions of MUC1, MUC2, MUC5AC, CEA, CA199, CK20, CDX-2 and Ki-67 were positive, MUC6, CK7 and p53 were negative. Whole-exome sequencing identified that the most significant genetic alteration is the exon10 missense mutation c. 1621A>C of KIT gene, the mutation abundance was 89.7%. Conclusion: PDX model of PMCA-S is successfully established, which displays the characters of high-degree malignancy, high proliferation and strong aggressiveness.

Next-generation sequencing in salivary gland carcinoma: Targetable alterations lead to a therapeutic advantage-Multicenter experience.

Salivary gland cancers (SGCs) are rare. The approach to metastatic patients is histology-dependent. There is little evidence on whether next-generation sequencing (NGS) findings translate to tumor control in SGCs. We analyzed all patients with histologically confirmed SGC who underwent NGS. Twenty-seven patients were identified, 14 (51.8%) had targetable findings in NGS: 5 ERBB2 amplifications, 3 PIK3CA mutations, 2 RUNX1 mutations, 1 TRIM33-RET fusion, 1 FGFR3-TACC3 fusion, 1 microsatellite instability-high, and 2 high mutational burden. Ten patients were treated accordingly. Median progression-free survival for targeted treatment was 8.4 months. Of five patients who achieved durable responses of 8.4 to 31.3 months, two are ongoing. The overall median survival was not reached for patients receiving targeted treatment and was 40.4 months for patients treated conventionally (P = .18). In the absence of a well-established therapeutic approach, NGS may detect clinically significant genetic alterations and benefit patients with advanced SGC.

Significantly dysregulated genes in osteoarthritic labrum cells identified through gene expression profiling.

The aim of the present study was to explore the molecular basis and identify significant genetic alterations in acetabular labrum cells associated with osteoarthritis (OA). Gene expression data of osteoarthritic and normal human labrum cells were downloaded from a public database and reanalyzed. Significant differentially expressed genes (DEGs) were acquired by performing a thorough analysis of microarray data between the OA acetabular labrum cells and control cells. Key genes in OA labrum cells were revealed by a combination of weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis. Literature mining and drug screening were further performed for these key genes. In total, 141 DEGs between OA and normal labrum cells were identified. In addition, WGCNA and PPI analysis identified 23 DEGs as key genes in the OA labrum. All the key genes were significantly downregulated in OA labrum cells and were grouped into two different WGCNA-PPI common subnetworks. Kinase insert domain receptor (KDR), CD34, cadherin 5 (CDH5), Fms related tyrosine kinase 1 (FLT1) and asporin were hub nodes in the PPI network of DEGs. These key genes were significantly enriched in functional clusters of transforming growth factor, alkaline phosphatase, bone morphogenic protein and extracellular matrix. Drug screening analysis identified several drugs targeting the key genes, including arachidonic acid, yohimbic acid and mimosine. The results of the present study indicate that the changes of FLT1, KDR, CD34 and CDH5 in acetabular labrum cells may be involved in the pathogenesis of OA and could serve as biomarkers and therapeutic targets of OA. Additionally, arachidonic acid, yohimbic acid and mimosine may act as potential drugs for OA.

Clinical Utility of GlioSeq Next-Generation Sequencing Test in Pediatric and Young Adult Patients With Brain Tumors

Brain tumors are the leading cause of death in children. Establishing an accurate diagnosis and therapy is critical for patient management. This study evaluated the clinical utility of GlioSeq, a next-generation sequencing (NGS) assay, for the diagnosis and management of pediatric and young adult patients with brain tumors. Between May 2015 and March 2017, 142 consecutive brain tumors were tested using GlioSeq v1 and subset using GlioSeq v2. Out of 142 samples, 63% were resection specimens and 37% were small stereotactic biopsies. GlioSeq sequencing was successful in 100% and 98.6% of the cases for the detection of mutations and copy number changes, and gene fusions, respectively. Average turnaround time was 8.7 days. Clinically significant genetic alterations were detected in 95%, 66.6%, and 66.1% of high-grade gliomas, medulloblastomas, and low-grade gliomas, respectively. GlioSeq enabled molecular-based stratification in 92 (65%) cases by specific molecular subtype assignment (70, 76.1%), substantiating a neuropathologic diagnosis (18, 19.6%), and diagnostic recategorization (4, 4.3%). Fifty-seven percent of the cases harbored therapeutically actionable findings. GlioSeq NGS analysis offers rapid detection of a wide range of genetic alterations across a spectrum of pediatric brain tumors using formalin-fixed, paraffin-embedded specimens and facilitates integrated molecular-morphologic classification and personalized management of pediatric brain tumors.

