Diagnostic Accuracy and Prognostic Relevance of Immunoglobulin Heavy Chain Rearrangement and 18F-FDG-PET/CT Compared With Unilateral Bone Marrow Trephination for Detecting Bone Marrow Involvement in Patients With Diffuse Large B-Cell Lymphoma.

BACKGROUND: In diffuse large B-cell lymphoma (DLBCL), bone marrow involvement (BMI) has an important clinical implication as a component of staging and International Prognostic Index. This study aimed to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and positron emission tomography-computed tomography (PET/CT) could overcome the limitation of defining morphologic BMI by trephination biopsy and could increase the diagnostic accuracy or prognostic prediction. METHODS: A total of 94 de novo patients with DLBCL underwent PET/CT, polymerase chain reaction (PCR) test for detection of IgH gene rearrangement, and unilateral bone marrow (BM) trephination at diagnosis. RESULTS: A total of 9 patients (9.6%) were confirmed to present morphologic BMI (mBMI) based on trephination biopsy. On the other hand, 21 patients (22.3%) were confirmed to have IgH clonality (IgH BMI), while 16 (17.0%) were classified with BMI based on the assessment of PET/CT (PET BMI). Each IgH rearrangement PCR and PET/CT showed the high negative predictive value of detecting the BMI. However, the combined assessment of IgH rearrangement and PET/CT could increase the diagnostic accuracy and specificity with 87.2% and 97.0%, respectively. The survival outcome of patients with double positive PET BMI and IgH BMI was significantly worse than that with either single positive PET BMI or IgH BMI, and even less than patients with neither PET BMI nor IgH BMI (3-year PFS: 50.0% vs. 75.4% vs. 97.9%, P = 0.007, 3-year OS: 50.0% vs. 75.6% vs. 80.1%, P = 0.035, respectively). CONCLUSION: This study suggests that the combined evaluation of PET/CT and IgH rearrangement could give additional information for predicting therapeutic outcomes in patients with negative morphologic BMI as an important part of the prognosis.

Exploiting bone niches: progression of disseminated tumor cells to metastasis.

Many solid cancers metastasize to the bone and bone marrow (BM). This process may occur even before the diagnosis of primary tumors, as evidenced by the discovery of disseminated tumor cells (DTCs) in patients without occult malignancies. The cellular fates and metastatic progression of DTCs are determined by complicated interactions between cancer cells and BM niches. Not surprisingly, these niches also play important roles in normal biology, including homeostasis and turnover of skeletal and hematopoiesis systems. In this Review, we summarize recent findings on functions of BM niches in bone metastasis (BoMet), particularly during the early stage of colonization. In light of the rich knowledge of hematopoiesis and osteogenesis, we highlight how DTCs may progress into overt BoMet by taking advantage of niche cells and their activities in tissue turnover, especially those related to immunomodulation and bone repair.

Alterations in the Transcriptional Programs of Myeloma Cells and the Microenvironment during Extramedullary Progression Affect Proliferation and Immune Evasion.

PURPOSE: In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites. EXPERIMENTAL DESIGN: Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed. RESULTS: Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells. CONCLUSIONS: These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion.

Comparison of Hematological Aspects between Neuroblastoma Patients with and without Bone Marrow Infiltration.

Bone marrow involvement in neuroblastoma indicates an advanced stage of the disease. In this study, we retrospectively analyzed the bone marrow infiltration status and hematological parameters of 111 cases of neuroblastoma. Among the 111 cases, 62 (55.9%) exhibited marrow infiltration. Erythrocytopenia, anemia, and thrombocytopenia were significantly (p<0.05) associated with marrow infiltration of >10% malignant cells. The evaluation of blood cell count parameters with receiver operating characteristic curves indicated that erythrocytopenia can substantially improve the accurate prediction of the risk of bone marrow involvement with a Youden index of 0.60. The sensitivity and specificity were 90% and 70%, respectively, which correspond to a cut-off value of 4.015×10^12/L. Hematological aspects with diagnostic value are different among neuroblastoma patients with and without bone marrow infiltration.

Biochemical Changes in the Niche Following Tumor Cell Invasion.

