Surface IgD is coexpressed with IgM on naive mature B cells. Still, the role of surface IgD remains enigmatic even 50 y after its initial discovery. In this study, we examined the in vivo role of surface IgD in human B cell homeostasis and Ab responses in four individuals with heterozygous nonsense mutations in IGHD All IGHD heterozygous individuals had normal numbers of B cells and serum Igs and did not show signs of immunodeficiency or immune dysregulation. IgD+ and IgD- naive mature B cells were present in equal numbers and showed similar immunophenotypes, except for decreased expression of CD79b in the IgD- subset. Furthermore, both IgD+ and IgD- naive mature B cells had normal replication histories and similar capacities to differentiate into plasma cells upon in vitro stimulation, and Ig class-switched memory B cells showed similar levels of somatic hypermutations. Thus, human B cells lacking IgD expression develop normally and generate immunological memory in vivo, suggesting that surface IgD might function more restrictedly in regulating of B cell activation to specific antigenic structures.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Diffuse large B-cell lymphoma (DLBCL) usually needs to be treated immediately after diagnosis from a single lymph node biopsy. However, several reports in other malignancies have shown substantial spatial heterogeneity within large tumours. Therefore, we collected multiple synchronous biopsies of twelve patients that had diagnostic or therapeutic resections of large lymphoma masses and performed next-generation sequencing of 213 genes known to be important for lymphoma biology. Due to the high tumour cell content in the biopsies, we were able to detect several mutations which were present with a stable allelic frequency across all the biopsies of each patient. However, ten out of twelve patients had spatially discordant mutations and similar results were found by the analysis of copy number variants. The median Jaccard similarity coefficient, a measure of the similarity of a sample set was 0.77 (range 0.47-1), and some of the involved genes such as CARD11, CD79B, TP53, and PTEN have a known prognostic or therapeutic relevance in DLBCL. This shows that single biopsies underestimate the complexity of the disease and might overlook possible mechanisms of resistance and therapeutic targets. In the future, the broader application of liquid biopsies will have to overcome these obstacles.

Keywords: clonal evolution; diffuse large B-cell lymphoma; lymphomagenesis; massively parallel sequencing; spatial heterogeneity.

Immunoglobulin (Ig) genes encode antibodies in jawed vertebrates. They are essential elements of the adaptive immune response. Ig exists in soluble form or as part of the B cell membrane antigen receptor (BCR). Studies of Ig genes in fish genomes reveal the absence of Ig genes in Gouania willdenowi by deletion of the entire Ig locus from the canonical chromosomal region. The genes coding for integral BCR proteins, CD79a and CD79b, are also absent. Genes exist for T α/β lymphocyte receptors but not for the T γ/δ receptors. The results of the genomic analysis are independently corroborated with RNA-Seq transcriptomes from other Gobiesocidae species. From the transcriptome studies, Ig is also absent from these other Gobiesocidae species, Acyrtus sp. and Tomicodon sp. Present evidence suggests that Ig is missing from all species of the Gobiesocidae family.

Keywords: B cell receptor; Gobiesocidae; Immunoglobulins.

Flow cytometry immunophenotyping has an essential role in distinguishing chronic lymphocytic leukemia from other B-chronic lymphoproliferative disorders. Recently, CD200 is considered as a relatively consistent marker in chronic lymphocytic leukemia. We retrospectively assessed CD200 expression in 252 patients with B chronic lymphoproliferative disorders with four-color flow cytometry. CD200 expression estimation included the proportion of positive cells (≥30%) and the mean fluorescence intensity ratio. Additionally, we have incorporated CD200 into Matutes score, also replaced FMC7 and CD79b in an attempt to improve the score discriminative power. Of 252 patients enrolled, 199(79%) patients were classified as chronic lymphocytic leukemia and 53 (21%) as other B-chronic lymphoproliferative disorders. All chronic lymphocytic leukemia cases and 20 of 53 (37.7%) of other B-chronic lymphoproliferative disorders demonstrated high CD200 expression (≥30%). Further, CD200 (≥30%) revealed a higher accuracy in comparison to other markers in Matutes score (range: 51%-92.5%). Also, CD200 addition to the Matutes score has correctly recognized all 199 chronic lymphocytic leukemia cases including 10 atypical chronic lymphocytic leukemia cases. As for non-CLL cases, 20 of 53 attained a higher score, yet keeping the original diagnosis. Moreover, CD200 enhanced the diagnostic accuracy of Matutes score to 100%, and when included in a simplified 4-markers score, showed an accuracy of 99.8% compared to 99.4% of Matutes score. In conclusion, CD200 is an accurate diagnostic marker for chronic lymphocytic leukemia, and can refine the modified Matutes score accuracy when added with other markers.

