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Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Patients with chronic lymphocytic leukemia (CLL) or monoclonal B-lymphocytosis (MBL) have impaired response to COVID-19 vaccination. A total of 258 patients (215 with CLL and 43 with MBL) had antispike antibody levels evaluable for statistical analysis. The overall seroconversion rate in patients with CLL was 94.2% (antispike antibodies ≥50 AU/mL) and 100% in patients with MBL after multiple vaccine doses. After 3 doses (post-D3) in 167 patients with CLL, 73.7% were seropositive, 17.4% had antispike antibody levels between 50 and 999 AU/mL, and 56.3% had antispike antibody levels ≥1000 AU/mL, with a median rise from 144.6 to 1800.7 AU/mL. Of patients who were seronegative post-D2, 39.7% seroconverted post-D3. For those who then remained seronegative after their previous dose, seroconversion occurred in 40.6% post-D4, 46.2% post-D5, 16.7% post-D6, and 0% after D7 or D8. After seroconversion, most had a progressive increase in antispike antibody levels. Neutralization was associated with higher antispike antibody levels, more vaccine doses, and earlier severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants; neutralizing antibody against early clade D614G was detected in 65.3%, against Delta in 52.0%, and against Omicron in 36.5%. SARS-CoV-2-specific T-cell production of interferon γ and interleukin 2 occurred in 73.9% and 60.9%, respectively, of 23 patients tested. After multiple vaccine doses, by multivariate analysis, immunoglobulin M ≥0.53 g/L, immunoglobulin subclass G3 ≥0.22 g/L and absence of current CLL therapy were independent predictors of positive serological responses. Multiple sequential COVID-19 vaccination significantly increased seroconversion and antispike antibody levels in patients with CLL or MBL., (© 2022 by The American Society of Hematology.)

Chronic lymphocytic leukaemia (CLL) is invariably accompanied by some degree of immune failure, and CLL patients have a high rate of second primary malignancy (SPM) compared to the general population. We comprehensively documented the incidence of all forms of SPM including skin cancer (SC), solid organ malignancy (SOM), second haematological malignancy (SHM) and separately Richter's syndrome (RS) across all therapy eras. Among the 517 CLL/small lymphocytic lymphoma (SLL) patients, the overall incidence of SPMs with competing risks was SC 31.07%, SOM 25.99%, SHM 5.19% and RS 7.55%. Of the 216 treated patients, 106 (49.1%) had at least one form of SPM, and 63 of 106 (29.2% of treated patients) developed an SPM 1.5 years (median) after treatment for their CLL. Melanoma accounted for 30.3% of SC. Squamous cell carcinoma (SCC), including eight metastatic SCCs, was 1.8 times more than basal cell carcinoma (BCC), a reversal of the typical BCC:SCC ratio. The most common SOMs were prostate (6.4%) and breast (4.5%). SHM included seven acute myeloid leukaemia (AML) and five myelodysplasia (MDS) of which eight (four AML, four MDS) were therapy-related. Any SPM occurred in 32.1% of 53 Monoclonal B-lymphocytosis (MBL) patients. Age-adjusted standardised rates of SPM (per 100,000) for CLL, MBL and the general Australian population were 2648, 1855 and 486.9, respectively. SPMs are a major health burden with 44.9% of CLL patients with having at least one SPM, and apart from SC, associated with significantly reduced overall survival. Dramatic improvements in CLL treatment and survival have occurred with immunochemotherapy and targeted therapies, but mitigating SPM burden will be important to sustain further progress., Competing Interests: The authors have no conflict of interest to declare., (© 2021 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre-clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC-212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53 ko CLL cell line (OSU-CLL- TP53 ko). ONC-212 induced dose-dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU-CLL- TP53 ko cells. The effects of ONC-212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B-cell receptor (BCR) (AKT and MAPK-ERK1/2) and a pro-apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC-212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro-survival effects of the BCL2-family proteins., Competing Interests: Edwin J. Iwanowicz and Henk Lang have financial interests in Madera Therapeutics, LLC.  In addition, Donald S. Karanewsky is a consultant for Madera Therapeutics, LLC., (© 2020 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

