Molecular Diagnostic Work-up Research Articles

Focus on human epidermal growth factor receptor 2 (HER2) positive metastatic colorectal cancer (HER2+ mCRC): A real world retrospective analysis.

3080 Background: HER2+ mCRCs, displaying HER2 overexpression or ERBB2 amplification, represent a rare molecular subset, accounting for 2- 6% of all mCRCs. HER2+ mCRCs are thought to be enriched in KRASwt pts and associated with distal primary tumor location. While its prognostic value is debated, HER2 seems a negative predictor for antiEGFR agents. Recently, its role as a potential actionable target has emerged, with promising results in refractory mCRC. Despite that, nowadays HER2 testing is not routinely included in the diagnostic workup of mCRC. Methods: This is an observational, retrospective, multicenter study, aiming to investigate and describe clinical and molecular features and prognostic value of HER2+ mCRCs from real world pts. Pts with mCRC tested for HER2 status who received at least one line of therapy (tx) at 11 Italian Institutions within the Lazio Region between Mar2011 and Jan2024 were enrolled. HER2 status was assessed either for HER2 overexpression using IHC according to the HERACLES diagnostic criteria or for ERBB2 amplification using NGS, followed by IHC confirmation for positive cases. Differences between groups for categorical variables were compared using the Chi Square test. Endpoint for prognostic assessment was OS, estimated with the Kaplan-Meier method and compared using log-rank test. Endpoints for predictive assessment were RR and DCR. Statistical significance was set at p .05. Results: 422 pts were included, of those 38 pts had a HER2+ mCRC (9%). HER2 positivity was more frequent among patients with RASwt tumor compared to RASmt (12.2 vs 6.1%; p .029), lung metastasis ( p .013) and synchronous metastatic disease ( p .044). No correlation with age, sex, primary tumor location, peritoneal spread, histologic grading and mucinous histology was observed. At a median FU of 19 months, OS was significantly shorter in HER2+ compared to HER2- mCRCs (HR 2.17, 95%CI 1.14-4.13; p .017). 104 pts with RASwt mCRC received an antiEGFR-based first line tx and were evaluable for response; of those 96 were HER2- and 12 HER2+. RR was higher for HER2- tumors (80 vs 42%; p .003), while no difference was observed for DCR (96 vs 83%; p .085). Of 38 HER2+ tumors, 14 received an antiHER2 tx, of those 11 were RASwt and 3 RASmt. RAS status did not impact on antiHER2 activity (DCR 64 vs 33% for RASwt and RASmt; p .347). Conclusions: We showed that HER2+ mCRCs are more frequently, although not exclusively, RASwt, display a lung tropism, present with synchronous metastases and have a negative prognosis. Moreover, in accordance with results from DESTINY-CRC02, the target actionability seems to be retained irrespective of RAS status. Given also the possible use in first line of antiHER2 tx in the near future, the baseline molecular diagnostic workup of mCRC must include HER2 status assessment, irrespective of RAS mutational status and primary tumor location.

Non-canonical BRAF variants and rearrangements in hairy cell leukemia.

Hairy cell leukemia (HCL) is an uncommon mature B-cell malignancy characterized by a typical morphology, immunophenotype, and clinical profile. The vast majority of HCL patients harbor the canonical BRAF V600E mutation which has become a rationalized target of the subsequently deregulated RAS-RAF-MEK-MAPK signaling pathway in HCL patients who have relapsed or who are refractory to front-line therapy. However, several HCL patients with a classical phenotype display non-canonical BRAF mutations or rearrangements. These include sequence variants within alternative exons and an oncogenic fusion with the IGH gene. Care must be taken in the molecular diagnostic work-up of patients with typical HCL but without the BRAF V600E to include investigation of these uncommon mechanisms. Identification, functional characterization, and reporting of further such patients is likely to provide insights into the pathogenesis of HCL and enable rational selection of targeted inhibitors in such patients if required.

