How I communicate with patients and families about germ line genetic information.

Because germ line genetic testing is increasingly integrated into the clinical care of patients with hematologic malignancies, it is important for hematologists to effectively communicate with patients and their families about the genetic testing process and to relay the results in a concise and understandable manner. Effective communication facilitates trust between patients and providers and allows patients to feel empowered to ask questions and actively participate in their health care. Especially for inherited conditions, the patient's understanding of germ line genetic information is critical because it enables them to share this information with relatives who are at risk, thereby promoting cascade testing and providing potentially life-saving information to family members who may be similarly affected. Accordingly, a hematologist's skills in understanding the importance and implications of germ line genetic information and the ability to convey this information in patient-friendly language is a critical first step and can have a far-reaching impact. In this article, we outline a straightforward approach to discussing genetic information and provide the reader with practical tips that can be used when consenting patients to germ line genetic testing and disclosing subsequent test results. We also review special considerations and ethical concerns arising when offering genetic evaluation and germ line testing to patients and related donors for allogeneic hematopoietic stem cell transplantation.

Proliferative Glomerulonephritis With Fibrils, Monoclonal κ Light Chain, and C3 Deposits.

There is increasing recognition of monoclonal gammopathy as a cause of proliferative glomerulonephritis (GN), including cases in which glomerular deposition of monoclonal immunoglobulin is demonstrated. Recently, proliferative GN with monoclonal immunoglobulin deposits (PGNMID) has incorporated a light chain variant of the disease (termed PGNMID-LC). Intriguingly, glomerular co-deposition of C3 is found in addition to monotypic light chain, implying complement activation via the alternative pathway (AP). We present a unique case of proliferative GN in a 42-year-old man who presented with nephrotic syndrome and was found to have κ light chain multiple myeloma. Immune staining of the glomerulus was positive only for κ light chain and C3, with the striking appearance of nonamyloid fibrils on electron microscopy. Following clonally targeted therapy for myeloma, the renal clinical abnormalities resolved completely. We present detailed molecular studies for light chain and complement and consider local mechanisms whereby monoclonal κ light chain fibrils may have triggered AP activation within the glomerulus.

Role of bone marrow biopsy for fever of unknown origin in the contemporary Australian context.

BACKGROUND: Bone marrow biopsy (BMB) is an accepted investigation in fever of unknown origin (FUO) to uncover haematological malignancies, such as lymphoma, and sometimes infections. With the advance in imaging modalities, such as 18-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) to identify the focus of lymphoma, BMB may not contribute to the diagnosis when there are no other clinical features to suggest an underlying haematological disease. AIM: To investigate the utility of BMB in determining the cause of FUO, when there are no other indications for BMB. METHODS: Medical records of adult patients who had BMB performed for FUO or febrile illness from 1 January 2005 to 31 December 2014 in four metropolitan tertiary hospitals in Melbourne, Australia were reviewed. Patients with other concurrent indications for BMB, known human immunodeficiency virus infection and previously diagnosed connective tissue diseases were excluded. RESULTS: Seventy-three patients were included in the study. Fifty-one patients had a final diagnosis for fever (systemic inflammatory diseases, infective, malignancy or other) while 22 patients had no diagnoses. In only 10 patients (13.7%) did BMB contribute to the diagnosis, finding either malignancy or granulomata. However, all these diagnoses could have been made without BMB. Two patients with diffuse large B-cell lymphoma had normal BMB. FDG-PET was helpful in making a diagnosis in eight (25%) out of 32 patients. CONCLUSION: Performing BMB in patients with FUO and no other haematological abnormalities is of very limited value, and other investigations, such as FDG-PET, may be more likely to help establish a definitive diagnosis.

Revisiting acquired aplastic anaemia: current concepts in diagnosis and management.