The B-cell receptor signaling pathway and dysregulation of the Bcl-2 family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Despite significant advances in the treatment of the disease, relapse and drug resistance are not uncommon. In the current study, we investigated the dual PI3/PIM kinase inhibitor IBL-202 in combination with venetoclax as a treatment option for CLL using both primary CLL cells and TP53-deficient OSU-CLL cells generated using the CRISPR-Cas9 system. IBL-202 and venetoclax were highly synergistic against primary CLL cells cocultured with CD40L fibroblasts (combination index [CI], 0.4, at a fractional effect of 0.9) and TP53-knockout (KO) OSU-CLL cells (CI, 0.5, at a fractional effect of 0.9). Synergy between the drugs was consistent, with a significant (P < .05) reduction in the 50% inhibitory concentration for both drugs. IBL-202 and venetoclax in combination induced cell-cycle arrest and slowed the proliferation of both wild-type and TP53-KO cell lines. The drug combination inhibited AKT phosphorylation, reduced expression of Bcl-xL and NF-κB, and increased the Noxa/Mcl-1 ratio. Downregulation of CXCR4 was consistent with inhibition of the SDF-1α-induced migratory capacity of CLL cells. Synergy between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53-KO OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for high-risk CLL., (© 2020 by The American Society of Hematology.)

The treatment of chronic lymphocytic leukaemia has been revolutionised in recent years, first by the introduction of chemoimmunotherapy regimens and subsequently by the development of drugs, including ibrutinib, idelalisib and venetoclax, that target components of the B-cell receptor signalling pathway or B-cell lymphoma 2 family of proteins. Despite high initial response rates in patients treated with chemoimmunotherapy or targeted agents, a significant proportion of patients relapse with progressive and refractory disease. In a subset of these patients, drug resistance has been associated with specific genetic lesions or activation of alternate pro-survival pathways. However, the mechanisms that confer drug resistance in the remainder of the patients with refractory disease have yet to be fully elucidated. In this review, we discuss our current understanding of the mechanics of drug resistance in chronic lymphocytic leukaemia and describe how this knowledge may aid in rationalising future treatment strategies to prevent the development of refractory or aggressive transformation of the disease., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2020.)

Ibrutinib, a once-daily oral inhibitor of Bruton's tyrosine kinase, is approved in the United States and Europe for treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). The phase 3 RESONATE study showed improved efficacy of single-agent ibrutinib over ofatumumab in patients with relapsed/refractory CLL/SLL, including those with high-risk features. Here we report the final analysis from RESONATE with median follow-up on study of 65.3 months (range, 0.3-71.6) in the ibrutinib arm. Median progression-free survival (PFS) remained significantly longer for patients randomized to ibrutinib vs ofatumumab (44.1 vs 8.1 months; hazard ratio [HR]: 0.148; 95% confidence interval [CI]: 0.113-0.196; P˂.001). The PFS benefit with ibrutinib vs ofatumumab was preserved in the genomic high-risk population with del(17p), TP53 mutation, del(11q), and/or unmutated IGHV status (median PFS 44.1 vs 8.0 months; HR: 0.110; 95% CI: 0.080-0.152), which represented 82% of patients. Overall response rate with ibrutinib was 91% (complete response/complete response with incomplete bone marrow recovery, 11%). Overall survival, censored for crossover, was better with ibrutinib than ofatumumab (HR: 0.639; 95% CI: 0.418-0.975). With up to 71 months (median 41 months) of ibrutinib therapy, the safety profile remained consistent with prior reports; cumulatively, all-grade (grade ≥3) hypertension and atrial fibrillation occurred in 21% (9%) and 12% (6%) of patients, respectively. Only 16% discontinued ibrutinib because of adverse events (AEs). These long-term results confirm the robust efficacy of ibrutinib in relapsed/refractory CLL/SLL irrespective of high-risk clinical or genomic features, with no unexpected AEs. This trial is registered at www.clinicaltrials.gov (NCT01578707)., (© 2019 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.)

Ibrutinib, a once-daily oral inhibitor of Bruton tyrosine kinase, has greatly improved outcomes for patients with chronic lymphocytic leukemia (CLL). The phase 3 RESONATE trial, which compared single-agent ibrutinib to ofatumumab in high-risk, relapsed patients with CLL, provided support for approval of ibrutinib in the United States and Europe. We describe long-term follow-up of patients treated in RESONATE, where continued superiority of progression-free survival (PFS) (hazard ratio [HR], 0.133; 95% confidence interval [CI], 0.099-0.178) was observed. Overall survival benefit continues (HR, 0.591; 95% CI, 0.378-0.926), although with decreased magnitude relative to that seen before crossover to ibrutinib was implemented for patients on ofatumumab (HR, 0.426; 95% CI, 0.220-0.823). Notably, overall response to ibrutinib increased over time, with 91% of patients attaining a response. The PFS benefit with ibrutinib was independent of baseline risk factors, although patients with ≥2 prior therapies had shorter PFS than those with <2 prior therapies, and the presence of TP53 or SF3B1 mutations showed a trend toward shorter PFS vs without these factors. Median duration of ibrutinib was 41 months, with 46% remaining on treatment at a median follow-up of 44 months. Grade ≥3 adverse events generally decreased over time, causing only a small proportion of patients to cease therapy. Ibrutinib was discontinued due to progressive disease in 27% of patients. This long-term study provides support for sustained efficacy and safety of ibrutinib in relapsed/refractory CLL and consideration of study provisions that allow crossover to investigational therapy when benefit has been clearly demonstrated. This trial was registered at www.clinicaltrials.gov as #NCT01578707., (© 2019 by The American Society of Hematology.)