BACKGROUND

The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency.

METHODS

Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients.

RESULTS

The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients.

CONCLUSIONS

This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common non Hodgkin lymphoma (NHL) in adults, and it accounts for about 30% of adult NHL cases. Newly diagnosed patients are treated with rituximab in combination with anthracycline-containing chemotherapy, but a significant number of patients relapse after initial treatment. New strategies for relapsed lymphomas are in development among which antibody-drug conjugates (ADCs) are currently in clinical trials. Polatuzumab vedotin is a novel ADC which binds to the commonly expressed B-cell antigen CD79b, and it delivers monomethyl auristatin E, a small molecule with anti-tubulin activity. Polatuzumab vedotin in combination with bendamustine and rituximab (BR) has been approved in the U.S. and the E.U. for use in patients with relapsed or refractory DLBCL ineligible for transplant. These approvals were based on a randomized study of patients treated with either polatuzumab vedotin plus BR or BR alone, where complete response was 40% in the polatuzumab vedotin + BR group versus 18% in the BR group. The most common adverse events of this treatment were cytopenias and peripheral neuropathy.

A recombinant chimeric fusion protein, muIgbeta-hugamma4.Fc, composed of the extracellular domain of mouse Igbeta (CD79b) and the CH2-CH3 domains of human IgGgamma4.Fc (hugamma4.Fc), linked via an immunologically inert flexible peptide, was prepared. The fusion protein was evaluated for its ability to induce specific auto-reactive immune response against Igbeta and to modulate B cell activity in Balb/c mice. Upon immunization with muIgbeta-hugamma4.Fc, mice developed immunoglobulin (IgG) against self-Igbeta, which could bind to the cells of a mouse B cell line expressing Igbeta on the cell surface. The proportion of B cells in mononuclear cells in the peripheral blood (PBMC) of treated mice decreased as compared to that of mice immunized with hugamma4.Fc without the Igbeta component. Furthermore, mice immunized against muIgbeta-hugamma4.Fc displayed a reduced antibody response against an irrelevant antigen. The implications of employing the present approach in developing a therapeutic strategy for regulating B cell activity has been discussed.

The current study aimed to explore the mechanisms associated with classic Hodgkin lymphoma (cHL) to identify novel diagnostic and therapeutic targets. The GES12453 microarray dataset was downloaded from the Gene Expression Omnibus database; the differentially expressed genes (DEGs) between cHL samples and normal B cell samples by were identified using the limma package. Gene ontology (GO) and pathway enrichment analysis of DEGs gene were performed. Furthermore, construction and analysis of protein‑protein interaction (PPI) network was performed, and co‑expression modules of DEGs were produced. A total of 450 DEGs were identified, comprising 216 upregulated and 234 downregulated genes in cHL compared with normal B cell samples. The DEGs were enriched in biological processes associated with immune response. The upregulated genes were mainly associated with the pathway of transcriptional misregulation in cancer, while downregulated genes were associated with B cell receptor signaling. PPI network analysis demonstrated that IL6 had the highest connectivity degree. Interleukin‑6 (IL6) and signal transducer and activator of transcription 1 (STAT1) were demonstrated to be involved with the response to cytokine GO term in co‑expression module 1. Spleen tyrosine kinase (SYK), B‑cell linker protein (BLNK), CD79B, phospholipase C γ2 (PLCG2) were enriched in the B cell receptor signaling pathway in module 2. Matrix metallopeptidase 9 (MMP9), protein tyrosine phosphatase receptor type C had the highest connectivity degrees in module 3 and module 4, respectively. The results suggested that DEGs, including IL6, STAT1, MMP9, SYK, BLNK, PLCG2 and CD79B, and the pathways of B cell receptor signaling, Epstein‑Barr virus infection and transcriptional misregulation in cancer have strong potential to be useful as targets for diagnosis or treatment of cHL.