Double-hit high-grade B-cell lymphoma with aberrant T-cell phenotype

Abstract Aberrant T-cell phenotype or aberrant B-cell phenotype in B-cell non-Hodgkin’s lymphoma or T-cell non-Hodgkin’s lymphoma, respectively, are uncommon phenomenon and a diagnostic challenge. Multiple T-cell antigen, including CD3 expression in large B-cell lymphomas, which is considered a specific marker for T cells, is extremely rare and have scattered instances in the literature; however, none of those showed myc and bcl2 rearrangement. This is a case of stage IV NHL, previously diagnosed as T-cell NHL, which on detailed immunohistochemical and molecular diagnostic workup, later was confirmed as “Double hit” B-cell lymphoma with aberrant T-cell phenotype. With the 1st ever-reported case, we wish to bring into the notice of such aberrancy so as to not misdiagnose or wrongly classify high-grade lymphomas.

A RARE CASE OF ACTINOMYCES TURICENSIS EMPYEMA IN AN IV DRUG USER

A RARE CASE OF ACTINOMYCES TURICENSIS EMPYEMA IN AN IV DRUG USER

EP11.01-004 An Effective Two-step Reflex Test for 10 Biomarkers Analysis in Non-small Cell Lung Cancer

The treatment of non-small cell lung cancer (NSCLC) has become increasingly biomarker-driven over the last decade, with a growing number of targeted therapies available for patients with specific genomic alterations. A fast-track screening of multiple molecular biomarkers is therefore required for first-line treatment selection in NSCLC, although challenging considering both turnaround time and sample quality. In an effort to improve the molecular diagnostic workup at our institution, we implemented a two-step reflex test (RT) at the time of pathological diagnosis of non-squamous NSCLC for EGFR, KRAS, BRAF, ERBB2, ALK, ROS1, PDL-1, RET, MET, and NTRK testing.

Abstract 5323: Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels

Abstract Liquid biopsy tests are an integral part of the molecular diagnostic workup of patients with multiple cancer types. We have extended validation studies of a new liquid biopsy test panel that uses an amplicon-based targeted next-generation sequencing (NGS) panel with a semiconductor-based system, the Ion GeneStudio S5 Prime. The workflow requires very low input (20 ng) of cell-free nucleic acids extracted from human plasma and has been demonstrated to yield results in less than 72 hours from sample receipt at the laboratory. This report details the analytic performance of the test panel for detecting copy number variations (CNVs), fusions and exon skipping variants in plasma. These data add to previously reported validation data for the detection of somatic nucleotide variants (SNV) and indels [97.7% - 100% concordant with orthogonal droplet digital PCR (ddPCR) tests]. The updated study sample sets included consented clinical and normal healthy, donor specimens, and reference cell lines to detect fusions and exon skipping [HCC78 (ROS1), CRL-5935 (EML4-ALK), KM12 (NTRK1) and HTB-178 (MET exon 14 skipping)], and CNVs [CRL-5928 (HER2 gain), SK-BR-3 (HER2, CDK6, EGFR and MYC gains), HCC-827 (EGFR gain) and CRL-5909 (MET gain)]. The limit of detection (LOD) for fusion/skipping was initially established at a molecular coverage of 42 copies and was then verified by detection of three additional fusion/skipping variants. The LOD for CNVs was initially established at 1.40 fold-change and was then verified with the detection of three additional CNVs. Verification was conducted with specimens near the initially established LOD. In the precision studies, all expected fusion/skipping variants were detected in all runs and all days of testing (n=18/18) generating a hit rate of 100%. An average CV of 20.5% (range 8.7% to 34.8%) for repeatability and 20.8% (range 15.7% to 30.5%) for reproducibility was established. Twenty eight of 29 CNVs were detected, generating a hit rate of 96.6%. An average CV of 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for reproducibility was established for detected CNVs. We conclude that the validated test panel meets the criteria for being rapid, highly sensitive and specific, and has utility for the reporting of clinically relevant variants of interest in multiple cancer types. Citation Format: Audrey Audetat, Cherie Tschida, Sarah Kreston, Leisa Jackson, Hestia Mellert, Adam Stephen, Gary A. Pestano. Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels [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 5323.