Metastatic cancer is the leading cause of all cancer related deaths. Prostate cancer (PCa) metastasizes preferentially to the bone marrow, specifically within the endosteal niche. Endosteal cells secrete homing molecules that may recruit PCa cells to the bone marrow. Once there, the biochemical signature of this niche regulates PCa fate including cellular dormancy or cell cycle arrest, reactivation and resistance to chemotherapeutics. Growth factors, interleukins, adhesion molecules, as well as extra-cellular matrix proteins can collectively change the phenotype of PCa cells. Understanding the biochemical signature of endosteal niche parasitism by PCa is imperative for the establishment of new and innovative therapeutic strategies. This review seeks to summarize these important niche signatures and the potential therapeutic approaches to target metastatic PCa within the bone marrow hematopoietic stem cell (HSC) niche. J. Cell. Biochem. 118: 1956-1964, 2017. © 2016 Wiley Periodicals, Inc.

The bone marrow niche in support of breast cancer dormancy.

Despite the success in detecting breast cancer (BC) early and, with aggressive therapeutic intervention, BC remains a clinical problem. The bone marrow (BM) is a favorable metastatic site for breast cancer cells (BCCs). In BM, the survival of BCCs is partly achieved by the supporting microenvironment, including the presence of immune suppressive cells such as mesenchymal stem cells (MSCs). The heterogeneity of BCCs brings up the question of how each subset interacts with the BM microenvironment. The cancer stem cells (CSCs) survive in the BM as cycling quiescence cells and, forming gap junctional intercellular communication (GJIC) with the hematopoietic supporting stromal cells and MSCs. This type of communication has been identified close to the endosteum. Additionally, dormancy can occur by soluble mediators such as cytokines and also by the exchange of exosomes. These latter mechanisms are reviewed in the context of metastasis of BC to the BM for transition as dormant cells. The article also discusses how immune cells such as macrophages and regulatory T-cells facilitate BC dormancy. The challenges of studying BC dormancy in 2-dimensional (2-D) system are also incorporated by proposing 3-D system by engineering methods to recapitulate the BM microenvironment.

Aggressive natural killer-cell neoplasm presenting in the marrow: a report of two cases including one with gains of chromosomes 4q and 9p.

Aggressive nature killer (NK)-cell neoplasm includes aggressive NK-cell leukemia (ANKL) and extranodal NK/T-cell lymphoma (ENKTL), nasal type. ANKL is rare and is characterized by a systemic neoplastic proliferation of NK-cells, usually with a leukemic presentation. ENKTL is a predominantly extranodal lymphoma, occurring mainly in the upper aerodigestive tract. Both are aggressive neoplasms strongly associated with Epstein-Barr virus (EBV). Here we report two patients with aggressive NK-cells neoplasms localized in the bone marrow (BM) who presented as prolonged fever, anemia, and thrombocytopenia. Both were treated initially as infectious disease. Imaging studies revealed splenomegaly without any nodular lesion or lymphadenopathy. BM examination revealed extensive involvement by EBV-positive NK-cells in both cases. Staging workup including nasal examination/biopsy was negative. Both patients passed away in a month. One case showed gains of chromosomes 4q and 9p by array comparative genomic hybridization. Both tumors were diagnostically challenging due to the unusual clinical presentation and absence of leukemic change, tumor mass or lymphadenopathy. Our cases demonstrate that lymphoma should be considered in patients with fever of unknown origin and bone marrow aspiration/biopsy should be performed as early diagnosis and novel therapeutic regimens may benefit these patients.

[Cerebral aspergillosis in an immunocompromised patient with follicular lymphoma].

We present a case of central nervous system aspergillosis in an immunocompromised 69-year-old male with a history of chemotherapeutic treatment for follicular lymphoma. The patient presented with aphasia, apraxia and confusion. An MRI of the central nervous system and Aspergillus antigen in the spinal fluid was suggestive for this invasive fungal infection. Despite treatment with voriconazole the patient succumbed to the infection. A rise in rare, severe infectious complications as presented is expected due to increasing dose-intensity of chemotherapy.

Zoledronate sensitizes neuroblastoma-derived tumor-initiating cells to cytolysis mediated by human γδ T cells.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of Vγ9Vδ2 T cells (γδ T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of γδ T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to γδ T-cell-mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to γδ T-cell-mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of γδ T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of γδ T cells. On the basis of these data, we conclude that ex vivo expanded γδ T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

CD20 antigen expression by lymphoproliferative disorders after kidney transplant is independently associated with a poor outcome: PTLD.Int survey.