A 70-year-old woman presented with an atypical erythematopapular zosteriform eruption of 3 weeks' duration. The patient had no history of previous vesicular eruption. She developed a painful burning sensation on the neck. Clinical examination revealed a cluster of small erythematous firm papules and plaques in a zosteriform distribution on the left ear, face, neck, and shoulder (Figure 1A). The lesions were unilateral and did not cross the midline. Multiple cervical and axillary lymph nodes were palpable. Laboratory tests revealed an increase in white blood cells of 25,000/mm3, with 17,910/mm3 lymphocytes and a normal range of hemoglobin, platelets, creatinine, and liver enzymes. Erythrocyte sedimentation rate was 87 mm. Blood smear results showed small, morphologically mature lymphocyte cells. In immune phenotyping, lymphocyte cells co-express CD5 and B-cell-surface antigens CD19 and CD23, as well as a restriction of kappa immunoglobulin light chains. The cells were CD22-, CD79b-, CD38-, CD10-, CD25- and FMC7-. Computed thoracoabominal tomography revealed cervical, mediastinal, abdominal, and pelvic adenopathy confirming the diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) stage B. Histology of a skin biopsy from a papule showed a dense nodular granulomatous infiltrate in the dermis (Figure 2A). The infiltrate contained epithelioid and giant cells surrounded by lymphocytes and plasma cells. Small monomorphic lymphocytes without mitotic figures predominated (Figure 2B). The epidermis was irregularly thickened. Immunohistology revealed a polymorphous infiltrate with a phenotype of reactive T lymphocytes (CD3, CD5 positive) (Figure 2C), B lymphocytes (CD20 positive) (Figure 2D). Epithelioid and giant cells were positive for CD68 (Figure 2E). A latent herpes zoster infection with granulomatous reaction at the site ofzoster lesions was highly suspected as the patient reported a unilateral burning sensation without a history of vesicular zosteriform eruption. She received treatment with intravenous acyclovir 10 mg/kg every 8 hours. The papular lesions resolved markedly (60%) on macular plaques at the end of the treatment. Following topical treatment with corticosteroids, the lesions healed completely within 4 weeks (Figure 1B). Concerning leukemia, our patient was monitored without therapy by the hematologist.

Primary cardiac diffuse large B cell lymphoma (PC-DLBCL) is a rare kind of hematological malignancy, and its clinical and pathologic characteristics, especially in Eastern countries, remain unclear. Moreover, genomic alterations in PC-DLBCL have not been studied previously. We describe a case of a 57-year-old man who presented with exertional dyspnoea due to a heart mass in April 2018 and was diagnosed with PC-DLBCL characterized by immunohistochemical markers of the activated B cell (ABC) subtype and double expression of c-MYC and Bcl-2. Mutations in a total of 11 genes-TBL1XR1, CD79B, IGLL5, ZMYM3, MYD88, TMSB4X, PIM1, BTK, NRXN3, CUX1, and CSMD1-were detected via next-generation sequencing (NGS), while 19 copy number variations (CNVs) such as 1q+, 3p+, 3q+(*2), 5p+, 6p-, 6q-, 7q+, +11, 12q-, 15q-, 17q+, 17p-, +18, 19q+, 19p-, 19q-, X q+, and -Y and 4 copy-neutral loss of heterozygosity (CN-LOH) lesions located at 1q21.1q44, 3p26.3q11.2, 3q13.11q29 and 6p22.2p21.32 were identified by single nucleotide polymorphism (SNP) array karyotyping. Some key gene alterations in lymphoma, such as PRDM1 deletion and Bcl-2 amplification, were identified using SNP array analysis. The patient received 6 courses of chemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, R-CHOP regimen) after surgery and is currently in remission. In summary, the present case was diagnosed as PC-DLBCL, ABC subtype by the Hans algorithm and double expression lymphoma, with co-occurrence of the MYD88^L265P and CD79B mutations (MCD) subtype by genetic alteration analysis. This study presents a unique PC-DLBCL case in which complex genomic alterations were revealed by NGS and SNP array analysis, which has never been reported in the literature, and these findings could provide new insight into the genomic characterization of PC-DLBCL.