Acquired aplastic anaemia is a rare, serious, immunologically mediated bone marrow failure syndrome, characterised by marrow hypoplasia of varying severity and significant pancytopenia. Careful attention and investigation, including molecular testing, is required to confirm the diagnosis and exclude other mimicking conditions, such as inherited bone marrow failure syndromes. In a proportion of patients, the disease evolves to myelodysplasia or acute myeloid leukaemia and in some there is an association with paroxysmal nocturnal haemoglobinuria. The disease has a major impact on patient quality of life. Haemopoietic stem/progenitor cell transplantation for eligible patients with an available donor is the only current curative therapy. Other patients may receive immunosuppression, most commonly with anti-thymocyte globulin and cyclosporin. An initial response to immunosuppression is often encouraging, but relapse is common. Supportive care, including management of transfusion requirements and infections, is central to management. Promising new diagnostic tools and emerging therapies will likely transform approaches to this important, chronic and life-threatening condition.

Consensus opinion on diagnosis and management of thrombotic microangiopathy in Australia and New Zealand.

Thrombotic microangiopathy (TMA) arises in a variety of clinical circumstances with the potential to cause significant dysfunction of the kidneys, brain, gastrointestinal tract and heart. TMA should be considered in all patients with thrombocytopenia and anaemia, with an immediate request to the haematology laboratory to look for red cell fragments on a blood film. Although TMA of any aetiology generally demands prompt treatment, this is especially so in thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS), where organ failure may be precipitous, irreversible and fatal. In all adults, urgent, empirical plasma exchange (PE) should be started within 4-8 h of presentation for a possible diagnosis of TTP, pending a result for ADAMTS13 (a disintegrin and metalloprotease thrombospondin, number 13) activity. A sodium citrate plasma sample should be collected for ADAMTS13 testing prior to any plasma therapy. In children, Shiga toxin-associated haemolytic uraemic syndrome due to infection with Escherichia coli (STEC-HUS) is the commonest cause of TMA, and is managed supportively. If TTP and STEC-HUS have been excluded, a diagnosis of aHUS should be considered, for which treatment is with the monoclonal complement C5 inhibitor, eculizumab. Although early confirmation of aHUS is often not possible, except in the minority of patients in whom auto-antibodies against factor H are identified, genetic testing ultimately reveals a complement-related mutation in a significant proportion of aHUS cases. The presence of other TMA-associated conditions (e.g. infection, pregnancy/postpartum and malignant hypertension) does not exclude TTP or aHUS as the underlying cause of TMA.

Consensus opinion on diagnosis and management of thrombotic microangiopathy in Australia and New Zealand.

Thrombotic microangiopathy (TMA) arises in a variety of clinical circumstances with the potential to cause significant dysfunction of the kidneys, brain, gastrointestinal tract and heart. TMA should be considered in all patients with thrombocytopenia and anaemia, with an immediate request to the haematology laboratory to look for red cell fragments on a blood film. While TMA of any aetiology generally demands prompt treatment, this is especially so in thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS), where organ failure may be precipitous, irreversible and fatal. In all adults, urgent, empirical plasma exchange (PE) should be started within 4-8 h of presentation for a possible diagnosis of TTP, pending a result for ADAMTS13 activity (a disintegrin and metalloprotease thrombospondin, number 13). A sodium citrate plasma sample should be collected for ADAMTS13 testing prior to any plasma therapy. In children, Shiga toxin-associated haemolytic uraemic syndrome due to infection with Escherichia coli (STEC-HUS) is the commonest cause of TMA, and is managed supportively. If TTP and STEC-HUS have been excluded, a diagnosis of aHUS should be considered, for which treatment is with the monoclonal complement C5 inhibitor, eculizumab. While early confirmation of aHUS is often not possible, except in the minority of patients in whom autoantibodies against factor H are identified, genetic testing ultimately reveals a complement-related mutation in a significant proportion of aHUS cases. The presence of other TMA-associated conditions (e.g. infection, pregnancy/postpartum and malignant hypertension) does not exclude TTP or aHUS as the underlying cause of TMA.