Next Generation Sequencing in Salivary Gland Carcinomas: Do Actionable Mutations Lead to a Therapeutic Advantage? A Multicenter Experience.

e17582 Background: Salivary gland cancers (SGCs) are rare tumors. The approach to recurrent or metastatic patients is histology dependent, as the clinical course is highly variable. Response rates to chemotherapy are modest and no second-line therapies have proven to be effective. The rarity and heterogeneity of SGCs make conducting clinical trials challenging. Recent reports of Next Generation Sequencing (NGS) in SGCs have identified unique genetic attributes of certain histological classes. However, despite the elaborate work done with genetic characterization, there is very little evidence on whether these findings translate to tumor control. In this study, we aimed to explore the treatment outcomes of patients with advanced SGCs who underwent a commercially available NGS. Methods: A retrospective analysis of all patients with histologically confirmed SGC treated in three major Israeli cancer centers who underwent NGS. Results: Twenty-seven patients were included. Eighteen patients were male (66.6%). The median age was 59 (range 38-81). Histologic type was adenoid cystic carcinoma in 11 (40.7%) patients, 8 (29.6%) mucoepidermoid carcinomas, 5 (18.5%) adenocarcinomas, 2 (7.4%) salivary duct carcinomas and 1 (3.7%) acinic cell carcinoma. Twenty-five (92.6%) had recurrent disease after initial curative treatment and 2 were initially diagnosed with metastatic disease. Median time to recurrence was 24.3 months. The median number of systemic treatment lines was 1 (range 0-5). Thirteen patients (48%) had targetable findings in NGS, including: 5 ERBB2 amplifications, 3 PIK3CA mutation, 1 TRIM33-RET fusion, 1 FGFR3-TACC3 fusion, 1 MSI-high and 2 high mutational burden. Nine patients were treated accordingly; 5 in the second-line and 4 as first-line treatment. Median progression-free survival for targeted treatment was 8.4 months. Of 4 patients who achieved durable responses to anti-RET/HER2 agents and immunotherapy of 8.4 – 31.1 months, two are ongoing. The median overall survival for all patients was 101.75 months, was not reached for patients receiving targeted treatment and was 40.4 months for patients treated conventionally (p = 0.26). Conclusions: In the absence of a well-established therapeutic approach, NGS may detect clinically significant genetic alterations and benefit patients with advanced SGCs.

Genetic changes caused by restocking and hydroelectric dams in demographically bottlenecked brown trout in a transnational subarctic riverine system.

Habitat discontinuity, anthropogenic disturbance, and overharvesting have led to population fragmentation and decline worldwide. Preservation of remaining natural genetic diversity is crucial to avoid continued genetic erosion. Brown trout (Salmo trutta L.) is an ideal model species for studying anthropogenic influences on genetic integrity, as it has experienced significant genetic alterations throughout its natural distribution range due to habitat fragmentation, overexploitation, translocations, and stocking. The Pasvik River is a subarctic riverine system shared between Norway, Russia, and Finland, subdivided by seven hydroelectric power dams that destroyed about 70% of natural spawning and nursing areas. Stocking is applied in certain river parts to support the natural brown trout population. Adjacent river segments with different management strategies (stocked vs. not stocked) facilitated the simultaneous assessment of genetic impacts of dams and stocking based on analyses of 16 short tandem repeat loci. Dams were expected to increase genetic differentiation between and reduce genetic diversity within river sections. Contrastingly, stocking was predicted to promote genetic homogenization and diversity, but also potentially lead to loss of private alleles and to genetic erosion. Our results showed comparatively low heterozygosity and clear genetic differentiation between adjacent sections in nonstocked river parts, indicating that dams prevent migration and contribute to genetic isolation and loss of genetic diversity. Furthermore, genetic differentiation was low and heterozygosity relatively high across stocked sections. However, in stocked river sections, we found signatures of recent bottlenecks and reductions in private alleles, indicating that only a subset of individuals contributes to reproduction, potentially leading to divergence away from the natural genetic state. Taken together, these results indicate that stocking counteracts the negative fragmentation effects of dams, but also that stocking practices should be planned carefully in order to ensure long‐term preservation of natural genetic diversity and integrity in brown trout and other species in regulated river systems.

CD30-positive primary cutaneous lymphoproliferative disorders: molecular alterations and targeted therapies.

Primary cutaneous CD30-positive T-cell lymphoproliferative disorders are the second most common subgroup of cutaneous T-cell lymphomas. They include two clinically different entities with some overlapping features and borderline cases: lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Molecular studies of primary cutaneous anaplastic large cell lymphoma reveal an increasing level of heterogeneity that is associated with histological and immunophenotypic features of the cases and their response to specific therapies. Here, we review the most significant genetic, epigenetic and molecular alterations described to date in primary cutaneous CD30-positive T-cell lymphoproliferative disorders, and their potential as therapeutic targets.