CD79α protein together with the related CD79β protein forms the B-cell antigen receptor (BCR). It remains present when B cells transform into active plasma cells, and is also present in virtually all B cell neoplasms. Monoclonal antibody (mAb) S3 (S3Ab) is a novel anti-CD79α antibody generated by using Raji cells as an immunogen. Herein, we conducted a study on S3Ab using various cellular and immunocytological techniques. The results showed that S3Ab could recognise CD79α in living cells. The molecular weights of the heavy and the light chains of S3Ab were 55 and 26 kDa, respectively. S3 antigen is only expressed on more mature B cells and negative on blast B cells. It could partially block the binding of anti-CD79α (Hm47, recognising the cytoplasmic domain of CD79α) to target cells. Immunoprecipitation experiment showed that S3 antigen is about 33 kDa and S3 can specifically bind to the recombinant extracellular segment of CD79α. The internalisation rate of S3Ab to the target cells was as high as 74.0% after incubation at for 3 h. In conclusion, S3Ab is probably a new target molecule for B cells and can be an excellent antibody in targeting treatment of haematopoietic malignancies, warranting further development of this agent.

Introduction: Lymphoid enhancer-binding factor 1 (LEF-1) overexpression has been recently remarkably reported in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and has shown utility in distinguishing CLL/SLL from other B-cell lymphomas. CLL has a well-defined immunophenotype, yet, some cases of CLL demonstrate atypical morphology/ phenotype reflected by low Matutes score (atypical CLL). Till date, LEF1 expression has not been systematically studied in cases of CLL with atypical features.

Methods: In this study, LEF-1 expression was assessed by two different techniques, (immunohistochemistry and flow cytometry), to investigate the expression profile of LEF-1 in cases of CLL/SLL, in comparison with other low-grade B-lymphomas and CLL with atypical features, including atypical immunophenotype and CLL with increased prolymphocytes or morphologically atypical cells.

Results: We found that LEF-1 expression is downregulated in CLL with atypical immunophenotype/features compared to classic CLL; Chi-Square P < .0001. The ratio for LEF-1 expression in malignant B-cells/NK (by flow cytometry) in CLL/SLL with classic immunophenotype was higher than atypical CLL and is significantly higher in other small B-cell lymphomas (P < .01). Absence of LEF-1 expression in CLL/SLL is correlated (P < .05) with downregulation of CD5, CD23, CD200, expression of FMC7, brighter expression of CD79b, brighter expression of surface light chain, increased prolymphocytes and lower Matutes score.

Conclusion: As downregulation of LEF-1 expression is well correlated with atypical CLL, we suggest adding LEF-1 to Matutes score as a beneficial marker to differentiate classic from atypical CLL LEF-1 could also serve as a potential prognostic indicator for CLL clinical course.

Keywords: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL); Flow cytometry; Immunohistochemistry; Lymphoid enhancer-binding factor 1 (LEF-1); atypical CLL.

OBJECTIVE

Hairy cell leukaemia (HCL) is a B cell neoplasm which constitutes around 2 per cent of all the lymphoid leukaemias. It has a characteristic morphology and immunophenotypic profile. It is important to distinguish HCL from other B cell lymphoproliferative disorders due to availability of different chemotherapeutic agents. This study presents clinical, haematological and immunophenotypic profile of patients with HCL seen over a period of four years in a tertiary care hospital in north India.

