Comprehensive Molecular Diagnostics Research Articles

Abstract C021: Racial and socioeconomic disparities in molecular diagnostics for NSCLC patients

Abstract Background: Targeted therapies have substantially changed the landscape of non-small cell lung cancer (NSCLC) outcomes. These drugs work by inhibiting specific molecular pathways, such as the epidermal growth factor receptor (EGFR) tyrosine kinase, the anaplastic lymphoma kinase (ALK), KRAS, and BRAF oncogenic pathways. Approximately 30% of NSCLC patients will have tumors with eligible targetable mutations, but a molecular test must be conducted to assess eligibility to these treatments. Disparities in molecular testing could be partially responsible for the disproportionate utilization of targeted therapy across race and socioeconomic status (SES). Methods: Surveillance, Epidemiology and End Results (SEER)-Medicare linked data was queried for NSCLC patients diagnosed from 2013 (the first year that standard guidelines for molecular testing in lung cancer were available) to 2016. Patients were limited to age over 66 years at diagnosis with continuous Part A and B coverage and no Part C coverage, for 1 year prior and 90 days post-diagnosis. The primary outcome was receipt of a molecular test based on claims data. Race, defined as white, Black or other, was the primary predictor variable. Demographic and clinical characteristics of those receiving vs not receiving a molecular test were compared using χ2 tests for categorical variables and t-tests for continuous variables. We also performed multivariable logistic regression to assess the association of molecular testing with race adjusting for sex, age, marital status, Charlson comorbidity index, histology, tumor site, stage, and census-level poverty indicator. Results: There were 28,511 NSCLC patients, of which 9,639 (33.8%) received some type of molecular testing. Black patients were significantly less likely than white patients to have received a molecular test (p<0.0001). Only 23.9% of Black patients underwent any molecular testing, compared to 34.6% of white patients. Patients living in census tracts with at least 10% of residents living in poverty were also less likely to receive a molecular test (p<0.0001). Patients who were female (p<0.0001), married (p<0.0001), with fewer comorbidities (p<0.0001), adenocarcinoma histology (p<0.0001), tumors located in the lower lobe (p<0.0001), diagnosed at a later stage (p=0.0001) were more likely to have molecular testing. After adjustment, Black patients (ORadj [odds ratio]: 0.65; 95% CI [confidence interval]: 0.59-0.72) and those living in areas with greater poverty (ORadj: 0.87; 95% CI: 0.82-0.91) were statistically significantly less likely to have received molecular testing. Conclusions: Disparities exist in comprehensive molecular diagnostics, and could explain, at least in part, the high mortality observed among Black NSCLC patients. Addressing barriers to molecular testing could increase uptake of targeted therapy treatment, decreasing disparities and improving patient outcomes. Citation Format: Stephanie Tuminello, Wiley Turner, Matthew Untalan, Raja Flores, Emaneula Taioli. Racial and socioeconomic disparities in molecular diagnostics for NSCLC patients [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C021.

Racial and socioeconomic disparities in NSCLC molecular diagnostics uptake.

Precision therapies, such as targeted and immunotherapies, have substantially changed the landscape of late-stage non-small cell lung cancer (NSCLC). Yet utilization of these therapies is disproportionate across strata defined by race and socioeconomic status (SES), possibly due to disparities in molecular diagnostic testing (or "biomarker testing"), which is a prerequisite to treatment. We extracted a cohort of NSCLC patients from the Surveillance, Epidemiology and End Results (SEER)-Medicare linked data. The primary outcome was receipt of a molecular diagnostic test, based on claims data. The primary predictors were race and SES. Likelihood of receiving a molecular diagnostic test, and overall survival (OS), were investigated using logistic and Cox proportional hazards regression, adjusted for sex, age, residence, histology, marital status, and comorbidity. Of the 28,511 NSCLC patients, 11,209 (39.3%) received molecular diagnostic testing. Compared to White patients, fewer Black patients received a molecular diagnostic test (40.4% vs 27.9%; p < .001). After adjustment, Black patients (ORadj [odds ratio]: 0.64; 95% CI [confidence interval]: 0.58-0.71) and those living in areas with greater poverty (ORadj: 0.85; 95% CI: 0.80-0.89) had statistically significant decreased likelihood of molecular diagnostic testing. Patients who did receive testing had a statistically significant decreased risk of death (HRadj [hazards ratio]: 0.74; 95% CI: 0.72-0.76). These results held in the stratified analysis of stage IV NSCLC patients. Disparities exist in comprehensive molecular diagnostics, which is critical for clinical decision making. Addressing barriers to molecular testing could help close gaps in cancer care and improve patient outcomes.