OBJECTIVES: Antigen expression by neoplastic cells is important because of its effects on the behavior and survival of patients. We sought to gather data on renal transplant recipients who had developed posttransplant lymphoproliferative disorders in their posttransplant era, and had a documented report on CD20 antigen testing. MATERIALS AND METHODS: A comprehensive search of the literature was done for reports that indicate test results for the CD20 antigen in kidney recipients having lymphoproliferative disorders after transplant. Their demographics, disease characteristics, and prognoses were analyzed. RESULTS: CD20-positive posttransplant lymphoproliferative disorder patients had a significantly shorter time from transplant to developing posttransplant lymphoproliferative disorder (P < .001). None of patients who had early onset posttransplant lymphoproliferative disorder was CD20 negative. Bone marrow involvement was significantly more prevalent among CD20-negative patients (P < .05) with no CD20-positive patient developing a bone marrow metastasis. Log-rank test showed a relatively worse survival for renal recipients expressing the CD20 antigen (P = .07). CONCLUSIONS: CD20-positive posttransplant lymphoproliferative disorder lesions in kidney transplant patients are significantly more likely to develop early after transplant and represent an inferior outcome. We suggest that all renal transplant recipients who develop posttransplant lymphoproliferative disorder within their early time after surgery should be given anti-CD20 therapy. Future prospective studies are required to confirm our conclusions.

Bone marrow and the control of immunity.

Bone marrow is thought to be a primary hematopoietic organ. However, accumulated evidences demonstrate that active function and trafficking of immune cells, including regulatory T cells, conventional T cells, B cells, dendritic cells, natural killer T (NKT) cells, neutrophils, myeloid-derived suppressor cells and mesenchymal stem cells, are observed in the bone marrow. Furthermore, bone marrow is a predetermined metastatic location for multiple human tumors. In this review, we discuss the immune network in the bone marrow. We suggest that bone marrow is an immune regulatory organ capable of fine tuning immunity and may be a potential therapeutic target for immunotherapy and immune vaccination.

Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

Tumor dormancy resulting from subcutaneous injection to SCID mice with cultured nasopharyngeal carcinoma cells is mediated via IFN-γ induction of a highly differentiated phenotype.

The mechanisms underlying tumor dormancy in human primary lesions and bone marrow metastases of nasopharyngeal carcinoma (NPC) are still not completely understood. The aim of this study was to determine differences in the fates of cultured primary NPC (P-NPC) cells, interferon-γ-transduced primary NPC (IFN-γ-P-NPC) cells, bone marrow metastatic NPC (BM-NPC), and IFN-γ-transduced BM-NPC (IFN-γ-BM-NPC) cells following xenotransplantation into these four groups of SCID mice through subcutaneous injection of 5×10(6) cells/site/animal (4 animals/group). The injected mice were monitored for tumor development at the sites of injection. In only the group injected with IFN-γ-P-NPC cells, the resulting nodules remained small throughout the 60-day observation period after injection, but gradually became palpably prickly. Histopathological examination revealed that these lesions invariably consisted of mostly structures of horny pearls and keratin bridges with occasional apoptotic and degenerative cells. In contrast, animals injected with nontransduced-P-NPC cells developed tumors progressively with occasional central necroses. In the two groups injected with IFN-γ-NPC-BM and NPC-BM cells, progressive growths of tumors were noted, with the latter being at slightly faster rates, whereas the xenografts of both groups showed a poorly differentiated phenotype with abundant vascularity. The study results highlight the high susceptibility of P-NPC but not BM-NPC following IFN-γ gene transfer to the induction of tumor dormancy, which is mediated via induced cell differentiation. Thus, induced cell differentiation could provide a new mechanism by which tumor dormancy is induced.

A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

BACKGROUND: Flow cytometry is the sole available technique for quantification of tumor plasma-cells in plasma-cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma-cells, validated in a multicenter manner. METHODS: CD36 was used to exclude red blood cell debris and erythroblasts, CD38 and CD138 to detect plasma-cells, immunoglobulin light chains, CD45, CD56, CD19, and CD117 + CD34 to simultaneously characterize abnormal plasma-cells and quantify bone marrow precursors. This approach was applied in nine centers to 229 cases, including 25 controls. RESULTS: Tumor plasma-cells were detected in 96.8% of cases, all exhibiting an immunoglobulin peak over 1g/L. Calculation of a plasma-cells/precursors (PC/P) ratio allowed quantification of the plasma-cell burden independently from bone marrow hemodilution. The PC/P ratio yielded the best results in terms of sensitivity (81%) and specificity (84%) for differential diagnosis between MGUS and myeloma, when compared with other criteria. Combination of both the PC/P ratio and percentage of abnormal plasma-cells allowed the best differential diagnosis, but these criteria were discordant in 25% cases. Indirect calculation of CD19 negative PC/R ratio gave the best results in terms of sensitivity (87%). CONCLUSION: This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution. This approach may also prove useful for the detection of minimal residual disease.