METHODS

Twenty one cases of hairy cell leukaemia were analyzed for their clinical details, haemogram, bone marrow examination and immunophenotypic findings.

RESULTS

Age of the patients ranged from 28-76 yr with male predominance. Weakness and fever were commonest presentations. Splenomegaly, hepatomegaly, lymphadenopathy were seen in decreasing order of frequency. Anaemia was noted in all 21 patients, leukopenia in 15 and thrombocytopenia in 19 cases. Fourteen patients were pancytopenic. Bone marrow examination showed typical hairy cells in all cases. Immunophenotyping showed expression of CD19, CD20, CD103, CD25 and CD11c in all cases, while positivity was seen for CD79b in 93.7 per cent, kappa light chain restriction in 60 per cent and lambda in 40 per cent cases. Notably, 20 per cent showed CD10 and 12 per cent showed CD23 expression.

CONCLUSIONS

This study reveals some unusual findings in otherwise classical disease entity, like absence of palpable spleen, presence of lymphadenopathy, normal or elevated leukocyte counts, expression of CD10, which at times could be diagnostically challenging.

B-cell commitment is characterized by the expression of specific membrane proteins and the rearrangement and expression of immunoglobulin (Ig) heavy (H) and light (L) chain genes. At early stages of B-cell development, unrearranged Ig loci are transcribed, which correlates with these regions becoming accessible for Ig gene rearrangement. Some germline transcripts can be translated into protein and potentially play a role in cell signaling during B-cell development. In this report an early stage in human B-cell development is characterized using Epstein-Barr virus (EBV)-transformed cell lines from patients with a severe combined immunodeficiency (SCID). These lines were shown to produce germline constant (C) gene transcripts from the IGH and IGK loci. We demonstrate here that these cells are committed to the B-cell lineage as substantiated by expression of CD79a and CD79b. No surrogate light chain (SLC) gene transcription was detected, indicative of a very early differentiation stage. From these cell lines two types of germline IgV gene transcripts were isolated: a transcript containing the IGKV4-1 gene and a germline IGHV-1 transcript nearly identical to IGHV1/OR15-1 (HC15-1, DP-1), an orphon VH gene on chromosome 15. Germline VH transcripts originating from the VH locus on chromosome 14 could not be detected. It is of interest that, apart from Ig V and C genes (non-functional), V genes that reside outside the Ig locus are a target for the transcription factors that are postulated to initiate Ig gene rearrangement early in B-cell development.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is often causally associated with asbestos exposure. Comparative genomic hybridization techniques and arrays demonstrated a complex set of copy number variations (CNVs) in the MPM-genome. These techniques however have a limited resolution, throughput and flexibility compared to next-generation sequencing platforms. In this study, the presence of CNVs in the MPM-genome was investigated using an MPM-cohort (N = 85) for which genomic microarray data are available through 'The Cancer Genome Atlas' (TCGA). To validate these results, the genomes of MPMs and matched normal samples (N = 21) were analyzed using low-pass whole genome sequencing on an 'Illumina HiSeq' platform. CNVs were detected using in-house developed analysis pipelines and frequencies of copy number loss and gain were calculated. In both datasets, losses on chromosomes 1, 3, 4, 6, 9, 13 and 22 and gains on chromosomes 1, 5, 7 and 17 were found in at least 25% and 15% of MPMs, respectively. Besides the well-known MPM-associated genes, CDKN2A, NF2 and BAP1, other interesting cancer-associated genes were listed as frequently involved in a copy number loss (e.g. EP300, SETD2 and PBRM1). Moreover, four cancer-associated genes showed a high frequency of copy number gain in both datasets (i.e. TERT, FCGR2B, CD79B and PRKAR1A). A statistically significant association between overall survival and the presence of copy number loss in the CDKN2A-containing region was observed in the TCGA-set. In conclusion, recurrent CNVs were detected in both datasets, occurring in regions harboring known MPM-associated genes and genes not previously linked to MPM.