Exploring oral bacterial compositional network in two oral disease groups using a convergent approach of NGS-molecular diagnostics.

Since most of the commonly known oral diseases are explained in link with balance of microbial community, an accurate bacterial taxonomy profiling for determining bacterial compositional network is essential. However, compared to intestinal microbiome, research data pool related to oral microbiome is small, and general 16S rRNA screening method has a taxonomy misclassification issue in confirming complex bacterial composition at the species level. Present study aimed to explore bacterial compositional networks at the species level within saliva of 39 oral disease patients (Dental Caries group: n = 26 and Periodontitis group: n = 13) through comparison with public Korean-specific healthy oral microbiome data. Here, we applied comprehensive molecular diagnostics based on qRT-PCR and Sanger sequencing methods to complement the technical limitations of NGS-based 16S V3-V4 amplicon sequencing technology. As a result of microbiome profiling at the genus level, relative frequencies of many nitrate-reducing bacteria within each oral disease group were found to be significantly low compared to the healthy group. In addition, the molecular diagnostics-based bacterial identification method allowed the determination of the correct taxonomy of screened primary colonizers (Streptococcus and Actinomyces unclassification clusters) for each oral disease. Finally, as with the results of microbiome profiling at the genus level, many core-species classified within the saliva of each oral disease group were also related to nitrate-reduction, and it was estimated that various pathogens associated with each disease formed a bacterial network with the core-species. Our study introduced a novel approach that can compensate for the difficulty of identifying an accurate bacterial compositional network at the species level due to unclear taxonomy classification by using the convergent approach of NGS-molecular diagnostics. Ultimately, we suggest that our experimental approach and results could be potential reference materials for researchers who intend to prevent oral disease by determining the correlation between oral health and bacterial compositional network according to the changes in the relative frequency for nitrate-reducing species.

Primary Results of Patients with Genitourinary Malignancies Presented at a Molecular Tumor Board

Introduction: Personalized medicine poses great opportunities and challenges. While the therapeutic landscape markedly expands, descriptions about status, clinical implementation and real-world benefits of precision oncology and molecular tumor boards (MTB) remain sparse, particularly in the field of genitourinary (GU) cancer. Hence, this study characterized urological MTB cases to better understand the potential role of MTB in uro-oncology. Methods: We analyzed patients with complete data sets being reviewed at an MTB from January 2019 to October 2022, focusing on results of molecular analysis and treatment recommendations. Results: We evaluated 102 patients with GU cancer with a mean patient age of 61.7 years. Prostate cancer (PCa) was the most frequent entity with 52.9% (54/102), followed by bladder cancer (18.6%, 19/102) and renal cell carcinoma (14.7%, 15/102). On average, case presentation at MTB took place 54.9 months after initial diagnosis and after 2.7 previous lines of therapy. During the study period, 49.0% (50/102) of patients deceased. Additional MTB-based treatment recommendations were achieved in a majority of 68.6% (70/102) of patients, with a recommendation for targeted therapy in 64.3% (45/70) of these patients. Only 6.7% (3/45) of patients – due to different reasons – received the recommended MTB-based therapy though, with 33% (1/3) of patients reaching disease control. Throughout the MTB study period, GU cancer case presentations and treatment recommendations increased, while the time interval between initial presentation and final therapy recommendation were decreasing over time. Conclusion: Presentation of uro-oncological patients at the MTB is a highly valuable measure for clinical decision-making. Prospectively, earlier presentation of patients at the MTB and changing legislative issues regarding comprehensive molecular testing and targeted treatment approval might further improve patients’ benefits from comprehensive molecular diagnostics.

