Diffuse large B-cell lymphoma at risk of secondary CNS involvement: The inefficacy of intravenous high-dose methotrexate CNS prophylaxis and the importance of baseline cerebrospinal fluid analysis.

High-dose intravenous methotrexate (HD-MTX) CNS prophylaxis in high-risk diffuse large B cell lymphoma (DLBCL) remains controversial. We describe real-world CNS relapse incidence following baseline cerebrospinal fluid (CSF) analysis to exclude asymptomatic leptomeningeal involvement in newly diagnosed high-risk DLBCL patients with versus without single-route HD-MTX CNS prophylaxis. Consecutively diagnosed high-risk systemic DLBCL patients without leptomeningeal involvement by CSF analysis (noCNS) were identified retrospectively. Five-year CNS relapse incidence and survival outcomes were examined, as stratified by receipt of HD-MTX prophylaxis. Secondary analysis of survival outcomes in patients with synchronous leptomeningeal involvement (CNSinv) by CSF analysis at diagnosis were compared with the noCNS group. No significant difference in 5-year CNS relapse incidence was observed following HD-MTX prophylaxis versus no prophylaxis (total n = 445) despite similar CNS-IPI risk; 6.2% versus 5.6%, adjusted HR 1.08 (95% CI 0.41-2.85), p = .88; nor in 5-year progression free survival (PFS) or overall survival (OS) risk. Of CNSinv patients, 93.3% had ≥1 extranodal site. Increased CNS relapse/progression risk (5-year risk; HR 10.7 [95% CI 5.35-21.37], p < .0001) and inferior PFS and OS were observed in CNSinv versus all noCNS patients. The CNSinv group had superior OS compared with noCNS patients who later experienced CNS relapse (HR 0.55, p = .052). HD-MTX prophylaxis does not reduce CNS relapse risk in high-risk systemic DLBCL without leptomeningeal involvement by CSF analysis at diagnosis. Asymptomatic patients with synchronous leptomeningeal involvement on baseline CSF examination are at increased risk of further CNS disease events and inferior survival compared to patients without CSF involvement.

Green neutrophil and monocyte inclusions - time to acknowledge and report.

Bright green inclusions in neutrophils have been reported as a sign of impending patient death. Here we report a series of 20 patients in whom green inclusions were identified in neutrophils or monocytes. Thirteen (65%) of the patients died within days of the detection of the inclusions. A common feature to almost all patients was ischaemic or hypoxic hepatitis which, in fatal cases, was associated with lactic acidosis. Light and electron microscopy indicated that the inclusions were lipid-rich, probably derived from lipofuscin-like material released from necrotic liver parenchymal cells. The majority of patients were known to be seriously ill at the time of detection of neutrophil inclusions. We recommend that the detection of green inclusions, which we refer to as 'critical green inclusions', is acknowledged and reported by laboratories and correlated with clinical findings.

A diagnostic mystery solved by electron microscopy: a case of an "atypical" lymphoproliferative disorder.

An elderly woman with a previous diagnosis of atypical chronic lymphocytic leukemia (CLL) was noted to have a strikingly abnormal blood film, with the lymphocytes displaying numerous large cytoplasmic granules. This appearance had not been described before in the literature to the best of the authors' knowledge. After a series of investigations, electron microscopy was eventually performed, which demonstrated that the abnormal granules were composed of immunoglobulin crystals. The immunofixation study confirmed that they were monoclonal IgM paraprotein. These results led to a change of diagnosis to lymphoplasmacytic lymphoma. This report illustrates how electron microscopy can be used as a valuable additional diagnostic tool in difficult cases.

Value of cerebrospinal fluid white cell count and protein level in predicting leptomeningeal involvement by systemic aggressive B-cell lymphoma.

INTRODUCTION: Diagnostic cerebrospinal fluid (CSF) analysis for patients with newly diagnosed aggressive B-cell lymphoma at risk of secondary central nervous system involvement typically includes multiparametric flow cytometry (MFC), cytology (CC), white cell count (WCC) and total protein. The strength of relationships between MFC results and the remaining variables has been disputed in small studies. We explored these relationships in a large homogeneous cohort of patient samples, aiming to establish the relationship between WCC and protein level and MFC results. METHODS: Adult patients with aggressive B-cell lymphoma at risk of CNS involvement who underwent staging CSF analysis by MFC were identified retrospectively from institutional electronic records between October 2011 and December 2020. RESULTS: Three hundred and seventy eight samples, including 45 (11.9%) MFC+ samples, were analysed. The relative sensitivity of CC for MFC positivity was 0.38, with PPV of 0.68. Significantly higher median WCC (p < .001) and protein levels (p = .011) were seen in MFC+ vs. MFC- samples. MFC + CC+ (vs. MFC + CC- samples) demonstrated higher median neoplastic events and neoplastic cell concentration. WCC ≥36 × 106 /L and protein ≥1.12 g/L cut-off values demonstrated the highest PPVs for MFC positivity (0.67 and 0.88, respectively). CONCLUSIONS: Statistically significant associations exist between elevated WCC and protein and MFC positivity, and selected WCC and protein cut-off values have PPVs comparable to that of cytological assessment. Whilst routine WCC and protein analysis may be unnecessary, WCC/protein values above these levels could be regarded as reasonable evidence of CSF involvement in the appropriate setting.