Successful multifold dose escalation of anti-GD2 monoclonal antibody 3F8 in patients with neuroblastoma: a phase I study.

PURPOSE: Pain can hinder immunotherapy with anti-G(D2) monoclonal antibodies (MoAbs) like 3F8. Heat-modified 3F8 (HM3F8) lacks effector functions and could mask G(D2) or cross-reactive epitopes on nerves, thereby preventing a subsequent dose of unmodified 3F8 from activating pain fibers. We hypothesized that 3F8 dose escalation is possible without increased analgesic requirements in patients pretreated with HM3F8. PATIENTS AND METHODS: Thirty patients with resistant neuroblastoma (NB) received one to two cycles of 3F8 plus granulocyte-macrophage colony-stimulating factor. 3F8 dosing began at 20 mg/m(2)/d and increased by 20 mg/m(2)/d in the absence of dose-limiting toxicity (DLT). Premedication included analgesics, antihistamines, and 5-minute infusions of HM3F8. On the basis of experience with 3F8 10 mg/m(2)/d in prior protocols, the DLT of pain was defined as more than seven doses of opioids administered within 2 hours. Opioid use was compared with a contemporary control group treated with 3F8 20 mg/m(2)/d but no HM3F8. Disease response was assessed. RESULTS: Treatment was administered in the outpatient setting. Dose escalation stopped at 160 mg/m(2)/d because of drug supply limitations; even through this dosage level, analgesic requirements were similar to historical controls, and there were no DLTs. Analgesic requirements at 3F8 dosage levels through 80 mg/m(2)/d were significantly less compared with controls. Anti-NB activity occurred at all dosages. CONCLUSION: Multifold dose escalation of 3F8 is feasible. The findings can be interpreted as compatible with the possibility that HM3F8 can modify toxicity without blunting anti-NB activity. This pain control strategy may help achieve dose escalation with other anti-G(D2) MoAbs.

High-dose busulfan and melphalan as conditioning regimen for autologous peripheral blood progenitor cell transplantation in high-risk neuroblastoma patients.

The aim of this retrospective study was to analyze the outcome and identify risk factors associated with progression-free survival (PFS) in 36 children with high-risk neuroblastoma who underwent autologous peripheral blood progenitor cell (PBPC) transplantation between 1994 and 2010. The conditioning regimen used in all cases consisted of high-dose of busulfan and melphalan. Median age at transplantation was 3 years (range: 0.7-14 years). The median times to neutrophil and platelet engraftment were 11 days (range: 9.16 days) and 13 days (range: 9.33), respectively. Twenty-one patients developed nonhematologic toxicity: 15 patients had mucositis, 4 patients developed an engraftment syndrome, and there were 2 cases of liver toxicity. No toxic deaths were observed. There were 15 patients who relapsed. The median time to relapse was 6 months after the transplant (range: 3-13 months). With a median follow-up of 55 months (range: 4-180 months), the PFS was 57% ± 8.5% for the whole group. In multivariate analysis, age below 3 years (P < .005), complete remission (CR) pretransplantation (P < .07) and 1p germline status (P < .01) were variables associated with better outcomes. Patients who were or achieved early CR following transplantation (3 months posttransplantation) had a probability of PFS of 91% ± 6% as compared to patients who did not (PFS 9% ± 8%) (P < .0001). This retrospective study shows that high dose of busulfan and melphalan as conditioning regimen in children with high-risk neuroblastoma is associated with very low morbidity and no mortality in the authors' hands. Younger patients with no 1p deletions and in first CR at transplantation had the better outcome.

Differential baseline and response profile to IFN-gamma gene transduction of IL-6/IL-6 receptor-alpha secretion discriminate primary tumors versus bone marrow metastases of nasopharyngeal carcinomas in culture.