Is Carbapenem Therapy Necessary for the Treatment of Non-CTX-M Extended-Spectrum β-Lactamase-Producing Enterobacterales Bloodstream Infections?

Investigations into antibiotics for extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) bloodstream infections (BSIs) have focused on blaCTX-M genes. Patient outcomes from non-CTX-M-producing ESBL-E BSIs and optimal treatment are unknown. A multicenter observational study investigating 500 consecutive patients with ceftriaxone-resistant Enterobacterales BSIs during 2018-2022 was conducted. Broth microdilution and whole-genome sequencing confirmed antibiotic susceptibilities and ESBL gene presence, respectively. Inverse probability weighting (IPW) using propensity scores ensured patients with non-CTX-M and CTX-M ESBL-E BSIs were similar before outcome evaluation. 396 patients (79.2%) were confirmed to have an ESBL-E BSI. ESBL gene family prevalence was as follows: blaCTX-M (n = 370), blaSHV (n = 16), blaOXY (n = 12), and blaVEB (n = 5). ESBL gene identification was not limited to Escherichia coli and Klebsiella species. In the IPW cohort, there was no difference in 30-day mortality or ESBL-E infection recurrence between the non-CTX-M and CTX-M groups (odds ratio [OR], 0.99; 95% confidence interval [CI], .87-1.11; P = .83 and OR, 1.10; 95% CI, .85-1.42; P = .47, respectively). In an exploratory analysis limited to the non-CTX-M group, 86% of the 21 patients who received meropenem were alive on day 30; none of the 5 patients who received piperacillin-tazobactam were alive on day 30. Our findings suggest that non-CTX-M and CTX-M ESBL-E BSIs are equally concerning and associated with similar clinical outcomes. Meropenem may be associated with improved survival in patients with non-CTX-M ESBL-E BSIs, underscoring the potential benefit of comprehensive molecular diagnostics to enable early antibiotic optimization for ESBL-E BSIs beyond just blaCTX-M genes.

Tailoring Therapeutic Strategies in Non-Small-Cell Lung Cancer: The Role of Genetic Mutations and Programmed Death Ligand-1 Expression in Survival Outcomes

Background: This study aims to assess the real-world impact of advancements in first-line systemic therapies for non-small-cell lung cancer (NSCLC), focusing on the role of driver gene mutations and programmed death-ligand 1 (PD-L1) expression levels. Methods: Conducted across eight medical facilities in Japan, this multicenter, retrospective observational research included 863 patients diagnosed with NSCLC and treated between January 2015 and December 2022. The patients were categorized based on the type of systemic therapy received: cytotoxic agents, molecular targeting agents, immune checkpoint inhibitors, and combination therapies. Comprehensive molecular and immunohistochemical analyses were conducted, and statistical evaluations were performed. Results: The median overall survival (OS) shows significant variations among treatment groups, with targeted therapies demonstrating the longest OS. This study also revealed that high PD-L1 expression was common in the group treated with immune checkpoint inhibitors. Multivariate analysis was used to identify the type of anticancer drug and the expression of PD-L1 at diagnosis as the impactful variables affecting 5-year OS. Conclusions: This study underscores the efficacy of targeted therapies and the critical role of comprehensive molecular diagnostics and PD-L1 expression in affecting OS in NSCLC patients, advocating for their integration into routine clinical practice.

