Surender Juneja

Value of bilateral bone marrow biopsy specimens in non-Hodgkin's lymphoma.

A study of 260 patients with non-Hodgkins lymphoma (NHL) who underwent bilateral bone marrow biopsy at initial diagnosis showed marrow disease in 99 (38%) cases. The highest incidence of disease (83%) was seen in small lymphocytic lymphoma (SLL) and the lowest (19%) in diffuse large cell lymphoma (DLCL). Among cases with positive marrows, disease was bilateral in all 15 cases of SLL but in only 10 of 20 (50%) of the DLCL cases. In 30 of 99 (30%) positive marrows disease was unilateral. Follicular lymphomas were strongly associated with a paratrabecular pattern, with 40 of 45 positive cases showing this. Discordant histology was seen in six of 20 positive cases of DLCL and two of 37 positive cases of follicular small cleaved cell lymphomas (FSCCL). A bone marrow aspirate was positive in only 56 of the 99 (57%) cases. Peripheral blood disease was present in 15% of the bone marrow positive cases and in 6% of the cases overall. The incidence of marrow disease varies with the histological subtype of lymphoma. The paratrabecular pattern is associated with follicular lymphoma, and bilateral biopsy specimens increase the positivity rate in most subtypes of NHL.

The prognostic impact of bone marrow involvement in patients with diffuse large cell lymphoma varies according to the degree of infiltration and presence of discordant marrow involvement.

Abstract:  Background: The prognostic significance of marrow involvement in diffuse large cell lymphoma (DLCL) is controversial. Factors that that have been reported to influence prognosis include the pattern and extent of marrow infiltration and histological discordance between the primary site and the bone marrow. Methods: Bone marrow biopsies from 172 patients with newly diagnosed DLCL entered in two consecutive trials of the Australasian Leukaemia and Lymphoma Group were analyzed. Progression‐free (PFS) and overall survival (OS) were calculated according to the absence or presence of bone marrow involvement (BMI), the extent of lymphomatous infiltration and the presence of histological discordance between the primary site and the bone marrow. Results: Of 172 patients with DLCL accrued between 1982 and 1990, who were treated with CHOP or CHOP‐like regimens, 47 (27%) demonstrated marrow involvement on examination of multiple levels. Seventy two percent (34/47) of patients had discordant marrow involvement (<50% large cells) and 28 had minimal (<10%) involvement; these latter patients with minimal marrow involvement (<10%) had similar PFS & OS to the 113 patients without involvement. Within the group of 47 patients with marrow involvement, an increasing percentage of BM involvement was significantly associated with an increasing percentage of concordant histology and a decreasing PFS & OS. Conclusions: Minimal BMI, seen in the majority of patients with DLCL with marrow infiltration, appears not to influence the PFS & OS. However, an increasing degree of marrow involvement is associated with an increasing component of large cells and a poorer prognosis in DLCL patients, independent of other risk factors.

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell‐capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93·9% (495/527 patients) for peripheral blood and 97·6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 × 109 cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Hairy cell leukemia has been shown to be strongly associated with the BRAF V600E mutation. We screened 59 unenriched archived bone marrow aspirate and peripheral blood samples from 51 patients with hairy cell leukemia using high resolution melting analysis and confirmatory Sanger sequencing. The BRAF V600E mutation was detected in 38 samples (from 36 patients). The BRAF V600E mutation was detected in all samples with disease involvement above the limit of sensitivity of the techniques used. Thirty-three of 34 samples from other hematologic malignancies were negative for BRAF mutations. A BRAF K601E mutation was detected in a patient with splenic marginal zone lymphoma. Our data support the recent finding of a disease defining point mutation in hairy cell leukemia. Furthermore, high resolution melting with confirmatory Sanger sequencing are useful methods that can be employed in routine diagnostic laboratories to detect BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Peripheral blood CD34 + cell count reliably predicts autograft yield

A reliable measure to predict peripheral blood progenitor cell (PBPC) autograft CD34+ cell content is required to optimize the timing of PBPC collection. We prospectively examined the peripheral blood (PB) CD34+ cell count in 59 consecutive patients with various malignancies and analyzed the correlation between the PB CD34+ cell count and various parameters in the PBPC autograft. Two hundred and thirty-five collections were performed with a median of 4.0 collections per patient (range, 2–10). The median PB CD34+ cell count at the time of collection was 39 × 106/l (range, 0.0–285.6). The PBPC autograft parameters measured were the CD34+ cell, colony-forming unit granulocyte–macrophage (CFU-GM) and mononuclear cell (MNC) content. There was a strong linear correlation between PB CD34+ cells/l and autograft CD34+ cells/kg (r = 0.8477). The correlation with CFU-GM/kg (r = 0.5512) was weaker. There was no correlation between autograft CD34+ cells/kg and PB WBC (r = 0.0684), PB MNC (r = 0.1518) or PB platelet count (r = 0.2010). At our institution we aim to obtain a minimum of 0.5 × 106 CD34+ cells/kg with each day of collection. We demonstrate that such a collection can be reliably obtained if the PB CD34+ cell count exceeds 5.0 × 106/l.

