Mast Cells and Angiogenesis in Human Plasma Cell Malignancies.

Bone marrow angiogenesis plays an important role in the pathogenesis and progression of hematological malignancies. It is well known that tumor microenvironment promotes tumor angiogenesis, proliferation, invasion, and metastasis, and also mediates mechanisms of therapeutic resistance. An increased number of mast cells has been demonstrated in angiogenesis associated with hematological tumors. In this review we focused on the role of mast cells in angiogenesis in human plasma cell malignancies. In this context, mast cells might act as a new target for the adjuvant treatment of these tumors through the selective inhibition of angiogenesis, tissue remodeling and tumor-promoting molecules, permitting the secretion of cytotoxic cytokines and preventing mast cell-mediated immune suppression.

Establishment of Immunoglobulin Heavy (IGH) Chain Clonality Testing by Next-Generation Sequencing for Routine Characterization of B-Cell and Plasma Cell Neoplasms.

Immunoglobulin heavy chain (IGH) clonality testing by next-generation sequencing (NGS) offers unique advantages over current low-throughput methods in the assessment of B-cell lineage neoplasms. Clinical use remains limited because assays are not standardized and validation/implementation guidelines are not yet developed. Herein, we describe our clinical validation and implementation of NGS IGH clonality testing and summarize our experience based on extensive routine use. NGS-based clonality testing targeting IGH FR1, FR2, FR3, and the conserved leader sequence upstream of FR1 was validated using commercially available kits. Data were analyzed by commercial and in-house-developed bioinformatics pipelines. Performance characteristics were evaluated directly comparing with capillary electrophoresis (CE) assays (BIOMED-2 primers). Assays were monitored after implementation (>1.5 years), concurrently testing by CE methods. A total of 1189 clinical samples were studied (94 validation, 1095 postimplementation). NGS showed superior performance compared with CE assays. For initial assessment, clonality detection rate was >97% for all malignancy types. Concordance with CE was 96%; discordances were related to higher sensitivity/resolution of NGS and improved detection in cases with high somatic hypermutation. Routine NGS clonality assessment is feasible and superior to existing assays, enabling accurate and specific index clone assessment and future tracking of all rearrangements in a patient sample. Successful implementation requires new standardization, validation, and implementation processes, which should be performed as a multicenter and multidisciplinary collaboration.

The Kaposi's sarcoma-associated herpesvirus viral interleukin 6 gene affects metastasis and expression of B cell markers in a murine xenograft model.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus in humans, primarily affecting AIDS patients. KSHV causes a range of cancers including Kaposi's sarcoma, pleural effusion lymphoma and multicentric Castleman's disease. Current methods available for treating these cancers are relatively ineffective, and new targets for therapy are needed. The KSHV viral homolog of interleukin-6 gene (vIL-6) may play a significant role in tumor development and may serve as a new anti-cancer target, but its role in tumor formation is only partially understood. Here, a novel animal model was used to study how vIL-6 affects tumor development. Highly immune-deficient Rag2-/-γc-/- mice were transplanted with an immortalized human B cell line (BJAB) harboring either wild-type (WT) KSHV or a mutant strain lacking vIL-6 ΔvIL-6). Solid tumors developed and total tumor mass and the number of tumors were characterized. The vIL-6 gene had no significant impact on tumor mass, but significantly more tumors were detected when vIL-6 was present. Significant differences in expression of B cell markers in cells from extracted tumors were detected based upon the presence of vIL-6. B cell markers in tumor cells were also compared to the same cell type in culture, prior to xenotransplantation; B cell markers were mostly downregulated during tumor formation and these changes did not differ based upon the presence of vIL-6. The only marker that significantly increased in expression during tumor development was CD30. Tumor blood vessels were quantified to determine if more angiogenesis occurred with vIL-6-expressing virus, but there was no significant difference. These data indicate that vIL-6 plays a role in KSHV tumor formation in B cells in vivo. Further investigation into how vIL-6 manipulates CD30 expression may shed insight into KSHV oncogenesis, and may identify how vIL-6 can be targeted.

Tumor suppressor CD99 is downregulated in plasma cell neoplasms lacking CCND1 translocation and distinguishes neoplastic from normal plasma cells and B-cell lymphomas with plasmacytic differentiation from primary plasma cell neoplasms.