Fumarate Hydratase Mutations and Alterations in Leiomyoma With Bizarre Nuclei

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

PO-335 EGFR and ALK mutation status in advanced lung adenocarcinoma: prevalence, gender and age-related peculiarities among patients in Ukraine

IntroductionLung adenocarcinoma is a heterogeneous group of tumours with distinct molecular characteristics. The most significant genetic alterations are EGFR mutations and ALK translocation, which prevalence, age and gender distribution are unknown in Ukrainian population of lung cancer patients. In addition, considering the appearance of new anti-ALK antibodies, the sensitivity and specificity of the IHC method for detecting ALK translocation were assessed.Material and methods267 patients with stage IV lung adenocarcinoma were included in the study. All patients underwent EGFR testing by Real-time PCR using EGFR Mutation Analysis Kit (Entrogen). 177 patients were tested for ALK translocation by FISH using ZytoLight SPEC ALK Dual Colour Break Apart Probe (Zytovision). Of these, 109 patients were co-tested for ALK by IHC using anti-ALK antibody clone 5A4 (Novocastra, Leica).Results and discussionsEGFR mutations and ALK translocation were mutually exclusive among observed patients. EGFR mutations were detected in 52 patients (19.5%). The most of cases demonstrated exon 19 deletion (61%); in 27% patients L858R mutation was detected: 4% showed exon 20 insertion; 4% - G719X mutation; 2% - G719X+exon 20 insertion; 2% L858R+T790M (during anti-EGFR TKI treatment). EGFR mutations were more often found in women rather than men (68.25% vs 7.3%; p<0,0001), with no relation to age. ALK translocation was found by FISH in 17 patients (9.6%). There was no relation to sex, however, the strong association with age was found (p=0.0018): ALK-translocation were more often among patients under 50 y.o. Moreover, this relationship was significant among females only (p=0.0175). Assessment of ALK testing by IHC and FISH revealed high concordance between these methods. The accuracy of IHC method was 92.66% (95% CI 86.05% to 96.78%). The negative predictive value was high (98.85%), however the positive predictive value (PPV) was only 68.18% due to false positive results, that requires an additional assessment of IHC-positive samples by FISH.ConclusionThe prevalence of EGFR mutations and ALK translocation among observed patients with advanced lung adenocarcinoma was quite high (19.5% and 9.6%, respectively). EGFR mutations were more common in women comparing with men, whereas ALK translocation was associated with younger age. High accuracy of ALK-detection by IHC allows using it for screening, however due to low PPV, ALK-positive IHC results should be verified by FISH.

Characterization, phylogenetic distribution and evolutionary trajectories of diverse hydrocarbon degrading microorganisms isolated from refinery sludge.

Phylogenic association between bacteria living under harsh conditions can provide important information on adaptive mechanism, survival strategy and their potential application. Indigenous microorganisms isolated from toxic refinery oily sludge with ability to degrade a diverse range of hydrocarbons were identified and characterized. The strains including Pseudomonas aeruginosa RS1, Microbacterium sp. RS2, Bacillus sp. RS3, Acinetobacter baumannii RS4 and Stenotrophomonas sp. RS5 could utilize n-alkanes, cycloalkanes, polynuclear aromatic hydrocarbons (PAHs) with 2-4 rings and also substituted PAHs as sole substrate. The phylogenetic position of Bacillus sp. RS3 and Pseudomonas sp. RS1 was tested by applying the maximum likelihood (ML) method to the aligned 16S rRNA nucleotide sequences of PAH and aliphatic hydrocarbon degrading strains belonging to the corresponding genus. The base substitution matrix created with each set of organisms capable of degrading aromatic and aliphatic hydrocarbons showed significant transitional event with high values of transition: transversion ratio (R) under all conditions. The guanine-cytosine (GC) content of the hydrocarbon degrading test strains was also found to be highest for the clade which harbored them. The test strains consistently occupied a distinct terminal end within the phylogenetic tree constructed by ML analysis. This study reveals that the refinery sludge imposed environmental stress on the bacterial strains which possibly caused significant genetic alteration and phenotypic adaptation. Due to the divergent evolution of the Pseudomonas and Bacillus strains in the sludge, they appeared distinctly different from other hydrocarbon degrading strains of the same genus.

37 - Utilization of a SNP Microarray for Chronic Lymphocytic Leukemia: Efficacy, Informative Findings and Prognostic Capabilities

Chronic lymphocytic leukemia is the most common form of adult-onset leukemia in the Western world. A total of 775 patients who were confirmed to have CLL were studied by microarray analysis which has the potential to identify significant genetic alterations that would remain uncharacterized not targeted by the DNA probes utilized in routine FISH analysis. FISH was negative in 21.2% of the patients but 55.4% of this FISH negative group had aberrations by array studies. Array abnormalities were detected in over 90% of the patients. The array revealed genome complexity in ~ 46% of the patients, compared to 9% by FISH analysis. The array analysis revealed genome complexity in 38% of 13q deletion patients suggesting a poorer outcome in this subgroup. Overall, it can be concluded from these studies that: (1) The array is clearly more effective in detecting abnormalities than FISH alone; (2) Although the array may be slightly less sensitive than FISH it could pick up abnormalities present in at least 10% of the cells; (3) The array delineated genome complexity ~90% of the time that FISH did not and was effective in subdividing 13q deletions into two groups; one with a much more likely chance of being an aggressive disease; (4) The array is effective in detecting anomalies such as CN-LOH (in 17.9% of patients) and chromothripsis (1.6%) that could not be delineated by FISH; (5) The whole genome array was effective in detecting a second disorder such as myelodysplasia that would not be seen by FISH.