Clinical validation of a single NGS targeted panel pipeline using the KAPA HyperChoice system for detection of germline, somatic and mitochondrial sequence and copy number variants

ABSTRACT Background Comprehensive molecular diagnostics are highly dependent on the technical performance of next-generation sequencing (NGS) pipelines, which are assessed by data quality, cost, turnaround time, and accuracy of detecting a range of sequence and copy number variants. Methods A dataset of 285 clinically validated cases (205 retrospective and 80 prospective), carrying complex sequence and copy number variants and thousands of genetic polymorphisms underwent a clinical validation of the KAPA HyperChoice target enrichment system with parallel sample fidelity assessment across a number of NGS panels. The analysis included assessment of peripheral blood, urine, muscle and FFPE tissues. Results High-quality and exceptionally uniform data with 100% coverage of all targeted panels were obtained, resulting in complete sensitivity and specificity for all variant types across nearly all panels and tissue types. Overall reduction in cost and turnaround times was obtained with the implementation of a parallel genotyping sample fidelity system. Conclusion Results of the laboratory quality improvement study focused on a single NGS pipeline that includes both nuclear and mitochondrial genomes demonstrated utility in the clinical setting to assess a range of referral reasons, necessary due to the complex molecular etiology of human genetic disorders, while reducing costs and turnaround times.

Rare embryonal and sarcomatous central nervous system tumours: State-of-the art and future directions

The introduction of molecular methods into the diagnostics of central nervous system (CNS) tumours and the subsequent deciphering of their molecular heterogeneity has resulted in a significant impact on paediatric neurooncology. Particularly in the field of rare embryonal and sarcomatous CNS tumours, novel tumour types have been delineated and introduced in the recent 5th edition of the WHO classification of CNS tumours. The rarity and novelty of these tumour types result in diagnostic and therapeutic challenges. Apart from distinct histopathological and molecular features, these tumour types exhibit characteristic clinical properties and require different therapeutic approaches for optimal patient management. However, based on the limited availability of clinical data, current therapeutic recommendations have to be based on data from small, predominantly retrospective patient cohorts. Within this article, we provide guidance for diagnostic work-up and clinical management of rare CNS embryonal tumours (‘embryonal tumour with multi-layered rosettes’, ETMR; ‘CNS neuroblastoma, FOXR2-activated’, CNS NB-FOXR2; ‘CNS tumour with BCOR-ITD, CNS BCOR-ITD) and rare CNS sarcomatous tumours (‘primary intracranial sarcoma, DICER1-mutant’, CNS DICER1; ‘CIC-rearranged sarcoma’, CNS CIC). By emphasizing the significant consequences on patient management in paediatric CNS tumours, we want to encourage wide implementation of comprehensive molecular diagnostics and stress the importance for joint international efforts to further collect and study these rare tumour types.

Abstract 810: HRD is inversely correlated with MSI and identifies immunologically cold tumors in most cancer types