The Epidemiology of Myeloproliferative Neoplasms in New Zealand between 2010 and 2017: Insights from the New Zealand Cancer Registry.

BACKGROUND: There is a paucity of data on ethnic disparities in patients with the classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs): polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). METHODS: This study analysed the demographic data for PV, ET and PMF collected by the New Zealand Cancer Registry (NZCR) between 2010 and 2017. RESULTS: We found that the NZCR capture rates were lower than average international incidence rates for PV and ET, but higher for PMF (0.76, 0.99 and 0.82 per 100,000, respectively). PV patients were older and had worse outcomes than expected, which suggests these patients were reported to the registry at an advanced stage of their disease. Polynesian patients with all MPN subtypes, PV, ET and PMF, were younger than their European counterparts both at the time of diagnosis and death (p < 0.001). Male gender was an independent risk factor for mortality from PV and PMF (hazard ratios (HR) of 1.43 and 1.81, respectively; p < 0.05), and Maori ethnicity was an independent risk factor for mortality from PMF (HR: 2.94; p = 0.006). CONCLUSIONS: New Zealand Polynesian patients may have increased genetic predisposition to MPN, thus we advocate for modern genetic testing in this ethnic group to identify the cause. Further work is also required to identify modifiable risk factors for mortality in MPN, in particular those associated with male gender and Maori ethnicity; the results may benefit all patients with MPN.

Deletion of five residues from the coiled coil of fibrinogen (Bbeta Asn167_Glu171del) associated with bleeding and hypodysfibrinogenemia.

Routine pre-surgical coagulation investigations led to the detection of a novel type of hypodysfibrinogenemia whose functional defect appears to result from an alteration in the spacing between the functional domains of the fibrinogen molecule. The detection, by reverse phase HPLC, of a minor isoform of Bbeta chain with a 554 Da decrease in mass led to the identification of a deletion of five amino acids (NVVNE) from the center of the coiled coil. The variant chain contributed only 10% of the total Bbeta material and the mutation (BbetaAsn167_Glu171del) was associated with both increased clotting times and low functional and physical fibrinogen concentrations in 3 family members. There was a significant history of pregnancy-associated bleeding and miscarriage within the first trimester. Mechanistically the 15-nucleotide deletion appears to arise from replication advancement during DNA synthesis caused by a flanking pentanucleotide repeat of AATGA.

A second case of Hb Fontainebleau [alpha21(B2)Ala-->Pro] in an individual with microcytosis.

The identification of a second case of Hb Fontainbleau [alpha21(B2)Ala-->Pro] allowed us to re-examine its association with microcytosis, explore the effects of the mutation on protein stability and define the mutation at a DNA level. Although slightly unstable, the variant was expressed at 28-29% of the total and was caused by a heterozygous mutation in the alpha2 gene. There was no evidence for concomitant alpha-thalassemia (alpha-thal); both alpha-globin gene deletion analysis and sequencing of the alpha-globin locus failed to detect any additional mutations that might explain the relatively high expression level.

Myeloblasts in normal bone marrows expressing leukaemia-associated immunophenotypes.

Measurable residual disease (MRD) status of patients undergoing treatment for acute myeloid leukaemia (AML) is important for prognosis and guides treatment. Multicolour flow cytometry (MCF) is a sensitive MRD method. The current approach relies on identification of blasts expressing leukaemia-associated immunophenotypes (LAIP) or by blasts expressing aberrant differentiation/maturation profiles compared to that seen in normal haematopoietic precursor cells at follow-up, i.e., different from normal (DFN). However, expression of LAIP on normal myeloblasts affects the specificity of the result, and the understanding of what is normal is important. Limited published data are currently available. We report findings from 14 normal adult bone marrows. MCF was performed on the residual normal marrow specimens from 14 adults. Expression of CD15, CD11b, CD7, CD4, and CD56 on CD34+ myeloblasts was assessed. Analysis of samples was performed using 4-colour flow cytometry which was the methodology used when this work was done, and is still being used in many clinical flow laboratories worldwide. LAIP is defined by lineage infidelity or asynchronous expression of differentiation markers. The cases of normal myeloblasts with LAIP involving the markers used and above the cut-off levels for MRD detection (0.01%) varies between 43% and 100%, limiting the specificity of the results for MRD. Even if the threshold is raised to 0.1%, there will still be false positive cases using aberrant CD15 or CD7. Our work provided useful information for AML MRD determination in our laboratory. A collaborative database of LAIP on normal myeloblasts using standardised analysis should be useful to determine the optimal diagnostic cut-off for AML MRD using LAIP.

Early treatment of acute promyelocytic leukaemia is accurately guided by the PML protein localisation pattern: real-life experience from a tertiary New Zealand centre.