Myoepithelial Carcinoma of Soft Tissue With an EWSR1-KLF15 Gene Fusion in an Infant

Overall, neonatal cancer is uncommon. Because of its rarity and heterogeneity, diagnosis can be challenging. We report a unique case of a myoepithelial carcinoma in a 7 week old girl. Molecular diagnostic workup revealed a EWSR1-KLF15 gene fusion which was previously described in only six cases of myoepithelial tumors so far. All cases occurred in children and adolescents. To our knowledge, this is the first report of a congenital EWSR1-KLF15 fusion positive myoepithelial tumor in an infant.

HMGA2 Variants in Silver-Russell Syndrome: Homozygous and Heterozygous Occurrence.

Silver-Russell syndrome (SRS) is a clinical and molecular heterogeneous disorder associated with short stature, typical facial gestalt, and body asymmetry. Though molecular causes of SRS can be identified in a significant number of patients, about one-half of patients currently remain without a molecular diagnosis. However, determination of the molecular cause is required for a targeted treatment and genetic counselling. The aim of this study was to corroborate the role of HMGA2 as an SRS-causing gene and reevaluate its mode of inheritance. Patients were part of an ongoing study aiming on SRS-causing genes. They were classified according to the Netchine-Harbison clinical scoring system, and DNA samples were investigated by whole exome sequencing. Common molecular causes of SRS were excluded before. Three novel pathogenic HMGA2 variants were identified in 5 patients from 3 SRS families, and fulfilling diagnostic criteria of SRS. For the first time, homozygosity for a variant in HMGA2 could be identified in a severely affected sibpair, whereas parents carrying heterozygous variants had a mild phenotype. Treatment with recombinant growth hormone led to a catch-up growth in 1 patient, whereas all others did not receive growth hormone and stayed small. One patient developed type 2 diabetes at age 30 years. Identification of novel pathogenic variants confirms HMGA2 as an SRS-causing gene; thus, HMGA2 testing should be implemented in molecular SRS diagnostic workup. Furthermore, inheritance of HMGA2 is variable depending on the severity of the variant and its consequence for protein function.

S2k guidelines for the diagnosis and treatment of herpes zoster and postherpetic neuralgia.

The present guidelines are aimed at residents and board-certified specialists in the fields of dermatology, ophthalmology, ENT, pediatrics, neurology, virology, infectious diseases, anesthesiology, general medicine and any other medical specialties involved in the management of patients with herpes zoster. They are also intended as a guide for policymakers and health insurance funds. The guidelines were developed by dermatologists, virologists, ophthalmologists, ENT physicians, neurologists, pediatricians and anesthesiologists/pain specialists using a formal consensus process (S2k). Readers are provided with an overview of the clinical and molecular diagnostic workup, including antigen detection, antibody tests and viral culture. Special diagnostic situations and complicated disease courses are discussed. The authors address general and special aspects of antiviral therapy for herpes zoster and postherpetic neuralgia. Furthermore, the guidelines provide detailed information on pain management including a schematic overview, and they conclude with a discussion of topical treatment options.

Development and feasibility of virtual molecular tumor board.

e18334 Background: Practicing precision oncology is challenging due to the complex molecular interpretation and its implications in deciding pertinent treatment options. This is addressed by institutional Molecular Tumor Board (MTB) which is presided by all oncology sub-specialists including molecular biologist and bioinformatician, doing a meticulous job in untwining the intricacies but fail to obtain experiences across institutes. Further, meeting of all MTB members is neither always feasible nor accessible for the community oncologists. We hereby offer a virtual formation of MTB that is easily accessible and remotely attended by various experts across institutes, giving their opinion objectively at their ease that can be documented and archived. Methods: Virtual MTB (vMTB), a mobile cloud-based application was developed for both android and iOS platforms. The vMTB process involves submission and review of cases, whereby assigned members can opine blindly and rate different treatment options. Followed by averaging of rating score and then a final recommendation generated by the vMTB case-convener within 7 days, based on clinical context. A pilot study (2 months duration) was carried out where clinical cases bearing molecular information were invited from various oncology centers across India. Results: 21 cases were submitted (19 relapsed refractory, 3 treatment naïve) that included Breast (6), Lung (5), Leukemia/Lymphoma (3), Gall bladder (2), Uterine (2), Urinary bladder (1), GBM (1) and Sarcoma (1) cancers. The molecular query was based on 18 somatic (8 FoundationOne & 10 Others) and 3 germline testing. Cases assigned to vMTB members (~7 members/case) had ~ 6 treatment options/case with a response turn-around time ~4 days. Therapeutic final recommendation(s) were given for all cases with 95% concordance among the members; 6 cases where multiple options were advised, discordance rate was 14%. Additional molecular diagnostic workup was advised in 3 cases. vMTB recommendations including 10 off label treatment options were accepted by majority of the submitting physicians (82%). Clinical benefit was observed in 5 (24%) cases within the stipulated analysis duration. Conclusions: vMTB is a user-friendly, feasible, acceptable mobile solution alternate to MTB for objectively generating clinically meaningful consensus document for complex molecular clinical case scenarios, where guidelines are limited. To best of our knowledge, this is a first smart mobile solution connecting various oncology experts and have an immediate utility for the practicing community oncologists globally.