CD99(MIC2) is a widely expressed cell surface glycoprotein and functions as a tumor suppressor involved in downregulation of SRC family of tyrosine kinase. CD99 expression is tightly regulated through B-cell development. The principal aims of this study were to investigate the clinical utility of CD99 expression (i) in distinguishing normal plasma cells from primary plasma cell neoplasms; (ii) in detection of minimal residual disease in primary plasma cell neoplasms; and (iii) in distinguishing plasma cell component of B-cell lymphomas from primary plasma cell neoplasms. We analyzed expression of CD99 by flow cytometry and immunohistochemistry in lymph nodes, peripheral blood, and bone marrow samples. CD99 showed stage-specific expression with highest expression seen in precursor B and plasma cells. In contrast to the uniform bright expression on normal plasma cells, CD99 expression on neoplastic plasma cells was lost in 39 out of 56 (69.6%) cases. Furthermore, 8 out of 56 samples (14%) showed visibly (>10-fold) reduced CD99 expression. Overall, CD99 expression was informative (absent or visibly dimmer than normal) in 84% of primary plasma cell neoplasm. In the context of minimal residual disease detection, CD99 showed superior utility in separating normal and abnormal plasma cells over currently established antigens CD117, CD81, and CD27 by principal component analysis. Preservation of CD99 expression was strongly associated with cyclin D1 translocation in myeloma (p < 0.05). B-cell lymphomas with plasma cell component could be distinguished from myeloma by CD99 expression. In summary, we established that tumor suppressor CD99 is markedly downregulated in multiple myeloma. The loss is highly specific for identification of abnormal cells in primary plasma cell neoplasms, and can be exploited for diagnostic purposes. The role of CD99 in myeloma pathogenesis requires further investigation.

8q24/MYC rearrangement is a recurrent cytogenetic abnormality in blastic plasmacytoid dendritic cell neoplasms.

8q24/MYC rearrangements resulting in MYC overexpression occur most frequently in lymphoid neoplasms. MYC rearrangements rarely have been described in blastic plasmacytoid dendritic cell neoplasm (BPDCN). Over an 8-year period in our hospital, 5 of 41 (12%) patients with BPDCN were shown 8q24/MYC rearrangements, including 2 with t(6;8)(p21;q24), 1 with t(8;14)(q24;q32), 1 with t(X;8)(q24;q24), and 1 with t(3;8)(p25;q24). 8q24/MYC rearrangement was present in the stemline in 4 patients and in the sideline in one; the latter was a patient with primary myelofibrosis who then developed BPDCN. MYC overexpression by immunohistochemistry was variable, but largely correlated with the percentage of blasts. Four patients were treated with acute lymphoblastic leukemia-type chemotherapy regimens and 3 had a good response; 1 patient was treated with acute myeloid leukemia-type regimens and was refractory to therapy. By the end of the follow-up, 3 patients died and 2 were alive in complete remission. We conclude that 8q24/MYC rearrangements occur in 10-15% of BPDCN, often partnered with non-immunoglobulin chromosomal loci, and may play a role in BPDCN pathogenesis. In this small patient sample, patients with BPDCN and MYC rearrangement often responded to therapy with acute lymphoblastic leukemia-type chemotherapy regimens.

Immunophenotypic and laboratory features of t(11;14)(q13;q32)-positive plasma cell neoplasms.

The t(11;14)(q13;32)-positive plasma cell neoplasms (PCNs) are common. In light of their lymphoplasmacytoid features and CD20 expression, we speculated that they may share laboratory features with B-cell lymphomas with plasmacytic differentiation (BCL-PCDs). We investigated flow cytometric CD19 and CD45 expression, DNA ploidy index and M-protein subtype in 416 t(11;14)-positive PCNs, as well as control groups (88 BCL-PCDs and 81 t(11;14)-negative PCNs). The plasma cells from the t(11;14)-positive PCNs were largely CD19-/CD45-, similar to the t(11;14)-negative PCNs and unlike the BCL-PCD plasma cells (p < .0001). 79% of the t(11;14)-positive PCNs were diploid, which was significantly more than in t(11;14)-negative PCNs (p < .0001) and significantly less than in the BCL-PCDs (p < .001). Although IgM secretion was common in BCL-PCDs (80%) and rare in PCNs (3%), most IgM PCNs (92%) were t(11;14)-positive. These findings may be helpful in evaluating specimens with clonal plasma cells in the reference laboratory setting.

Neoplastic plasma cells generate an inflammatory environment within bone marrow and markedly alter the distribution of T cells between lymphoid compartments.

Monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) are characterised by the accumulation of malignant plasma cells within bone marrow and lead to a range of abnormalities in the peripheral blood T cell repertoire. We investigated the level of inflammatory chemokines within the bone marrow and blood of patients with MGUS and MM and related this to the pattern of chemokine receptor expression on T cells in both compartments.The expression of a wide range of chemokine ligands for CXCR3 and CCR4 was markedly increased within the bone marrow of patients with MGUS and MM compared to healthy donors. The most marked effects were seen for CCL4 and CXCL9 which were increased by 4 and 6 fold respectively in the bone marrow of patients with myeloma. The expression of CXCR3 and CCR4, the major TH1 and TH2-associated chemokine receptors, was increased substantially on T cells within the bone marrow of patients whereas the percentage of CXCR3-expressing T cells within blood was correspondingly decreased. The presence of even small numbers of neoplastic plasma cells or associated stroma can therefore generate an inflammatory chemokine tumour microenvironment. This leads to the selective recruitment or retention of specific T cell subsets which is likely to underlie many of the features regarding the peripheral T cell repertoire in myeloma and may also contribute to the immune suppression associated with this disease. This local inflammatory reaction may represent a tumour-specific immune response or may itself play an important role in tumour progression and as such may offers a potential novel target for therapeutic intervention.

Autophagy in Plasma Cell Ontogeny and Malignancy.

Autophagy is a highly conserved pathway that recycles cytosolic material and organelles via lysosomal degradation. Once simplistically viewed as a non-selective survival strategy in dire straits, autophagy has emerged as a tightly regulated process ensuring organelle function, proteome plasticity, cell differentiation and tissue homeostasis, with key roles in physiology and disease. Selective target recognition, mediated by specific adapter proteins, enables autophagy to orchestrate highly specialized functions in innate and adaptive immunity. Among them, the shaping of plasma cells for sustainable antibody production through a negative control on their differentiation program. Moreover, memory B cells and long-lived plasma cells require autophagy to exist. Further, the plasma cell malignancy, multiple myeloma deploys abundant autophagy, essential for homeostasis, survival and drug resistance.

Accuracy of Bone Marrow Flow Cytometry Analysis in Patients With Plasma Cell Neoplasm in Thailand: A Single Institutional Study.

BACKGROUND: Plasma cell neoplasm is a common hematologic malignancy. Treatment with novel agents results in favorable outcomes. Reliable investigations are required to monitor the residual disease, especially after such effective treatments. Flow cytometric analysis is a speedy and accurate method to detect abnormal cells. The aim of this study was to determine diagnostic performance of flow cytometry in the detection of abnormal plasma cells in bone marrow specimens. MATERIALS AND METHODS: We included bone marrow samples taken from patients suspected to harbor plasma cell neoplasm at the time of diagnosis or follow-up after treatment from 2013 to 2015. Flow cytometric analyses, using cluster of differentiation (CD)19/CD20/CD27/CD38/CD45/CD56/CD117/CD138 and cytoplasmic κ/λ, were done and results compared with morphologic evaluation of marrow aspirate smear, histology, and immunohistochemistry of marrow biopsy and protein electrophoretic analyses. RESULTS: A total of 154 specimens were included. Plasma cell neoplasm was detected in 56 samples (36.4%). Most abnormal plasma cells in this study were CD19-negative (CD19(-))/CD20(-)/CD27(+)/CD38(+)/CD45(-) (or weakly positive)/CD56(+)/CD117(+)/CD138(+). Light chain restriction was found only in 18 samples (32.1%). Sensitivity and specificity of flow cytometric analysis were 91.1% and 96.9%, respectively. For the follow-up cohort, sensitivity and specificity were 86.7% and 66.7%, respectively. CONCLUSION: Analysis of plasma cell neoplasm using flow cytometry has high sensitivity and specificity. As an adjunct to marrow histology and immunohistochemistry, flow cytometry can be used in diagnosis of plasma cell neoplasm and more importantly in monitoring the disease after treatment. We propose a limited panel of CD19/CD38/CD45/CD56/CD117/CD138 for detecting minimal residual disease in Thai patients.

Clonality of neutrophilia associated with plasma cell neoplasms: report of a SETBP1 mutation and analysis of a single institution series.

A rare but well-known association between plasma cell neoplasms and neutrophilia is known to exist. Whether the neutrophilia is secondary to the plasma cell neoplasm or this convergence represents two independent clonal disorders is unclear. We reviewed five consecutive cases from a single institution over a 3-year period, applying molecular, cytogenetic and cytokine-profiling approaches to determine whether neutrophilia associated with plasma cell neoplasms represents a reactive or clonal process. We report, for the first time, the occurrence of a SETBP1 mutation in two cases, as well as changes in G-CSF and IL-6 in SETBP1 wild type vs. mutated patients that are supportive of a hypothesis that neutrophilia associated with plasma cell neoplasms may sometimes be reactive and may sometimes represent a second clonal entity. Finally, using an ex vivo drug screening platform we report the potential efficacy of the multi-kinase inhibitor dasatinib in select patients.