Abstract Homologous recombination deficiency (HRD) leads to DNA double strand breaks and can be exploited by the use of PARP inhibitors to induce synthetic lethality of cancer cells. Over the last years, the clinical utility of this therapeutic concept was successfully demonstrated in ovarian, breast, prostate and pancreatic cancer. Currently, the role of HRD is investigated in trials testing immune checkpoint blockers alone or in combination with PARP inhibitors. But the relationship between HRD and immune cell contexture in cancer is incompletely understood. Here, we analyzed the association of HRD with tumor mutational burden (TMB), immune cell composition and gene expression in 10,000 tumors of 32 solid cancer types from the TCGA project. For each of the tumors, the HRD sum score (HRDsum) was calculated from allele-specific copy numbers (derived from genotyping data) and the TMB was calculated from the missense mutation calls (derived from WES data). HRDsum and tumor mutational burden (TMB) correlated positively pan-cancer (R=0.42) and within most cancer types. By contrast, HRD was absent in ultra-hypermutated (TMB &amp;gt;= 100 mut/Mb) tumors. Ultra-hypermutation was typically associated with microsatellite instability (MSI) or POLE/POLD1 mutation. Significant positive correlation of the HRDsum and immune cell infiltration was observed pan-cancer, but only within a few cancer types. Significant positive correlation of HRDsum and the T-cell inflamed gene expression profile was observed only in 7 of 32 cancer types: in breast cancer, ovarian cancer, low grade glioma, testicular germ cell tumors and three kinds of kidney cancer. A functional genomics analysis was carried out by correlating genome-wide gene expression data (derived from RNA-Seq) with HRDsum. The resulting lists of significantly correlating genes were analyzed for enrichment of 50 hallmark gene sets (catalog H, MSigDB). We detected simultaneous enrichment of two proliferation-related categories, E2F_TARGETS and G2M_checkpoint, for 13 of the 32 cancer types (ACC, BLCA, BRCA, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, PRAD, SARC, THYM). The study shows that HRD is associated with immunological activation of the tumor microenvironment only in a minority of cancer types. Our data support further studies of immune activating therapies in combination with immune checkpoint blockade in the not intrinsically activated cancer types and advocate to combine different genomic and transcriptomic biomarkers (including HRD and TMB) for comprehensive molecular diagnostics and therapy guidance. Citation Format: Jan Budczies, Klaus Kluck, Susanne Beck, Iordanis Ouralidis, Michael Allgäuer, Michael Menzel, Eugen Rempel, Daniel Kazdal, Lukas Perkhofer, Alexander Kleger, Peter Schirmacher, Thomas Seufferlein, Albrecht Stenzinger. HRD is inversely correlated with MSI and identifies immunologically cold tumors in most cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 810.

Quantum Cascade Laser-Based Infrared Imaging as a Label-Free and Automated Approach to Determine Mutations in Lung Adenocarcinoma

Therapeutic decisions in lung cancer critically depend on the determination of histologic types and oncogene mutations. Therefore, tumor samples are subjected to standard histologic and immunohistochemical analyses and examined for relevant mutations using comprehensive molecular diagnostics. In this study, an alternative diagnostic approach for automatic and label-free detection of mutations in lung adenocarcinoma tissue using quantum cascade laser-based infrared imaging is presented. For this purpose, a five-step supervised classification algorithm was developed, which was not only able to detect tissue types and tumor lesions, but also the tumor type and mutation status of adenocarcinomas. Tumor detection was verified on a data set of 214 patient samples with a specificity of 97% and a sensitivity of 95%. Furthermore, histology typing was verified on samples from 203 of the 214 patients with a specificity of 97% and a sensitivity of 94% for adenocarcinoma. The most frequently occurring mutations in adenocarcinoma (KRAS, EGFR, and TP53) were differentiated by this technique. Detection of mutations was verified in 60 patient samples from the data set with a sensitivity and specificity of 95% for each mutation. This demonstrates that quantum cascade laser infrared imaging can be used to analyze morphologic differences as well as molecular changes. Therefore, this single, one-step measurement provides comprehensive diagnostics of lung cancer histology types and most frequent mutations.

1788. The Utility of Next-Generation Sequencing for Detection of Candidate Pathogens in Bronchoalveolar Lavage Fluid from Pediatric Patients with Respiratory Failure

BackgroundIn the field of infectious diseases, identification of etiologic pathogen is essential for definitive diagnosis and decisions regarding appropriate management. Bronchoalveolar lavage fluid (BALF) is considered a sterile type of specimen that is suitable for detecting pathogens of respiratory infections. Recently, next-generation sequencing (NGS) has been applied in the field of infectious diseases and has enabled us to identify pathogenic microorganisms comprehensively. The aim of this study was to comprehensively identify pathogens using NGS in BALF samples from immunocompetent pediatric patients with respiratory failure.MethodsTen patients hospitalized in the pediatric intensive care unit with respiratory failure were included. BALF samples obtained in the acute phase were used to prepare DNA- and RNA-sequencing libraries. The libraries were sequenced on MiSeq, and the sequence data were analyzed using metagenome analysis tools.ResultsA mean of 2,041,216 total reads were sequenced for each library. A significant number of four types of bacterial reads was detected in three BALF samples with DNA-sequencing, whereas pathogenic respiratory viruses were detected in seven of 10 patients with RNA-sequencing.Candidate pathogens were detected in three patients in whom etiologic agents were not identified by conventional methods. A summary of the detected pathogens is listed in Table 1. Sequence coverage and depth of each reference bacterial and viral genome are shown in Figures 1 and 2, respectively. The complete genome of enterovirus D68 was identified in two patients without underlying diseases, and phylogenetic analysis suggested that both strains belong to subclade B3, which is an epidemic strain that has spread worldwide in recent years.ConclusionWe demonstrated the utility of the NGS-based approach for detection of candidate pathogens in BALF from pediatric patients with severe respiratory failure. Our results suggest that NGS can be applied for comprehensive molecular diagnostics as well as surveillance of pathogens in the field of infectious diseases. Disclosures All authors: No reported disclosures.