BACKGROUND: Understanding of immunobiology of bone marrow metastases (designated BM-NPC) versus primary tumors (P-NPC) of the nasopharynx is far from complete. The aim of this study was to determine if there would be differences between cultured P-NPCs and BM-NPCs with respect to (i) constitutive IL-6 and the IL-6 receptor gp80 subunit (IL-6Ralpha) levels in the spent media of nontransduced cells, and (ii) IL-6 and IL-6Ralpha levels in the spent media of cells transduced with a retroviral vector containing the IFN-gamma gene. METHODS: A panel of NPC cell lines were transduced with the IFN-gamma gene through a retroviral vector. Four clonal sublines were isolated via limiting dilution methods. Cytofluorometric analysis was performed for the detection of cell surface antigens of HLA class I, HLA class II and ICAM-1. ELISA was used to assay for IFN-gamma, IL-6 and IL-6Ralpha in the spent media of cultured cell lines. RESULTS: Our results showed that in day 3 culture supernatants, low levels of soluble IL-6 were detected in 5/5 cultured tumors derived from P-NPCs, while much higher constitutive levels of IL-6 were detected in 3/3 metastasis-derived NPC cell lines including one originated from ascites; the difference was significant (p = 0.025). An inverse relationship was found between IL-6Ralpha and IL-6 in their release levels in cultured P-NPCs and metastasis-derived NPCs. In IFN-gamma-transduced-P-NPCs, IL-6 production increased and yet IL-6Ralpha decreased substantially, as compared to nontransduced counterparts. At variance with P-NPC cells, the respective ongoing IL-6 and IL-6Ralpha release patterns of BM-NPC cells were not impeded as much following IFN-gamma transduction. These observations were confirmed by extended kinetic studies with representative NPC cell lines and clonal sublines. The latter observation with the clonal sublines also indicates that selection for high IL-6 or low IL-6Ralpha producing subpopulations did not occur as a result of IFN-gamma-transduction process. P-NPCs, which secreted constitutively only marginal levels of IFN-gamma (8.4 ~ 10.5 pg/ml), could be enhanced to produce higher levels of IFN-gamma (6.8- to 10.3-fold increase) after IFN-gamma transduction. Unlike P-NPCs, BM-NPCs spontaneously released IFN-gamma at moderate levels (83.8 ~ 100.7 pg/ml), which were enhanced by 1.3- to 2.2-fold in the spent media of their IFN-gamma-transduced counterparts. CONCLUSION: Our results showed that cultured P-NPCs and BM-NPCs could be distinguished from one another on the basis of their differential baseline secretion pattern of IFN-gamma, IL-6 and IL-6Ralpha, and their differential response profiles to IFN-gamma gene transfer of the production of these three soluble molecules. These results suggest that the IL-6 and IFN-gamma pathways in a background of genetic instability be involved in the acquisition of metastatic behaviour in BM-NPCs.

Novel immunophenotypic features of marrow lymphoplasmacytic lymphoma and correlation with Waldenström's macroglobulinemia.

Lymphoplasmacytic lymphoma involving the bone marrow can be difficult to diagnose, and pathological features that predict the presence of associated Waldenström's macroglobulinemia have yet to be identified. To address these issues, marrow histology, immunohistochemistry, and flow cytometry were studied from 35 lymphoplasmacytic lymphoma cases that had comprehensive clinical assessment for Waldenström's macroglobulinemia. In all cases, the plasma cells were analyzed by a novel 6-color flow method. Both immunohistochemistry and flow cytometry were useful in identifying the lymphoid and plasmacytic disease components. In 19 cases, immunohistochemistry revealed an earlier unrecognized pattern of plasma cell infiltration in which they were physically separate from the lymphoid infiltrates. B-cell flow cytometry revealed monotypic cells in 96% of the cases. Approximately half of these were CD5 and/or CD23 positive, although none had features of chronic lymphocytic leukemia, and none of the B cells had flow cytometric features suggesting plasmacytic differentiation. In contrast, highly sensitive 6-color plasma cell flow cytometry revealed monotypic cells in 32 of the 35 cases; in 20 cases, the pattern of CD38 and CD138 coexpression detected was identical to that seen in plasma cell malignancies such as multiple myeloma. In 18 of these 20 lymphoplasmacytic lymphoma cases, these plasma cells were CD19 positive, distinguishing them from those of true plasma cell neoplasms, which are CD19 negative. It is interesting that the two lymphoplasmacytic lymphoma cases with CD19-negative plasma cells had an IgG isotype serum paraprotein. Apart from this, no other pathological correlates of the clinical or laboratory features of symptomatic Waldenström's macroglobulinemia were identified.