ATR-16 syndrome: mechanisms linking monosomy to phenotype.

BACKGROUND: Deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual's clinical phenotype is challenging. METHODS: Here, as an example of this common phenomenon, we analysed 41 patients with simple deletions of ~177 to ~2000 kb affecting one allele of the well-characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised deletion extents and screened for genetic background effects, telomere position effect and compensatory upregulation of hemizygous genes. RESULTS: We find the risk of developmental and neurological abnormalities arises from much smaller distal chromosome 16 deletions (~400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes; however, genetic background effects substantially modify phenotypic abnormalities. CONCLUSIONS: Using ATR-16 as a general model of disorders caused by CNVs, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling.

Screening for anaplastic lymphoma kinase (ALK) gene rearrangements in non-small-cell lung cancer in New Zealand.

BACKGROUND: Lung cancer is a major cause of death in New Zealand. In recent years, targeted therapies have improved outcomes. AIM: To determine the uptake of anaplastic lymphoma kinase (ALK) testing, and the prevalence, demographic profile and outcomes of ALK-positive non-small-cell lung cancer (NSCLC), in New Zealand, where no national ALK-testing guidelines or subsidised ALK tyrosine kinase inhibitor (TKI) therapies are available. METHODS: A population-based observational study reviewed databases to identify patients presenting with non-squamous NSCLC over 6.5 years in northern New Zealand. We report the proportion tested for ALK gene rearrangements and the results. NSCLC samples tested by fluorescence in situ hybridisation were retested by next generation sequencing and ALK immunohistochemistry. A survival analysis compared ALK-positive patients treated or not treated with ALK TKI therapy. RESULTS: From a total of 3130 patients diagnosed with non-squamous NSCLC, 407 (13%) were tested for ALK gene rearrangements, and patient selection was variable and inequitable. Among those tested, 34 (8.4%) had ALK-positive NSCLC. ALK-positive disease was more prevalent in younger versus older patients, non-smokers versus smokers and in Maori, Pacific or Asian ethnic groups than in New Zealand Europeans. Fluorescence in situ hybridisation, ALK immunohistochemistry and next generation sequencing showed broad concordance for detecting ALK-positive disease under local testing conditions. Among patients with ALK-positive metastatic NSCLC, those treated with ALK TKI survived markedly longer than those not treated with ALK TKI (median overall survival 5.12 vs 0.55 years). CONCLUSION: Lung cancer outcomes in New Zealand may be improved by providing national guidelines and funding policy for ALK testing and access to subsidised ALK TKI therapy.

Phase 1 study of selinexor plus carfilzomib and dexamethasone for the treatment of relapsed/refractory multiple myeloma.

Selinexor, an oral Selective Inhibitor of Nuclear Export, targets Exportin 1 (XPO1, also termed CRM1). Non-clinical studies support combining selinexor with proteasome inhibitors (PIs) and corticosteroids to overcome resistance in relapsed/refractory multiple myeloma (RRMM). We conducted a phase I dose-escalation trial of twice-weekly selinexor in combination with carfilzomib and dexamethasone (SKd) to determine maximum tolerated dose in patients with RRMM (N = 21), with an expansion cohort to assess activity in carfilzomib-refractory disease and identify a recommended phase II dose (RP2D). During dose escalation, there was one dose-limiting toxicity (cardiac failure). The RP2D of twice-weekly SKd was selinexor 60 mg, carfilzomib 20/27 mg/m2 and dexamethasone 20 mg. The most common grade 3/4 treatment-emergent adverse events included thrombocytopenia (71%), anaemia (33%), lymphopenia (33%), neutropenia (33%) and infections (24%). Rates of ≥minimal response, ≥partial response and very good partial response were 71%, 48% and 14%, respectively; similar response outcomes were observed for dual-class refractory (PI and immunomodulatory drug)/quad-exposed (carfilzomib, bortezomib, lenalidomide and pomalidomide) patients (n = 17), and patients refractory to carfilzomib in last line of therapy (n = 13). Median progression-free survival was 3·7 months, and overall survival was 22·4 months in the overall population. SKd was tolerable and re-established disease control in RRMM patients, including carfilzomib-refractory patients. Registered at ClinicalTrials.gov (NCT02199665).

Validation of a molecular assay to detect SARS-CoV-2 in saliva.