Background: Diffuse large B-cell lymphoma (DLBCL) is a spectrum of disease comprising more than 30% of non-Hodgkin lymphomas. Although studies have identified several molecular subgroups, the heterogeneous genetic background of DLBCL remains ambiguous. In this study we aimed to develop a novel approach and to provide a distinctive classification system to unravel its molecular features.

Method: A cohort of 342 patient samples diagnosed with DLBCL in our hospital were retrospectively enrolled in this study. A total of 46 genes were included in next-generation sequencing panel. Non-mutually exclusive genetic signatures for the factorization of complex genomic patterns were generated by random forest algorithm.

Results: A total of four non-mutually exclusive signatures were generated, including those with MYC-translocation (MYC-trans) (n = 62), with BCL2-translocation (BCL2-trans) (n = 69), with BCL6-translocation (BCL6-trans) (n = 108), and those with MYD88 and/or CD79B mutations (MC) signatures (n = 115). Comparison analysis between our model and traditional mutually exclusive Schmitz's model demonstrated consistent classification pattern. And prognostic heterogeneity existed within EZB subgroup of de novo DLBCL patients. As for prognostic impact, MYC-trans signature was an independent unfavorable prognostic factor. Furthermore, tumors carrying three different signature markers exhibited significantly inferior prognoses compared with their counterparts with no genetic signature.

Conclusion: Compared with traditional mutually exclusive molecular sub-classification, non-mutually exclusive genetic fingerprint model generated from our study provided novel insight into not only the complex genetic features, but also the prognostic heterogeneity of DLBCL patients.

Keywords: Classification; DLBCL; Random forest; Sequencing; Signature.

Over the past year, progress has been made in understanding of the physiology and disease associations of CD5+ (B1) B cells, although their exact role in pathogenesis remains unclear. Earlier studies on the negative function of CD5 within the B-cell receptor complex have been substantiated, and it seems likely that soon the signaling pathways used by this coreceptor will be elucidated. Progress in diagnosis, physiology, and etiopathogenesis of CD5+ malignancies has been made, particularly in B-cell chronic lymphocytic leukemia. The low-level expression of surface immunoglobulin has been explained by the mutations that occur in the associated CD79b. Two new potential tumor-suppressor genes have been identified in the hot spot of chromosome 13q, which provides an exciting step forward in understanding of the etiopathogenesis of some B-cell chronic lymphocytic leukemia. Activated signal transducers for activation of transcription factors molecules have been shown to be phosphorylated on different amino acids in B1 and chronic lymphocytic leukemia tumors, although the significance of this is, as yet, unclear. Finally, aberrant expression of CD40L by chronic lymphocytic leukemia T cells may contribute to the immunodeficiency that develops in these patients.

Morphological differentiation between benign and malignant lymphoproliferative disorders (LPDs) can be challenging. Immunophenotyping (IPT) by either technique, flow cytometry or immunohistochemistry (IHC), is an important step in solving such difficulty. Thirty-five newly diagnosed patients with chronic B-cell neoplasms (11 chronic lymphocytic leukemia, 22 non Hodgkin lymphoma and 2 hairy cell leukemia) were included in this study with age range from 20 to 70 years. Monoclonal antibodies surface expression using lymphoproliferative disorders panel (CD45, CD19, CD5, CD10, CD11c, CD20, CD22, CD23, CD38, CD79b, FMC7, CD103, CD25, kappa and lambda light chains) by flow cytometry was done on bone marrow samples. CD20, CD5, CD23, Bcl-2, Bcl-6, kappa and lambda light chain immunostaining were performed on fixed bone marrow trephine biopsy specimen. The sensitivity of IHC was 81.8% in chronic lymphocytic leukemia (CLL) and 100% in non Hodgkin lymphoma (NHL) as regards CD20, 100% in both groups as regards CD5, 46% in CLL and 66.7% in NHL as regards CD23, 33.3% in CLL and 50% in NHL as regards kappa chain, 20% in CLL and 33.3% in NHL as regards lambda chain. We found that IHC and flow cytometry are equally effective in diagnosing CLL; however, IHC might be slightly more sensitive than flow cytometry in detecting bone marrow infiltration in NHL and hairy cell leukemia (HCL).