HER2 Positivity Predicts Unresponsiveness to EGFR-Targeted Treatment in Metastatic Colorectal Cancer.

HER2 amplification is detected in 3% of patients with colorectal cancer (CRC), making tumors in the metastatic setting vulnerable to double pharmacological HER2 blockade. Preclinical findings show that it also might impair response to anti-epidermal growth factor receptor (EGFR) treatment. Patients with KRAS exon 2 wild-type metastatic CRC underwent molecular screening of HER2 positivity by HERACLES criteria (immunohistochemistry 3+ or 2+ in ≥50% of cells, confirmed by fluorescence in situ hybridization). A sample of consecutive HER2-negative patients was selected as control. A regression modeling strategy was applied to identify predictors explaining the bulk of HER2 positivity and the association with response to previous anti-EGFR treatment. From August 2012 to April 2018, a total of 100 HER2-positive metastatic CRC tumors were detected out of 1,485 KRAS exon 2 wild-type screened patients (6.7%). HER2-positive patients show more frequently lung metastases (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.15-3.61; p = .014) and higher tumor burden (OR, 1.48; 95% CI, 1.10-2.01; p = .011), and tumors were more likely to be left sided (OR, 0.50; 95% CI, 0.22-1.11; p = .088). HER2-positive patients who received treatment with anti-EGFR agents (n = 79) showed poorer outcome (objective response rate, 31.2% vs. 46.9%, p = .031; progression-free survival, 5.7 months vs. 7 months, p = .087). Testing for HER2 should be offered to all patients with metastatic CRC because the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Patients with HER2-amplified metastatic CRC are less likely to respond to anti-EGFR therapy. Patients with HER2-amplified/overexpressed metastatic colorectal cancer (mCRC) harbor a driver actionable molecular alteration that has been shown in preclinical models to hamper efficacy of the anti-epidermal growth factor receptor (EGFR) targeted therapies. The present study confirmed that this molecular feature was associated with worse objective tumor response and shorter progression-free survival in response to previous anti-EGFR therapies. Moreover, it was found that the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Therefore, HER2 status assessment should be included in the molecular diagnostic workup of all mCRC for speedy referral to clinical trials encompassing HER2-targeted double blockade independently of previous anti-EGFR treatment.

Frequency of molecular detection of equine coronavirus in faeces and nasal secretions in 277 horses with acute onset of fever.