Molecular Mechanisms of Disease Progression in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type during Ibrutinib Therapy.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is one of the well-recognized extranodal lymphomas commonly addicted to the B-cell receptor-MYD88 superpathway. We aimed to describe the genomic changes in a patient who progressed through treatment with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor. An 80-year-old woman presented with multiply relapsed PCDLBCL-LT after multiple lines of chemoimmunotherapy and radiotherapy. Pre-treatment testing of the localized cutaneous tumor lesion on a lymphoid amplicon panel demonstrated an MYD88 p.L265P mutation. Ibrutinib therapy was subsequently commenced, resulting in complete resolution of the skin disease. Despite an ongoing skin response, the patient developed progressive nodal disease at two months. Genomic analysis of the cutaneous tumor sample at baseline was compared to that of the inguinal lymph node upon progression, and revealed the acquisition of multiple genomic changes. These included several aberrations expected to bypass BTK inhibition, including two CARD11-activating mutations, and a deleterious mutation in the nuclear factor kappa B (NF-κB) negative regulator, NFKBIE. In addition, an IgH-IRF8 translocation was detected (which brings the IRF8 transcription factor under control of the immunoglobulin heavy chain locus), representing a third plausible mechanism contributing to ibrutinib resistance. Several copy-number changes occurred in both samples, including an amplification of 18q, which encodes the anti-apoptotic protein BCL2. We describe the first case of novel genomic changes of PCDLBCL-LT that occurred while on ibrutinib, providing important mechanistic insights into both pathogenesis and drug resistance.

The Australian Aplastic Anaemia and other Bone Marrow Failure Syndromes Registry.

The bone marrow failure syndromes (BMFS) are a diverse group of acquired and inherited diseases which may manifest in cytopenias, haematological malignancy and/or syndromic multisystem disease. Patients with BMFS frequently experience poor outcomes, and improved treatment strategies are needed. Collation of clinical characteristics and patient outcomes in a national disease-specific registry represents a powerful tool to identify areas of need and support clinical and research collaboration. Novel treatment strategies such as gene therapy, particularly in rare diseases, will depend on the ability to identify eligible patients alongside the molecular genetic features of their disease that may be amenable to novel therapy. The Australian Aplastic Anaemia and other Bone Marrow Failure Syndromes Registry (AAR) aims to improve outcomes for all paediatric and adult patients with BMFS in Australia by describing the demographics, treatments (including supportive care) and outcomes, and serving as a resource for research and practice improvement.

Functional interaction between compound heterozygous TERT mutations causes severe telomere biology disorder.

Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.

Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions.

Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

Diagnostic evaluation and considerations in hypocellular bone marrow failure-A focus on genomics.

Hypocellular bone marrow failure (BMF) has myriad differential diagnoses, most simply considered as acquired and inherited disorders, which are frequently indistinguishable upon morphologic examination of the blood and bone marrow. Accurate diagnosis is critical to optimization of management and begins with a detailed history (including family history) and physical examination. Next-generation sequencing technologies complement traditional testing techniques (such as chromosomal fragility and telomere length assessment) and have a broad application in the diagnosis and prognostication of BMF, with the importance of detection of both germline changes and also somatic variants increasingly well understood and appreciated. There is increasing awareness of germline predisposition to haematological malignancy, which incorporates but is not limited to the traditional inherited BMF syndromes and which raises challenges for counselling, monitoring and treatment of people who harbour a germline lesion. There are many benefits to both patients and their kindred of accurate determination of the precise germline change underlying heritable bone marrow diseases, along with its associated mode of inheritance. While individually, these diseases are rare, collectively they are not so and there are many collaborative efforts underway to document the natural history of these disorders, the associated phenotypes and the ever-increasing list of variants which have sufficient evidence to warrant the ascription of a pathogenic classification. We describe the many diagnostic considerations when evaluating newly presenting patients with hypocellular BMF, with a focus on genomic assessment, which is relevant in both germline and acquired diseases.