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

Ibrutinib, an oral inhibitor of Bruton's tyrosine kinase (BTK), at a once-daily dose of 420 mg achieved BTK active-site occupancy in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) that was maintained at 24 hours. It is unknown if intermittent interruption of ibrutinib therapy contributes to altered clinical outcomes. We therefore evaluated the effect of ibrutinib dose adherence on patient outcomes in the phase 3 RESONATE trial. The overall mean dose intensity (DI) was 95% with median treatment duration of ∼9 months. Pharmacokinetic assessment of ibrutinib exposure at 420-mg dose suggested similar exposure regardless of patient weight or age. As assessed by independent review committee, patients with higher DI experienced longer median progression-free survival (PFS) compared with those with lower DI regardless of del17p and/or TP53 status. Of 79 patients requiring a drug hold, treatment was restarted at the original dose in 73 (92%) patients. Mean duration of a missed-dose event was 18.7 days (range, 8-56). Patients missing ≥8 consecutive days of ibrutinib had a shorter median PFS vs those missing <8 days (10.9 months vs not reached). These results support sustained adherence to once-daily ibrutinib dosing at 420 mg as clinically feasible to achieve optimal outcomes in patients with previously treated CLL. The trial was registered at www.clinicaltrials.gov as #NCT01578707., (© 2017 by The American Society of Hematology.)

Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Despite significant advances in therapy over the last decade CLL remains incurable. Current front-line therapy often consists of chemoimmunotherapy-based regimens, most commonly the fludarabine, cyclophosphamide plus rituximab combination, but rates of relapse and refractory disease are high among these patients. Several key signaling pathways are now known to mediate the survival and proliferation of CLL cells in vivo, the most notable of which are the pathways mediated by the B-cell receptor (BCR) and cytokine receptors. A better understanding of the pathogenesis of the disease, the underlying biology of the CLL-cell and the roles of the tumour microenvironment has provided the rationale for trials of a range of novel, more targeted therapeutic agents. In particular, clinical trials of ibrutinib and idelalisib, which target the Brutons tyrosine kinase and the delta isoform of phosphoinositol-3 kinase components of the BCR signaling pathway respectively, have shown extremely promising results. Here we review the current literature on the key signaling pathways and interactions of CLL cells that mediate the survival and proliferation of the leukemic cells. For each we describe the results of the recent clinical trials and in vitro studies of novel therapeutic agents.

Purpose: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, some patients may survive for many years, while 20-30% of patients progress and may die within several years. Currently, there is not a single procedure that enables accurate prognosis and triaging of those patients who need immediate and aggressive treatment. All CLL cells are characterised by the expression of the B-cell antigens CD19, CD20, CD21, CD22 and CD23, with aberrant expression of the T-cell antigen CD5., Methods: We have developed a CD antibody microarray (DotScan) containing 182 immobilised CD antibodies that has been used to obtain extensive surface profiles of CLL cells obtained from 96 patients., Results: Of these 182 antigens, 27 were significantly differentially expressed between stable, stable-progressive and progressive CLL. Some of these antigens are not expressed on normal B-cells and may be targets for therapeutic antibodies against CLL. Unsupervised hierarchical clustering of the surface profiles from 96 patients showed that those with progressive CLL could be distinguished based solely upon this 'disease signature'. The sensitivity (proportion of actual positives correctly identified) was 67.9%, the specificity (proportion of negatives correctly identified) was 77.5%, and the accuracy was 71.9%., Conclusions: Considerable effort by a number of research groups has resulted in identification of individual markers for progressive CLL, but their collective use is yet to provide a test that identifies CLL patients at risk. Data presented here provide a basis for development of a simple test using an antibody microarray.