AIM: To validate a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay to detect SARS-CoV-2 in saliva in two independent Aotearoa New Zealand laboratories. METHODS: An RT-qPCR assay developed at University of Illinois Urbana-Champaign, USA, was validated in two New Zealand laboratories. Analytical measures, such as limit of detection (LOD) and cross-reactivity, were performed. One hundred and forty-seven saliva samples, each paired with a contemporaneously collected nasal swab, mainly of nasopharyngeal origin, were received. Positive (N=33) and negative (N=114) samples were tested blindly in each laboratory. Diagnostic sensitivity and specificity were then calculated. RESULTS: The LOD was <0.75 copy per µL and no cross-reactivity with MERS-CoV was detected. There was complete concordance between laboratories for all saliva samples with the quantification cycle values for all three genes in close agreement. Saliva had 98.7% concordance with paired nasal samples: and a sensitivity, specificity and accuracy of 97.0%, 99.1% and 99.1%, respectively. CONCLUSION: This saliva RT-qPCR assay produces reproducible results with a low LOD. High sensitivity and specificity make it a reliable option for SARS-CoV-2 testing, including for asymptomatic people requiring regular screening.

Impact of an Oncology Clinical Pharmacist Specialist in an Outpatient Multiple Myeloma Clinic.

INTRODUCTION: Improvements in cancer treatment and supportive care, as well as the approval of oral chemotherapy medications over the past decade, have resulted in an increasing number of cancer patients being treated in outpatient settings. Transitioning cancer treatments to the outpatient setting places greater emphasis on proper medication counseling and optimal management of adverse effects. We therefore evaluated the clinical and financial impact of an oncology clinical pharmacist specialist in an interdisciplinary multiple myeloma clinic by using a validated scoring tool. METHODS: The oncology clinical pharmacist specialist was available for consult by the multiple myeloma clinic staff. The pharmacist may be consulted for any medication-related inquiry. On the basis of the consult, the pharmacist categorized interventions into 12 predefined intervention categories. RESULTS: Implementation of a clinical pharmacy specialist into a multiple myeloma clinic over 39 clinic days resulted in 241 patient consults and 474 interventions made by the pharmacist. The most frequent interventions made by the pharmacist were medication teaching (n = 97), dose adjustments (n = 82), and medication reconciliation (n = 63). The value of interventions made by the pharmacist during the study period was $189,441, with a predicted annual value of $757,764. CONCLUSION: The addition of an oncology pharmacist to an outpatient multiple myeloma clinic can improve clinical and financial outcomes.

Assessing copy number aberrations and copy neutral loss of heterozygosity across the genome as best practice: An evidence based review of clinical utility from the cancer genomics consortium (CGC) working group for myelodysplastic syndrome, myelodysplastic/myeloproliferative and myeloproliferative neoplasms.

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow).

Assessing copy number abnormalities and copy-neutral loss-of-heterozygosity across the genome as best practice in diagnostic evaluation of acute myeloid leukemia: An evidence-based review from the cancer genomics consortium (CGC) myeloid neoplasms working group.

Structural genomic abnormalities, including balanced chromosomal rearrangements, copy number gains and losses and copy-neutral loss-of-heterozygosity (CN-LOH) represent an important category of diagnostic, prognostic and therapeutic markers in acute myeloid leukemia (AML). Genome-wide evaluation for copy number abnormalities (CNAs) is at present performed by karyotype analysis which has low resolution and is unobtainable in a subset of cases. Furthermore, examination for possible CN-LOH in leukemia cells is at present not routinely performed in the clinical setting. Chromosomal microarray (CMA) analysis is a widely available assay for CNAs and CN-LOH in diagnostic laboratories, but there are currently no guidelines how to best incorporate this technology into clinical testing algorithms for neoplastic diseases including AML. The Cancer Genomics Consortium Working Group for Myeloid Neoplasms performed an extensive review of peer-reviewed publications focused on CMA analysis in AML. Here we summarize evidence regarding clinical utility of CMA analysis in AML extracted from published data, and provide recommendations for optimal utilization of CMA testing in the diagnostic workup. In addition, we provide a list of CNAs and CN-LOH regions which have documented clinical significance in diagnosis, prognosis and treatment decisions in AML.

Detection of complex genomic signatures associated with risk in plasma cell disorders.

Plasma cell disorders (PCD) range from benign to highly malignant disease. The ability to detect risk-stratifying aberrations based on cytogenetic and molecular genetic assays plays an increasing role in therapeutic decision making. In this study, 58 patients were chosen for screening by comparative genomic hybridisation microarray (aCGH) to identify the new high-risk prognostic markers of chromothripsis and chromoanasynthesis. All patients had an unequivocal clinical diagnosis of a plasma cell disorder (plasma cell myeloma (PCM)(n = 51) or monoclonal gammopathy of undetermined significance (MGUS)(n = 7)) and an abnormal FISH result. There were a total of 17 complex genomic events identified across 9 patient samples, which were selected for further investigation by high definition single nucleotide polymorphism (HD-SNP) microarray. Each event was analysed and characterised for chromothripsis, chromoanasynthesis or a complex step-wise chromosomal event. We describe an effective method to identify the new high-risk prognostic markers of chromothripsis and chromoanasynthesis in plasma cell disorders.