Background: There are no immunophenotypic guidelines for the investigation of MYC-rearranged lymphomas. We aimed to identify simple immunophenotypic features that would help to differentiate between MYC-rearranged lymphomas and guide cytogenetic analysis.

Methods: We reviewed diagnostic samples from patients diagnosed with Burkitt lymphoma (BL), double-hit lymphoma (DHL), MYC-rearranged diffuse large B-cell lymphoma (MYC-DLBCL), and standard (non-MYC-rearranged) DLBCL over the last decade in our Institution. Using flow cytometry (with antibodies CD20, CD10, CD38, bcl-2, Ki-67, FMC-7, CD43, CD27, CD79b, CD23, and CD22) we determined antigen% expression and median-fluorescence intensity ratios (MFIR). The forward scatter (FS) and side scatter (SS) characteristics of tumor B-cells were compared with normal T-cells (B/T ratios) for patients with MYC-rearranged lymphomas.

Results: We identified 51 patients of whom 14 had BL, 10 had DHL (6 MYC+/BCL2+; 4 MYC+/BCL6+), 8 MYC-DLBCL, and 19 standard DLBCL. The significant differences (p <.05) were: higher CD38% in BL than standard DLBCL; higher CD10% in BL and DHL versus MYC-DLBCL and standard DLBCL; higher CD10MFIR in BL than MYC-DLBCL and standard DLBCL; higher Ki-67% in BL than DHL and MYC-DLBCL; higher bcl-2% in DHL than BL; higher FMC-7% in BL than MYC-DLBCL and standard DLBCL; and lower SS (B/T) ratio in DHL than MYC-DLBCL.

Conclusions: The combination of CD38% > 90, CD10% > 80, CD10MFIR > 10, bcl-2% < 30, and Ki-67% > 70 was characteristic of BL. "Deviation" from these cut-offs should raise suspicion for DHL and, therefore, BCL2 and/or BCL6 FISH is required. We also found that a diagnosis of DHL rather than of MYC-DLBCL was significantly associated with CD10% > 60, Ki-67% > 50, and SS (B/T) <1.5.

Keywords: Burkitt lymphoma; DLBCL; MYC rearrangement; double-hit lymphoma; immunophenotype.

BACKGROUND

Chronic lymphocytic leukemia (CLL) is a rare B-lymphoid malignancy in Southeast Asia. We evaluated whether a scoring system based on the expression of CDS, CD23, FMC7, CD79b and surface immunoglobulin (Slg) could be utilized to distinguish CLL from other types of lymphoid neoplasms in the Thais.

METHODS

One-hundred and forty-five samples with a clinical suspicion for CLL were analysed by flow cytometry. A score of one was assigned if the following marker was identified: CD5+, CD23+, FMC7-, CD79b- and SIg(-/weak). A cut-off score of>> or =3 was required for the definitive diagnosis of B-CLL.

RESULTS

Only 50 cases (34.5%) were confirmed as B-CLL (scores>> or =3). Cases with scores>> or =3 had significantly higher leukocyte counts and marrow/blood lymphocytes than cases with scores < or =2. Dual CD5/CD23 expression was found in 87.5% of CLL cases. In 81 cases with scores < or =2, a variety of non-CLL disorders predominated, such as marginal zone lymphoma, splenic lymphoma with villous lymphocytes, mantle cell lymphoma, and prolymphocytic leukemia.

CONCLUSIONS

A score of 3 and dual CD5/CD23 expression are essential for the diagnosis of CLL while a score of 2 mostly indicative of non-CLL. The majority of clinical cases of CLL turned out to be non-CLL by flow cytometry. Increased utilization of this scoring system should increase the accuracy of diagnosis of this rare type of leukemia in the Thai population.