POEMS syndrome: update on diagnosis, risk-stratification, and management.

DISEASE OVERVIEW: POEMS syndrome is a paraneoplastic syndrome due to an underlying plasma cell neoplasm. The major criteria for the syndrome are polyradiculoneuropathy, clonal plasma cell disorder (PCD), sclerotic bone lesions, elevated vascular endothelial growth factor, and the presence of Castleman disease. Minor features include organomegaly, endocrinopathy, characteristic skin changes, papilledema, extravascular volume overload, and thrombocytosis. Diagnoses are often delayed because the syndrome is rare and can be mistaken for other neurologic disorders, most commonly chronic inflammatory demyelinating polyradiculoneuropathy. POEMS syndrome should be distinguished from the Castleman disease variant of POEMS syndrome, which has no clonal PCD and typically little to no peripheral neuropathy but has several of the minor diagnostic criteria for POEMS syndrome. DIAGNOSIS: The diagnosis of POEMS syndrome is made with three of the major criteria, two of which must include polyradiculoneuropathy and clonal plasma cell disorder, and at least one of the minor criteria. RISK STRATIFICATION: Because the pathogenesis of the syndrome is not well understood, risk stratification is limited to clinical phenotype rather than specific molecular markers. The number of clinical criteria is not prognostic, but the extent of the plasma cell disorder is. Those patients with an iliac crest bone marrow biopsy that does not reveal a plasma cell clone are candidates for local radiation therapy; those with a more extensive or disseminated clone will be candidates for systemic therapy. RISK-ADAPTED THERAPY: For those patients with a dominant sclerotic plasmacytoma, first line therapy is irradiation. Patients with diffuse sclerotic lesions or disseminated bone marrow involvement and for those who have progression of their disease 3-6 months after completing radiation therapy should receive systemic therapy. Corticosteroids are temporizing, but alkylators are the mainstay of treatment, either in the form of low dose conventional therapy or high dose with stem cell transplantation. Lenalidomide shows promise with manageable toxicity. Thalidomide and bortezomib also have activity, but their benefit needs to be weighed against their risk of exacerbating the peripheral neuropathy. The benefit of anti-VEGF antibodies is conflicting. Prompt recognition and institution of both supportive care measures and therapy directed against the plasma cell result in the best outcomes.

Practical diagnostic approaches to composite plasma cell neoplasm and low grade B-cell lymphoma/clonal infiltrates in the bone marrow.

Composite plasma cell neoplasm (PCN) and low grade B-cell lymphoma (B-NHL) in the bone marrow are uncommon and raise the differential diagnosis of B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. This can be a challenging differential diagnosis, and the distinctions are important because of differences in management. We report five cases of composite PCN with B-NHL or clonal B-cell infiltrates involving the bone marrow. By using multiple different diagnostic modalities, including immunophenotyping by flow cytometry and immunohistochemistry, cytogenetic analysis and IGH gene rearrangement studies by polymerase chain reaction, we were able to distinguish two distinct clonally unrelated neoplasms in all cases. We describe the utility and pitfalls of these different diagnostic modalities. Flow cytometric analysis with a panel of antibodies that includes CD19, CD56, CD138, CD45 and other aberrant markers commonly expressed by PCN will allow identification of clonally unrelated PCN and B-NHL in a composite neoplasm, and distinguish them from B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. Cytogenetic and molecular analyses can give false-negative or false-positive results. In summary, a multimodal approach utilizing these different tools, including clinical data, should be used to arrive at the correct diagnosis.

Plasma cell neoplasms presenting with masses: a study on morphology, expression of CD56 and cyclin D1, and presence of Epstein-Barr virus in 39 Thai patients in SiriraJ Hospital.