Metagenomic analysis using next-generation sequencing of pathogens in bronchoalveolar lavage fluid from pediatric patients with respiratory failure

Next-generation sequencing (NGS) has been applied in the field of infectious diseases. Bronchoalveolar lavage fluid (BALF) is considered a sterile type of specimen that is suitable for detecting pathogens of respiratory infections. The aim of this study was to comprehensively identify causative pathogens using NGS in BALF samples from immunocompetent pediatric patients with respiratory failure. Ten patients hospitalized with respiratory failure were included. BALF samples obtained in the acute phase were used to prepare DNA- and RNA-sequencing libraries. The libraries were sequenced on MiSeq, and the sequence data were analyzed using metagenome analysis tools. A mean of 2,041,216 total reads were sequenced for each library. Significant bacterial or viral sequencing reads were detected in eight of the 10 patients. Furthermore, candidate pathogens were detected in three patients in whom etiologic agents were not identified by conventional methods. The complete genome of enterovirus D68 was identified in two patients, and phylogenetic analysis suggested that both strains belong to subclade B3, which is an epidemic strain that has spread worldwide in recent years. Our results suggest that NGS can be applied for comprehensive molecular diagnostics as well as surveillance of pathogens in BALF from patients with respiratory infection.

SwissMTB: establishing comprehensive molecular cancer diagnostics in Swiss clinics

BackgroundMolecular precision oncology is an emerging practice to improve cancer therapy by decreasing the risk of choosing treatments that lack efficacy or cause adverse events. However, the challenges of integrating molecular profiling into routine clinical care are manifold. From a computational perspective these include the importance of a short analysis turnaround time, the interpretation of complex drug-gene and gene-gene interactions, and the necessity of standardized high-quality workflows. In addition, difficulties faced when integrating molecular diagnostics into clinical practice are ethical concerns, legal requirements, and limited availability of treatment options beyond standard of care as well as the overall lack of awareness of their existence.MethodsTo the best of our knowledge, we are the first group in Switzerland that established a workflow for personalized diagnostics based on comprehensive high-throughput sequencing of tumors at the clinic. Our workflow, named SwissMTB (Swiss Molecular Tumor Board), links genetic tumor alterations and gene expression to therapeutic options and clinical trial opportunities. The resulting treatment recommendations are summarized in a clinical report and discussed in a molecular tumor board at the clinic to support therapy decisions.ResultsHere we present results from an observational pilot study including 22 late-stage cancer patients. In this study we were able to identify actionable variants and corresponding therapies for 19 patients. Half of the patients were analyzed retrospectively. In two patients we identified resistance-associated variants explaining lack of therapy response. For five out of eleven patients analyzed before treatment the SwissMTB diagnostic influenced treatment decision.ConclusionsSwissMTB enables the analysis and clinical interpretation of large numbers of potentially actionable molecular targets. Thus, our workflow paves the way towards a more frequent use of comprehensive molecular diagnostics in Swiss hospitals.

Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

High-throughput parallel amplicon sequencing of common driver mutations from FFPE lung cancer samples in molecular pathologic routine diagnostics for a regional health care provider network.

e12517 Background: Treatment paradigms for non–small-cell lung cancer have shifted from histology based towards incorporation of molecular subtypes involving particular genetic alterations such as mutations in EGFR or translocations of ALK. The list of targetable lesions is rapidly increasing including mutations in genes such as EGFR, HER2, KRAS, ALK, BRAF, PIK3CA, AKT1, ROS1, NRAS, FGFR1 and MAP2K1. Analysis of these potential targets is becoming a challenge in terms of work load, tissue availability as well as cost. Within the Network Genomic Medicine Lung Cancer (NGM), a regional molecular screening network of the Center for Integrated Oncology Köln Bonn, we aimed to improve the sequential analysis of a set of 9 target amplicons by Sanger sequencing using bench top ultra-deep parallel sequencing platforms. We aimed to reduce 1) the time requirement for comprehensive molecular diagnostics, 2) the minimal amount of formalin fixed paraffin embedded (FFPE) derived input DNA, 3) while at the same time increasing the number of target regions analysed. Methods: We established a multiplex PCR to amplify up to 640 lung cancer relevant target regions from at least 20ng of FFPE derived tumor DNA. The amplicon libraries were ligated to adapters encompassing medical identifier sequences that allowed multiplexing of up to 48 patients. The resulting libraries were sequenced on a benchtop Illumina platform (MiSeq). Mutations identified by parallel sequencing were confirmed by Sanger sequencing. Results: 330 patients were analyzed by traditional Sanger sequencing of 9 amplicons and the newly established parallel sequencing protocol. The time needed to complete the mutation screening was significantly reduced to 7 working days from previously 21 days. A total of at least 300ng of DNA was needed to complete the analysis of 9 amplicons by Sanger sequencing compared to 20 to 100ng of DNA needed for up to 640 amplicons analyzed by parallel sequencing. Conclusions: Newly multiplex PCR based parallel sequencing allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small transbronchial biopsies.

Complete metabolic response in a patient with repeatedly relapsed non-small cell lung cancer harboring ROS1 gene rearrangement after treatment with crizotinib

A 55-year-old Caucasian woman with lung adenocarcinoma stage IV presented with repeated relapse after treatment with cytotoxic chemotherapy (carboplatin, gemcitabine, docetaxel, pemetrexed) and targeted agents (erlotinib, cetuximab, sunitinib). Comprehensive molecular diagnostics (EGFR-, ALK-, RAS-, BRAF-, PIK3CA-, HER2- and DDR2-aberrations) were performed and failed initially to detect any driver mutation. While the patient suffered from rapid deterioration of her general condition, in particular from progressive dyspnea due to lung metastases, we implemented screening for RET- and ROS1 translocations into our molecular diagnostic program based on recent reports of these new molecular subgroups in lung adenocarcinoma. On retesting the patient's tumor sample was found to harbor a ROS1-translocation. The patient was subsequently treated with crizotinib and experienced a pronounced clinical improvement corresponding to a complete metabolic response in 18F-FDG-PET and a good and confirmed partial response in CT (RECIST 1.1). Our case exemplifies the need for rapid implementation of newly discovered rare genetic lung cancer subtypes in routine molecular diagnostics.

Extended mutation spectrum of Usher syndrome in Finland

The Finnish distribution of clinical Usher syndrome (USH) types is 40% USH3, 34% USH1 and 12% USH2. All patients with USH3 carry the founder mutation in clarin 1 (CLRN1), whereas we recently reported three novel myosin VIIA (MYO7A) mutations in two unrelated patients with USH1. This study was carried out to further investigate the USH mutation spectrum in Finnish patients. We analysed samples from nine unrelated USH patients/families without known mutations and two USH3 families with atypically severe phenotype. The Asper Ophthalmics USH mutation chip was used to screen for known mutations and to evaluate the chip in molecular diagnostics of Finnish patients. The chip revealed a heterozygous usherin (USH2A) mutation, p.N346H, in one patient. Sequencing of MYO7A and/or USH2A in three index patients revealed two novel heterozygous mutations, p.R873W in MYO7A and c.14343+2T>C in USH2A. We did not identify definite pathogenic second mutations in the patients, but identified several probably nonpathogenic variations that may modify the disease phenotype. Possible digenism could not be excluded in two families segregating genomic variations in both MYO7A and USH2A, and two families with CLRN1 and USH2A. We conclude that there is considerable genetic heterogeneity of USH1 and USH2 in Finland, making molecular diagnostics and genetic counselling of patients and families challenging.