Due to the inconsistent development of enteric signs associated with ECoV infection in adult horses, many practitioners collect nasal secretions rather than feces for the molecular diagnostic work-up of such horses. ECoV infection should be considered in horses presenting with acute onset of fever, especially when nasal discharge is absent as one of the cardinal clinical sign. A total of 277 adult horses with acute onset of fever were enrolled in this study. Feces were tested for ECoV and nasal secretions for common respiratory pathogens (equine herpesvirus (EHV)-1, EHV-4, equine influenza virus (EIV), equine rhinitis viruses (ERVs) and Streptococcus equi ss. equi) and ECoV by qPCR. Each submission was accompanied by a questionnaire requesting information pertaining to signalment, use, recent transportation, number of affected horses on the premise and presence of clinical signs at the time of sample collection. The total number of horses testing qPCR-positive for ECoV in feces was 20 (7.2%), 4 of which also tested qPCR-positive for ECoV in nasal secretions. In the same population 9.0% of horses tested qPCR-positive for EHV-4, 6.1% for EIV, 4.3% for Streptococcus equi ss. equi, 3.2% for ERVs and 0.7% for EHV-1. Draft horses, pleasure use, multiple horses affected on a premise and lack of nasal discharge were significantly associated with ECoV qPCR-positive horses. The present study results showed that 7.2% of horses with acute onset of fever tested qPCR-positive for ECoV in feces, highlighting the importance of testing such horses for ECoV in feces. The various prevalence factors associated with ECoV qPCR-positive status likely relate to the high infectious nature of ECoV and breed-specific differences in management and husbandry practices. ECoV infection should be suspected and tested for in horses presenting with acute onset of fever, lethargy and anorexia with no respiratory signs. A two-step approach should be consider in which respiratory secretions and feces should be collected from such horses and submitted to a diagnostic laboratory. If the respiratory secretions test negative by qPCR for a panel of respiratory pathogens, feces already submitted to the laboratory should be tested for ECoV.

Universal genetic testing for inherited susceptibility in children and adults with myelodysplastic syndrome and acute myeloid leukemia: are we there yet?

Comprehensive genomic profiling of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cases have enabled the detection and differentiation of driver and subclonal mutations, informed risk prognostication, and defined targeted therapies. These insights into disease biology, and management have made multigene-acquired mutation testing a critical part of the diagnostic assessment of patients with sporadic MDS and AML. More recently, our understanding of the role of an increasing number of inherited genetic factors on MDS/AML risk and management has rapidly progressed. In recognition of the growing impact of this field, clinical guidelines and disease classification systems for both MDS and AML have recently incorporated familial MDS/AML predisposition syndromes into their diagnostic algorithms. In this perspective piece, we contemplate the advantages, disadvantages, and barriers that would need to be overcome to incorporate inherited MDS/AML genetic testing into the upfront molecular diagnostic work-up of every MDS/AML patient. For centers already performing panel-based tumor-only testing, including genes associated with familial forms of MDS/AML (e.g., RUNX1, CEBPA, GATA2, TP53), we advocate optimizing these tests to detect all types of germline variants in these genes and moving toward upfront paired tumor/germline testing to maximize detection and streamline patient care.

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup.

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

Individuals With Normal GLA Gene Sequence May Present Abnormally Spliced Alpha-Galactosidase mRNA Transcripts

Background: Deficient lysosomal α-galactosidase activity leads to intracellular accumulation of globotriaosylceramide (Gb3), which is the pathologic hallmark of Fabry disease (FD). There are over 750 pathogenic variants identified in the α-galactosidase gene (GLA). In rare patients, the cause of α-galactosidase deficiency is the overexpression of a GLA transcript with a cryptic exon in intron 4, which is physiologically present at trace levels. Objectives: We aim to report abnormally spliced alpha-galactosidase mRNA transcripts found with a cDNA-based GLA genotyping protocol performed in 482 patients. Patients and Methods: Genomic DNA and total RNA specimens were obtained from peripheral blood leukocytes of patients with premature stroke prospectively enrolled in the PORTYSTROKE study, or of patients with possible clinical manifestations of FD who have been referred for molecular diagnostic workup. Results: Approximately 20% of the patients expressed alternatively spliced transcripts of GLA mRNA involving exon 3. We additionally report that such non-canonical transcripts are physiologically expressed at trace levels in healthy individuals, and that their expression in leukocytes markedly increased in blood samples kept at room-temperature for 48 hours before RNA extraction. Conclusions: Production of alternatively spliced GLA transcripts might be involved in the regulation of GLA gene expression, and its deregulated overexpression, particularly if restricted to specific cells or tissues, might be the cause of organ-limited Gb3 pathology. Elucidation of the molecular mechanisms underlying the production of the non-canonical GLA transcripts warrants further investigation, as it may contribute important new data to the understanding of the molecular pathology of FD and Gb3-related disorders.

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing

ObjectivesRecurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. Materials and methodsWe developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5′ and 3′ ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. ResultsThe H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5′ and 3′ ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3′ ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. ConclusionMultiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.