Current guidelines recommend that a rapid test be used to assist diagnosis of acute promyelocytic leukaemia (APL), but the choice of an assay is discretionary. PML immunofluorescence (PML IF) identifies the microparticulate pattern of the PML protein localisation, highly specific for APL. The aim of this study was to evaluate clinical utility of PML IF in a real-life setting based on a retrospective records review for all patients who had PML IF performed in our centre between 2000 and 2017. Final analysis included 151 patients, 70 of whom had APL. PML IF was reported on average 3 days faster than cytogenetics. Compared with genetic results, PML IF showed sensitivity of 96% and specificity of 100%. PML IF accurately predicted APL in four APL cases with cryptic karyotype/FISH and excluded APL in 98% cases tested based on the suspicious immunophenotype alone, 21/28 of whom had mutated NPM1. Results of PML IF influenced decision to start ATRA in 25 (36%) APL patients and led to its termination in six non-APL patients. In conclusion, PML IF is a fast and reliable test that facilitates accurate treatment decisions when APL is suspected. This performance of PML IF remains hard to match in a real-life setting.

Aggressive Natural Killer-Cell Leukemia: report of five cases and review of the literature.

We report 5 cases of Aggressive Natural Killer-Cell Leukemia (ANKL) diagnosed and treated in our institution within a period of 5 years. Presented are the clinical, hematological, immunophenotypic, immunogenotypic and cytogenetic data. ANKL is a very rare disorder. On review of the English-language literature only 68 published cases were identified. Analysis was performed on a total number of 73 cases (68 from the literature and the series of 5 presented hereby). Presented and discussed are the epidemiology, clinical picture, morphology, cell marker, immunogenotype, cytogenetics, and survival data of the analysis, as well as the associations with the Epstein-Barr virus (EBV). To our knowledge this is the largest series of cases of ANKL analyzed and therefore it is hoped to contribute towards a better characterization of the disorder.

Impact of concordant and discordant bone marrow involvement on outcome in diffuse large B-cell lymphoma treated with R-CHOP.

PURPOSE: In diffuse large B-cell lymphoma (DLBCL), prior studies suggest that concordant bone marrow involvement with DLBCL portends a poorer prognosis, whereas discordant bone marrow involvement with small B-cell lymphoma does not. We examined the significance of bone marrow involvement in patients treated in the current era of therapy including rituximab. PATIENTS AND METHODS: We performed a retrospective analysis of the prognostic impact of bone marrow involvement in an unselected population of patients with newly diagnosed DLBCL treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone in British Columbia and Auckland, New Zealand, with complete clinical information and evaluable staging bone marrow biopsies. RESULTS: In total, 795 patients were identified. Six hundred seventy (84.3%) of 795 had a negative bone marrow, 67 patients (8.4%) had concordant and 58 (7.3%) had discordant involvement. Median follow-up was 41 months (range, 1 to 115). Progression-free survival (PFS) was inferior in those with concordant (P < .001) and discordant (P = .019) involvement while overall survival (OS) was inferior in those with concordant involvement (P < .001) only. In a multivariate analysis controlling for the International Prognostic Index (IPI) score, concordant involvement remained an independent predictor of PFS (P < .001) and OS (P = .007). Discordant involvement was associated with older age, elevated lactate dehydrogenase, advanced stage, and increased number of extranodal sites and was not a negative prognostic factor independent of the IPI score. CONCLUSION: The negative prognostic impact of discordant involvement is adequately represented by the IPI score, while the risk with concordant involvement is greater than that encompassed by this predictor. The results emphasize the need for accurate staging assessment of bone marrow involvement in DLBCL.

Novel hemoglobin alpha chain elongation resulting from a 15-residue insertion and tandem duplication of the F helix.

OBJECTIVES: To determine the cause of an unusual hemoglobin pattern with two novel components eluting after HbA(2) on cation exchange HPLC. This variant was detected during HbA1c measurement and was associated with a normal blood count and a positive isopropanol test. DESIGN AND METHOD: Whole hemolysate and isopropanol precipitates were analysed by ESI MS, and individual components were purified by reverse phase and cation exchange HPLC. Tryptic peptide mapping of isopropanol precipitates was used to detect the molecular lesion and DNA sequencing was used to characterise the precise rearrangement. RESULTS: ESI MS showed a mass increase of 1614Da in 9% of the alpha globin chains and sequence analysis of the alpha2 gene revealed the heterozygous insertion of 45 nucleotides after codon 93. The predicted in phase incretion of ALSALSDLHAHKLRV (+ 1613Da) is a direct repeat of residues alpha79-93 and signature ions from the new peptide were clearly visible in peptide maps of the unstable hemoglobin. CONCLUSION: The insertion probably results from replication slippage during DNA synthesis and the 15-residue repeat results in full repetition of the heme-linked F helix. The nature of the inserted sequence explains the molecular instability and the electrophoretic mobility, but not the twin peaks observed on cation exchange HPLC. These components had the same chain composition, (alpha(L) beta), the same number of heme groups per chain, were not in rapid equilibrium with each other, and probably represent hemoglobin species with different conformers of the elongated alpha(L) chain.