NRASQ61R Mutation-specific Immunohistochemistry is Highly Specific for Either NRASQ61R or KRASQ61R Mutation in Colorectal Carcinoma.

Anti-epidermal growth factor receptor-targeted therapy is only indicated in RAS wild-type colorectal carcinomas (CRCs). It is recommended that both NRAS and KRAS mutation testing to be performed before a CRC is considered RAS wild-type. Given that mutation-specific immunohistochemistry (IHC) has been shown to be sensitive and specific for the detection of NRAS mutations in melanoma, we assessed the specificity of NRAS mutation-specific IHC in CRC. IHC was performed on tissue microarrays containing 2823 consecutive CRC undergoing surgery with curative intent using a novel mutation-specific antibody to the protein produced by the NRAS mutation (clone SP174). Tissue microarrays were assessed by 2 observers and all IHC-positive or equivocal cases were repeated on whole sections to confirm the result. Positive cases then underwent molecular testing by matrix-assisted laser desorption/ionization-time of flight polymerase chain reaction. In total, 22 of 2823 (0.8%) CRCs demonstrated confirmed positive staining with complete interobserver concordance. RAS mutations were confirmed in all IHC-positive CRCs. In total, 11 cases harbored the NRASQ61R mutation. Surprisingly, 11 cases demonstrated the KRASQ61R mutation. We conclude that mutation-specific IHC with this currently available NRASQ61R antibody is highly specific for the presence of either NRASQ61R or KRASQ61R mutations in CRC. We caution that we did not assess the sensitivity of IHC and that this antibody does not detect other RAS mutations. Therefore, negative staining does not exclude a clinically significant RAS mutation. However, positive staining confirms the presence of an NRASQ61R or KRASQ61R mutation without the need for further molecular testing.

Emerging technologies in paediatric leukaemia.

Genetic changes, in particular chromosomal aberrations, are a hallmark of acute lymphoblastic lymphoma (ALL) and accurate detection of them is important in ensuring assignment to the appropriate drug protocol. Our ability to detect these genetic changes has been somewhat limited in the past due to the necessity to analyse mitotically active cells by conventional G-banded metaphase analysis and by mutational analysis of individual genes. Advances in technology include high resolution, microarray-based techniques that permit examination of the whole genome. Here we will review the current available methodology and discuss how the technology is being integrated into the diagnostic setting.

MicroRNA detection in bone marrow cells by LNA-FISH.

The protocol reported in this chapter describes a method for the detection and spatial localisation of microRNAs (miRNAs) in cryopreserved primary leukaemic suspension cells using digoxigenin (DIG)-labelled, Locked Nucleic Acid (LNA)-modified probes, and fluorescence in situ hybridisation (FISH). The LNA probe hybridisation yields highly accurate signals able to discriminate between single nucleotide differences and hence between closely related miRNA family members. DIG-labelled LNA probes for mature miRNAs are detected using an anti-DIG fluorescein isothiocyanate (FITC) conjugated antibody and the fluorescent signals visualised with a confocal microscope, which permits the spatial localisation of the miRNAs. Using LNA-FISH, we visualised the spatial localisation of two mature miRNAs, miR-127 and miR-154, in primary acute myeloid leukaemia (AML) suspension cells, and thus, we confirmed their expression in a specific leukaemic subtype as measured by real-time PCR.

Distinctive patterns of microRNA expression associated with karyotype in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia.

Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2.

CEBPA encodes the transcription factor C/EBPalpha and is specifically up-regulated during granulocytic differentiation. The gene is mutated in approximately 20% of patients with acute myeloid leukemia (AML) FAB type M2 and occurs in the absence of the t(8;21). In much the same way as specific translocations are associated with a particular AML FAB type, the identification of non-random associations of gene mutation with karyotype or FAB type may be helpful in elucidating the molecular basis of certain forms of leukemia. To confirm these initial findings, 99 patients with AML FAB type M1 or M2 were screened for CEBPA mutations by use of a PCR-single-strand conformational polymorphism and sequencing approach. Nine CEBPA mutations were identified in eight patients. The mutations were clustered toward the COOH terminal of the protein and occurred exclusively in the intermediate cytogenetic risk group (8/64, 12.5%). Two patients with biallelic mutation, one homozygous for 1137Ins (57 bp) and another with two CEBPA mutations, 1096Ins (27 bp) and 363Ins (GGCC), were observed. There was no evidence for deletion of this region in the other six mutated samples analyzed by fluorescence in situ hybridization with a BAC clone spanning the CEBPA locus. CEBPA mutation status was not demonstrated to be of prognostic importance in this patient group, although this may reflect the selection and size of the AML population studied. In conclusion, mutation of CEBPA is a recurrent finding in AML and appears specific to the intermediate cytogenetic risk group patients.