De novo 13q deletions in two patients with mild anorectal malformations as part of VATER/VACTERL and VATER/VACTERL‐like association and analysis of EFNB2 in patients with anorectal malformations

Anorectal malformations (ARMs) comprise a broad spectrum of conditions ranging from mild anal anomalies to complex cloacal malformations. In 40-50% of cases, ARM occurs within the context of defined genetic syndromes or complex multiple congenital anomalies, such as VATER/VACTERL (vertebral defects [V], ARMs [A], cardiac defects [C], tracheoesophageal fistula with or without esophageal atresia [TE], renal malformations [R], and limb defects [L]) association. Here, we report the identification of deletions at chromosome 13q using single nucleotide polymorphism-based array analysis in two patients with mild ARM as part of VATER/VACTERL and VATER/VACTERL-like associations. Both deletions overlap the previously defined critical region for ARM. Heterozygous Efnb2 murine knockout models presenting with mild ARM suggest EFNB2 as an excellent candidate gene in this region. Our patients showed a mild ARM phenotype, closely resembling that of the mouse. We performed a comprehensive mutation analysis of the EFNB2 gene in 331 patients with isolated ARM, or ARM as part of VATER/VACTERL or VATER/VACTERL-like associations. However, we did not identify any disease-causing mutations. Given the convincing argument for EFNB2 as a candidate gene for ARM, analyses of larger samples and screening of functionally relevant non-coding regions of EFNB2 are warranted. In conclusion, our report underlines the association of chromosome 13q deletions with ARM, suggesting that routine molecular diagnostic workup should include the search for these deletions. Despite the negative results of our mutation screening, we still consider EFNB2 an excellent candidate gene for contributing to the development of ARM in humans.

Rapid and highly specific screening for NPM1 mutations in acute myeloid leukemia

NPM1 mutations, the most frequent molecular alterations in acute myeloid leukemia (AML), have become important for risk stratification and treatment decisions for patients with normal karyotype AML. Rapid screening for NPM1 mutations should be available shortly after diagnosis. Several methods for detecting NPM1 mutations have been described, most of which are technically challenging and require additional laboratory equipment. We developed and validated an assay that allows specific, rapid, and simple screening for NPM1 mutations. FAST PCR spanning exons 8 to 12 of the NPM1 gene was performed on 284 diagnostic AML samples. PCR products were visualized on a 2% agarose E-gel and verified by direct sequencing. The FAST PCR screening method showed a specificity and sensitivity of 100%, i.e., all mutated cases were detected, and none of negative cases carried mutations. The limit of detection was at 5-10% of mutant alleles. We conclude that the FAST PCR assay is a highly specific, rapid (less than 2h), and sensitive screening method for the detection of NPM1 mutations. Moreover, this method is inexpensive and can easily be integrated in the routine molecular diagnostic work-up of established risk factors in AML using standard laboratory equipment.

EFNS guidelines for the molecular diagnosis of neurogenetic disorders: motoneuron, peripheral nerve and muscle disorders

These EFNS guidelines on the molecular diagnosis of motoneuron disorders, neuropathies and myopathies are designed to summarize the possibilities and limitations of molecular genetic techniques and to provide diagnostic criteria for deciding when a molecular diagnostic work-up is indicated. To collect data about planning, conditions and performance of molecular diagnosis of these disorders, a literature search in various electronic databases was carried out and original papers, meta-analyses, review papers and guideline recommendations reviewed. The best level of evidence for genetic testing recommendation (B) can be found for the disorders with specific presentations, including familial amyotrophic lateral sclerosis, spinal and bulbar muscular atrophy, Charcot-Marie-Tooth 1A, myotonic dystrophy and Duchenne muscular dystrophy. For a number of less common disorders, a precise description of the phenotype, including the use of immunologic methods in the case of myopathies, is considered as good clinical practice to guide molecular genetic testing. These guidelines are provisional and the future availability of molecular-genetic epidemiological data about the neurogenetic disorders under discussion in this article will allow improved recommendation with an increased level of evidence.

Targeted Next-Generation Sequencing and Genome-Wide High-Resolution Copy Number DNA Arrays Allow the Identification of Five Novel RUNX1 Fusions In Hematological Malignancies.