Detection of NPM1 exon 12 mutations and FLT3 – internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia

BackgroundMolecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples.ResultsWe developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results.ConclusionHRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.

The role of molecular studies in lymphoma diagnosis: a review

Lymphoma classification is based on a multiparametric approach to diagnosis, in which clinical features, morphology, immunophenotype, karyotype and molecular characteristics are important to varying degrees. While in most cases, a diagnosis can be confidently established on the basis of morphology and immunophenotype alone, a small proportion of diagnostically difficult cases will rely on molecular studies to enable a definitive diagnosis. This review discusses the various molecular techniques available including Southern blotting (SB), polymerase chain reaction (PCR), fluorescence in situ hybridisation (FISH)--including multicolour-FISH/spectral karyotyping and comparative genomic hybridisation--and also gene expression profiling using cDNA microarray technology. Emphasis is given to the analysis of antigen receptor gene rearrangements and chromosomal translocations as they relate to lymphoma diagnosis and also in the setting of minimal residual disease (MRD) detection and monitoring. Laboratories performing these tests need to have expertise in these areas of testing, and there is a need for greater standardisation of molecular tests. It is important to know the sensitivity and specificity of each test as well as its limitations and the pitfalls in the interpretation of results. Above all, results of molecular testing should never be considered in isolation, and must always be interpreted in the context of clinical and other laboratory data.

Secondary acute myeloid leukemia with inv(16): report of two cases following paclitaxel-containing chemotherapy and review of the role of intensified ara-C therapy

Acute myeloid leukemia developing secondary to prior cytotoxic chemotherapy (s-AML) encompasses a range of distinct entities. We report two cases of s-AML with inv(16)(p13q22) who had prior exposure to paclitaxel. Additionally, two previously reported cases of s-AML with inv(16) had prior paclitaxel exposure raising the possibility that the taxanes may predispose to this specific syndrome of s-AML. One of our patients received escalated-dose ara-C chemotherapy, achieving a complete remission (12+ months). We therefore examined the prognosis of previously reported cases of s-AML with inv(16) and analyzed the influence of escalated-dose ara-C (⩾400 mg/m2/day). A total of 25 evaluable cases were identified, with 96% attaining CR independent of ara-C dose. The estimated median remission duration was 40 months and the median survival has not been reached (actuarial 5-year survival 52 ± 18%). Although not achieving statistical significance, patients treated with escalated dose ara-C (n = 15) had longer remission duration and overall survival than those treated with standard dose ara-C (n = 10) (P = 0.063 and 0.20, respectively). In univariate analysis, younger age, male gender, and the presence of additional cytogenetic abnormalities were associated with a tendency towards adverse outcomes (P< 0.1). age and gender were equally distributed between ara-c dose cohorts, but more patients treated with standard-dose ara-c had additional cytogenetic abnormalities (P = 0.048). Within the limitations of this retrospective study, this analysis suggests that, similar to de novo AML with inv(16), secondary cases may also potentially benefit from treatment with escalated-dose ara-C. This is consistent with the premise that the underlying molecular defect, rather than the presence of prior cytotoxic drug exposure, may be the most important determinant of disease behavior and chemotherapy responsiveness in AML.

Bone marrow immunohistology of plasma cell neoplasms.

The application of immunohistology to the spectrum of plasma cell disorders has yet to be incorporated widely into routine haematology practice. This technique enables the direct visualisation of specific surface and cytoplasmic antigens in the context of the individual cell and the surrounding anatomical neighbourhood. This review outlines the role of bone marrow immunohistology in the laboratory evaluation of patients with suspected and established plasma cell neoplasms and its emerging role in understanding myeloma biology for possible future therapeutic application.

DNA ploidy patterns and cytokinetics of non-Hodgkin's lymphoma.

Flow cytometry studies for cellular DNA analysis were performed in 115 cases of non-Hodgkins lymphoma, 53 of which had not received any prior chemotherapy or radiotherapy. DNA content was measured in ethanol fixed cells stained with chromomycin A3. According to the criteria of the International Working Formulation there were 43 low grade, 58 intermediate grade, and eight high grade lymphomas; six cases were in the miscellaneous group. Seventy seven (67%) had only diploid DNA content. Thirty eight (33%) showed DNA aneuploidy; 20 of these had been previously treated with chemotherapy or radiotherapy, or both. DNA aneuploidy was seen as hyperdiploidy in all cases except one, and it varied from slightly hyperdiploid to tetraploid. The incidence of aneuploidy increased significantly with increasing histological grade (p = 0.0002) and was not related to previous treatment. The low, intermediate, and high grade lymphomas had 14% (six of 43), 47% (27 of 58), and 62.5% (five of eight) cases, respectively, that showed DNA aneuploidy. The percentage of cells in S phase increased significantly with a higher histological grade (p less than 0.0001). The median S fraction in the low, intermediate, and high grade lymphomas was 1.0 (0.5 to 10)% 4 (0.4 to 35)%, and 27 (4.6-56)%, respectively. There is a significant correlation between histological grade and S fraction and the presence or absence of aneuploidy. There is heterogeneity, however, within both histological grade and a histological subtype.