BACKGROUND: Plasma cell neoplasms (PCNs) presenting with masses are not common. Variable morphology, expression of CD56 and cyclin D1, and Epstein-Barr virus (EBV)-encoded small RNA (EBER) status have been described with a promising diagnostic role. There is no data of these findings in Thai patients. OBJECTIVE: To study morphology, CD56 and cyclin D1 expression and EBER status in PCNs presenting with masses. MATERIAL AND METHOD: Thirty-nine mass-forming PCNs with available materials between 2006 and 2010 were identified from Siriraj Hospital pathology database. H&E slides were reviewed for morphologic grade according to Bartl grading system. Immunohistochemistryfor CD56 and cyclin D1 and EBER in situ hybridization were analyzed on tissue microarray sections of the included cases. RESULTS: Of 39 cases, it comprised 31 (79.5%) plasma cell myelomas (PCMs), five (12.8%) osseous plasmacytomas (OPs), and three (7.7%) extramedullary plasmacytomas (EMPs). Intermediate-grade morphology was common to all types of PCNs. CD56 and cyclin D1 positivity were more often in PCMs comparing with OPs and EMPs; however differences in expression of these markers among different types of PCNs were insignificant (p > 0.05). An EBER-positive EMP was identified. CONCLUSION: The majority of mass-forming plasma cell tumors in the studied population are PCM-related. Intermediate-grade morphology is common in all types of PCNs. A value of CD56 and cyclin D1 immunostains in discrimination between types of PCNs cannot be confirmed in the current study. Identification of the EBER-positive EMP suggests that EBV association in plasma cell tumor can be encountered in Thais.

ASK1 promotes apoptosis of normal and malignant plasma cells.

Although the overproduction of immunoglobulins by short-lived plasma cells accompanying an immune response links with their apoptosis, how long-lived plasma cells adapt to ensure their longevity in this context is obscure. Here, we show that apoptosis signal-regulating kinase 1 (ASK1) contributes to apoptosis of plasma cells because ASK1 activity was induced during differentiation of short-lived plasma cells, and, when produced by ASK1-deficient mice, these cells survived better than those of control mice. Moreover, antigen-specific long-lived plasma cells generated by immunization accumulated in ASK1-deficient mice, suggesting ASK1 also plays a negative role in survival of long-lived plasma cells. In malignant plasma cells, ASK1 transcription was directly suppressed by B lymphocyte-induced maturation protein-1 (Blimp-1). The expression of ASK1 and Blimp-1 showed an inverse correlation between normal human mature B cells and bone marrow plasma cells from patients with multiple myeloma (MM). Suppression of ASK1 is crucial for cell survival because its enforced expression in MM cells caused apoptosis in vitro and lowered MM load in a xenograft animal model; furthermore, alteration of ASK1 activity affected MM cell survival. Our findings indicate a novel mechanism underlying the regulation of survival in normal and malignant plasma cells by ASK1.

Risk stratification of plasma cell neoplasm: insights from plasma cell-specific cytoplasmic immunoglobulin fluorescence in situ hybridization (cIg FISH) vs. conventional FISH.

UNLABELLED: We directly compared the results of routine fluorescence in situ hybridization (FISH) and plasma cell-specific cytoplasmic immunoglobulin (cIg) FISH from 75 paired samples for myeloma risk stratification. CIg FISH improves test specificity and sensitivity and tends to eliminate borderline results. It proves that most plasma cells (PCs) consistently carry the abnormality in myelomas with an IGH translocation, whereas routine FISH detects these cells only at variably low levels. BACKGROUND: Routine cytogenetic analysis of plasma cell neoplasms (PCNs) has a low sensitivity. Conventional fluorescence in situ hybridization (FISH) is not plasma cell (PC) specific and results are diluted by other cells in the sample. Although PC-specific FISH testing has been recommended for multiple myeloma (MM) risk stratification, eg, by combining cytoplasmic immunoglobulin (cIg) staining with FISH, the benefits of cIg FISH have never been directly demonstrated in a controlled study. PATIENTS AND METHODS: Seventy-five samples from patients with PCNs were analyzed by concomitant conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13, 17p-, and +3. The results were compared for their reliability, specificity, and consistency. RESULTS: Apart from marginally improving detection threshold in samples with low PC burden, cIg FISH identified more abnormal cases (50 vs. 47 cases) and more chromosome abnormalities (113 vs. 103 events) than did conventional FISH. It differentiated del(13q) in myelodysplasia from MM. Remarkably, cIg FISH consistently identified a high percentage of abnormal PCs in all cases. It detected IGH translocation in 78% to 100% of PCs in all but 2 positive cases, whereas conventional FISH detected 0% to 46% in these cases (median, 91% vs. 9%). The abnormal cells found in patients with 17p- were 19% to 96% by cIg FISH vs. 0% to 13% by conventional FISH (median, 54% vs. 9%). Cases with insufficient PCs for cIg FISH had only normal conventional FISH results. CONCLUSION: CIg FISH improves reliability of FISH testing for PCNs by eliminating borderline results. In myelomas with an IGH translocation, myeloma cells invariably carry the abnormality.