AbstractAbstract 1193RUNX1 is a crucial transcription factor involved in cell lineage differentiation during hematopoiesis. It contains a “Runt homology domain” (RHD; exons 3–5, amino acids 50–177) and a transactivation domain (TAD; exon 8, amino acids 291–371). RUNX1 can act as an activator or repressor of target gene expression and thus far two different mechanisms of somatically acquired alterations have been recognized: intragenic mutations and translocations. Most of the translocations involving RUNX1 lead to the formation of a fusion gene consisting of the 5' part of RUNX1 fused to sequences on partner chromosomes. We here present data on 5 cases, 4 acute myeloid leukemias (AML) and 1 chronic myelomonocytic leukemia (CMML) patient, respectively, where previous cytogenetic and FISH analyses revealed reciprocal translocations involving RUNX1. However, even sophisticated molecular diagnostic work-up failed to identify the corresponding RUNX1 fusion partners. Therefore, we used a combination of 454 shotgun pyrosequencing and long-oligonucleotide sequence capture microarrays to reveal these unknown RUNX1 partner genes in four cases. In detail, we performed DNA sequence enrichment using microarrays containing capture probes that were covering a contiguous region on chr. 21 (36,160,098 – 36,421,641), thereby allowing a specific enrichment by hybridization for genomic DNA where the RUNX1 gene is located (Roche NimbleGen 385K chip, Penzberg, Germany). This targeted next-generation sequencing (NGS) assay enabled to capture and sequence single reads mapping to both RUNX1 and other genomic regions (Burrows-Wheeler Aligner's Smith-Waterman algorithm). In median, 324 bp per patient (170,000 reads) with an 18-fold coverage were sequenced and in all cases chimeric reads were detectable, thereby confirming the presence of RUNX1 translocations and, moreover, identifying and characterizing 4 novel fusions on a molecular level. In one AML case, KCNMA1 was fused to RUNX1. KCNMA1, a potassium large conductance calcium-activated channel family member on chromosome 10q22.3, had recently been described to play a role in breast cancer invasion and metastasis to brain. In our case, as confirmed by RT-PCR and Sanger sequencing, the chimeric RUNX1-KCNMA1 fusion led to the disruption of the RHD of RUNX1. In the three additional cases, RUNX1 was fused to genomic regions on chromosomes 10q22, 17q21, and 5q13.3, respectively. The RUNX1-10q22 and the reciprocal 10q22-RUNX1 fusion were confirmed by PCR from genomic DNA and subsequent Sanger sequencing. According to its genomic structure the translocation RUNX1-chr.10q22 will result into the translation of a truncated RUNX1 protein with an intact RHD, but without TAD. Notably, in the remaining two cases, chr.17q21-RUNX1 and chr.5q13.3-RUNX1, only the reciprocal fusion events were detectable by PCR. In case chr.17q21-RUNX1 the translocation would disrupt RUNX1 after the RHD. In chr.5q13.3-RUNX1 the predicted fusion would not impact the RHD and TAD domains because the breakpoint is located before exon 1. In the fifth patient, we performed an analysis using a high-resolution genome-wide cytogenetic copy number DNA microarray to resolve a novel t(X;21)(p11;q22). In this case, the derivative chromosome × was duplicated, leading to a partial trisomy 21q and a partial trisomy X. On chr. 21 the breakpoint was mapped to be located in intron 6–7 within the RUNX1 gene. The breakpoint on the X-chromosome mapped to Xp11.23, thus leading to a truncated RUNX1 protein without the TAD domain. In summary, RUNX1 rearrangements either led to RUNX1 with an intact RHD and TAD (n=1), RUNX1 with an intact RHD but without TAD (n=3, dominant negative effect; similar to RUNX1-RUNX1T1), or to RUNX1 with a disrupted RHD and without TAD domains, leading to haploinsufficiency (n=1). In conclusion, the RUNX1 recombinome is an interesting target to understand pathogenetic heterogeneity in hematological malignancies. Here, we demonstrated that NGS and copy number DNA microarrays allow the identification of novel RUNX1 fusion partners not detectable by standard molecular techniques and reveals that cytogenetic reciprocal translocations lead to different types of RUNX1 alterations. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.