The interleukin (IL)-7 receptor (IL-7R) is expressed on human pre-B but not mature B-cells. We hypothesised that aberrant expression of IL-7R contributes to B-cell oncogenesis. Surface expression of IL-7R components CD127 and CD132, and intracellular Ki-67 and Bcl-2 were examined by flow cytometry on peripheral blood or bone marrow mononuclear cells (PBMC; BMMC) from patients with B-cell derived neoplasms, chronic human immunodeficiency virus type-1 (HIV-1) infection alone, or healthy volunteers. Plasma IL-7, IL-2, IL-4, IL-6, IL-10, IL-21, TNF-alpha, IFN-gamma and BAFF were measured by enzyme-linked immuno-sorbent assay or bead array. The effects of exogenous IL-7 on PBMC and BMMC were examined. CD127 expression was elevated in pre-B-cell acute lymphoblastic leukemia (pre-B-ALL) and in some cases of Non-Hodgkin's Lymphoma (B-NHL). Plasma IL-7 levels were higher in pre-B-ALL, B-cell chronic lymphocytic leukemia (B-CLL) and HIV-1 associated B-NHL (HIV-B-NHL) compared with control groups. CD127+ pre-B-ALL cells had higher expression of Ki-67, Bcl-2 and CD132 than CD127- counterparts. Unlike T-lineage cells, CD127+ pre-B-ALL cells did not down-regulate CD127 in response to exogenous IL-7. Patients with B-cell derived neoplasms had elevated circulating IL-10 and decreased BAFF. These findings support a role for the IL-7/IL-7R system in B-cell oncogenesis., (2009 Elsevier Ltd. All rights reserved.)

The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.

Cluster of differentiation (CD) antigens are defined when a surface molecule found on some members of a standard panel of human cells reacts with at least one novel antibody, and there is good accompanying molecular data. Monoclonal antibodies to surface CD antigens on leukocytes have been used for flow cytometry, and more recently to construct microarrays that capture live cells. These DotScan microarrays enable the rapid and highly parallel characterization of repertoires of CD antigens whose expression patterns may be correlated with discrete leukaemia subtypes, or used to define biomarker 'signatures' for non-hematological diseases. DotScan with fluorescence multiplexing enables profiling of CD antigens for minor subsets of cells, such as colorectal cancer cells and tumour-infiltrating lymphocytes from a surgical sample.

Cluster of differentiation (CD) antigens are expressed on cells of myeloid and lymphoid lineages. As most disease processes involve immune system activation or suppression, these antigens offer unique opportunities for monitoring host responses. Immunophenotyping using limited numbers of CD antigens enables differentiation states of immune system cells to be determined. Extended phenotyping involving parallel measurement of multiple CD antigens may help identify expression pattern signatures associated with specific disease states. To explore this possibility we have made a CD monoclonal antibody array and scanner, enabling the parallel immunophenotyping of leukocyte cell suspensions in a single and rapid analysis. To demonstrate this approach, we used the specific example of patients infected with human immunodeficiency virus type-1 (HIV-1). An invariant HIV-induced CD antigen signature has been defined that is both robust and independent of clinical outcome, composed of a unique profile of CD antigen expression levels that are both increased and decreased relative to internal controls. The results indicate that HIV-induced changes in CD antigen expression are disease specific and independent of outcome. Their invariant nature indicates an irreversible component to retroviral infection and suggests the utility of CD antigen expression patterns in other disease settings.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

We undertook a study to determine the specificity of the monoclonal antibody, B-ly-7, for hairy cell leukemia (HCL) by examining the expression in 150 samples from B-cell lymphoproliferative diseases as well as screening for reactivity in a number of other hematologic malignancies. Within the B-cell lineage we found that the expression of B-ly-7 was highly specific for HCL and reacted with all 28 cases examined, as well as with 3 of 9 cases of a variant form of HCL. Cells of other closely related B-cell disorders, prolymphocytic leukemia, and splenic lymphoma with villous lymphocytes were negative. Investigation of the peripheral blood and bone marrow of patients with HCL before and after treatment with alpha-interferon or deoxycoformycin suggests that B-ly-7 may be useful in the assessment of minimal disease after therapy. In addition to HCL, we found that B-ly-7 was positive with cells of three mature, CD4+ T-cell malignancies. In view of the reactivity with malignancies of activated B and T cells, we searched for the expression of B-ly-7 on activated, normal B and T cells and found that B-ly-7 reacted specifically with activated normal peripheral blood CD8+ T cells. B-ly-7 has a number of applications, including the precise classification of mature B-cell neoplasia and the diagnosis HCL and its assessment after treatment. In addition, B-ly-7 recognizes a small subset of T-cell disorders. Its expression on these malignancies and on in vitro activated peripheral blood CD8+ T cells suggests that B-ly-7 detects a lymphocyte activation antigen.