Survey of fungi and yeast in polymicrobial infections in chronic wounds

To assess the incidence, abundance and species diversity of fungi in chronic wounds, as well as to describe the associations of major fungi populations. Comprehensive molecular diagnostic reports were evaluated from a total of 915 chronic wounds in a retrospective study. Of the 915 clinical specimens, 208 (23%) were positive for fungal species. These samples were further compared in a compiled dataset, and sub-classified among the four major chronic wound types (decubitus ulcer, diabetic foot ulcer, non-healing surgical wound, and venous leg ulcer). The most abundant fungi were yeasts in the genus Candida; however, Curvularia, Malessezia, Aureobasidium, Cladosporium, Ulocladium, Engodontium and Trichtophyton were also found to be prevalent components of these polymicrobial infections. A notable bacterial/fungal negative correlation was found to be apparent between Staphylococcus and Candida. There were also significant relationships between both bacterial and fungal genera and patient metadata including gender, diabetes status and cardiovascular comorbidities. This microbial survey shows that fungi are more important wound pathogens and opportunistic pathogens than previously reported, exemplifying the impact of these under-reported pathogens. With the application of modern cost-effective and comprehensive molecular diagnostics, clinicians can now identify and address this significant component of chronic wound bioburden with targeted therapies, thereby improving healing trajectories.

Healing and healing rates of chronic wounds in the age of molecular pathogen diagnostics

To compare healing outcomes at a wound healing centre both before and after the introduction of molecular pathogen diagnostics. An IT consultant was recruited to analyse the medical records of patients at a wound healing centre, comparing patient groups from 2007 and 2009 - before and after the introduction of comprehensive molecular pathogen diagnostic methods. Before the implementation of molecular diagnostics, 244/503 patients (48.5%) healed completely, while after implementation 298/479 patients (62.4%) healed. Furthermore, based on survival analysis and after controlling for potential confounding factors, time to healing was significantly shorter in 2009 than 2007 (p<0.05). Specifically, biofilm-based wound care, along with the implementation of comprehensive molecular diagnostics for venous leg ulcers, pressure ulcers and diabetic foot ulcers and all wounds combined showed, respectively, 21%, 23%, 25% and 22% reductions in the time to healing. In addition, after implementing molecular diagnostics, the use of expensive fi rst-line antibiotics also declined in 2009, while a broader range of targeted antibiotics was used. The results of modern molecular pathogen diagnostic applications allow comprehensive evaluation of the microbial bioburden in chronic wounds. This comprehensive diagnostic in turn has led to a more precise and targeted therapeutic approach to wound care. With the comprehensive nature of molecular diagnostics future advances in topical patient specific therapeutics are now possible.

Current molecular diagnostics of breast cancer and the potential incorporation of microRNA

Although comprehensive molecular diagnostics and personalized medicine have sparked excitement among researchers and clinicians, they have yet to be fully incorporated into today’s standard of care. This is despite the discovery of disease-related oncogenes, tumor-suppressor genes and protein biomarkers, as well as other biological anomalies related to cancer. Each year, new tests are released that could potentially supplement or surpass standard methods of diagnosis, including serum, protein and gene expression analyses. All of these novel approaches have shown great promise, but initial enthusiasm has diminished as difficulties in reproducibility, expense, standardization and proof of significance beyond current protocols have emerged. This review will focus on current and novel molecular diagnostic tools applied to breast cancer with special attention to the exciting new field of microRNA analysis.