A Novel Integrated Approach for Cytogenomic Evaluation of Plasma Cell Neoplasms.

Plasma cell neoplasm (PCN) is associated with characteristic chromosomal aberrations of diagnostic and prognostic significance. The presence of a small percentage of neoplastic cells is a drawback in the application of karyotyping and fluorescence in situ hybridization for the evaluation of bone marrow aspirate. The analysis of samples enriched for CD138+ cells has improved the detection rate. However, fluorescence in situ hybridization requires several probes and may not be completed due to a limited number of isolated cells. To address the issues experienced with the conventional approach, a novel integrated protocol that consists of whole-genome amplification of DNA isolated from CD138+ cells, followed by microarray as well as one fluorescence in situ hybridization assay for balanced IGH gene rearrangements, has been developed. In the present study in a cohort of 56 patients with clinical suspicion for PCN, compared to conventional cytogenetic analysis, this approach provided higher yield in the detection of PCN-related abnormalities, irrespective of the initial percentage of plasma cells. Whole-genome profiling uncovered recurrent chromosomal abnormalities of prognostic value, including unbalanced alterations within the MYC locus, 16q loss, and hypodiploidy, that were not otherwise detectable by conventional methods. The proposed approach is cost-efficient and provides a superior detection rate, required for proper risk stratification and differential diagnosis of PCN regardless of initial plasma cell percentage.

The success rate of interphase fluorescence in situ hybridization in plasma cell disorders can be improved using unconventional sources of plasma cells.

BACKGROUND: Immunomagnetic cell sorting (IMCS) is a preferred technique for the enrichment of plasma cells (PC) before fluorescence in situ hybridization (FISH). Here, we share our real-world experience regarding the success rate of IMCS, its limitations, and the utility of alternate sources to obtain a successful FISH in various PC disorders. MATERIALS AND METHODS: A retrospective analysis was performed in patients with a PC neoplasm, who underwent bone marrow (BM) examination, and FISH testing over 30 months. In all cases with an unsuccessful IMCS, an attempt was made to identify the cause of failure. RESULTS: Immunomagnetic cell sorting of PCs was successful in 395/450 cases (87.8%; 77/98 cases (78.6%) with <10% PCs and 318/352 (90.3%) with ≥10% PCs in BM aspirate; P = .003). Among cases with unsuccessful IMCS (<10% PCs; n = 21 and ≥10% PCs; n = 34), an alternate source could be used successfully in 34 (62%) patients and includes air-dried trephine biopsy imprint smears (n = 28) with aggregates or sheets of PCs, fine-needle aspiration smears/biopsy from plasmacytoma (n = 5), and ascitic fluid (n = 1). 284/395 (71.9%) patients with successful IMCS and all 34 cases with an alternate source of PCs showed at least one cytogenetic abnormality on four-probe FISH. CONCLUSION: Variations in the sample quality together with significant variation in the number of PCs between BM aspirate and the trephine biopsy imprint smears/biopsy reduce the success rate of IMCS in a real-world scenario and necessitate utilization of patient-specific alternate sources of PCs like a trephine biopsy imprint or cytology smears from extramedullary sources for successful FISH testing in PC neoplasms.

Increased complexity of t(11;14) rearrangements in plasma cell neoplasms compared with mantle cell lymphoma.

Plasma cell neoplasms (PCN) and mantle cell lymphoma (MCL) can both harbor t(11;14)(q13;q32) (CCND1/IGH), usually resulting in cyclin D1 overexpression. In some cases, particularly at low levels of disease, it can be morphologically challenging to distinguish between these entities in the bone marrow (BM) since PCN with t(11;14) are often CD20-positive with lymphoplasmacytic cytology, while MCL can rarely have plasmacytic differentiation. We compared the difference in CCND1/IGH by fluorescence in situ hybridization (FISH) in PCN and MCL to evaluate for possible differentiating characteristics. We identified 326 cases of MCL with t(11;14) and 279 cases of PCN with t(11;14) from either formalin-fixed, paraffin-embedded tissue or fresh BM specimens. The "typical," balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals, three total IGH signals, and two total fusion signals. Any deviation from the "typical" pattern was defined as an "atypical" pattern, which was further stratified into "gain of fusion" vs "complex" patterns. There was a significantly higher proportion of cases that showed an atypical FISH pattern in PCN compared with MCL (53% vs 27%, P < .0001). There was also a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL (47% vs 17%, P < .0001). We confirmed these findings using mate-pair sequencing of 25 PCN and MCL samples. PCN more often have a complex CCND1/IGH FISH pattern compared with MCL, suggesting possible differences in the genomic mechanisms underlying these rearrangements in plasma cells compared with B cells.

miR-15a/16-1 deletion in activated B cells promotes plasma cell and mature B-cell neoplasms.

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

Initial Whole-Genome Sequencing of Plasma Cell Neoplasms in First Responders and Recovery Workers Exposed to the World Trade Center Attack of September 11, 2001.

PURPOSE: The World Trade Center (WTC) attack of September 11, 2001 created an unprecedented environmental exposure to known and suspected carcinogens. High incidence of multiple myeloma and precursor conditions has been reported among first responders to the WTC disaster. To expand on our prior screening studies, and to characterize the genomic impact of the exposure to known and potential carcinogens in the WTC debris, we were motivated to perform whole-genome sequencing (WGS) of WTC first responders and recovery workers who developed a plasma cell disorder after the attack. EXPERIMENTAL DESIGN: We performed WGS of nine CD138-positive bone marrow mononuclear samples from patients who were diagnosed with plasma cell disorders after the WTC disaster. RESULTS: No significant differences were observed in comparing the post-WTC driver and mutational signature landscapes with 110 previously published WGSs from 56 patients with multiple myeloma and the CoMMpass WGS cohort (n = 752). Leveraging constant activity of the single-base substitution mutational signatures 1 and 5 over time, we estimated that tumor-initiating chromosomal gains were windowed to both pre- and post-WTC exposure. CONCLUSIONS: Although limitations in sample size preclude any definitive conclusions, our findings suggest that the observed increased incidence of plasma cell neoplasms in this population is due to complex and heterogeneous effects of the WTC exposure that may have initiated or contributed to progression of malignancy.

Implementation of cytogenomic microarray with plasma cell enrichment enables better abnormality detection and risk stratification in patients with plasma cell neoplasia than conventional cytogenetics and fluorescence in situ hybridization.

The detection of chromosomal abnormalities is important in the diagnosis, prognosis and disease monitoring in plasma cell neoplasia (PCN). However, the gold standard diagnostic techniques of conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) are hampered by culture difficulties and probe availability. Cytogenomic microarray (CMA), however, is able to surmount such limitations and generate a comprehensive genomic profile with the implementation of plasma cell (PC) enrichment. In this study, we examined 89 bone marrow specimens with CC and FISH without PC enrichment, 35 of which were examined with CMA after PC enrichment. Results revealed that after PC enrichment, CMA was able to detect chromosomal abnormalities in 34 of 35 specimens tested (97.1%), compared to 21 and 32 specimens (60% and 91.4%, respectively) achieved by CC and FISH, respectively, which were similar to the abnormality detection rates among all 89 specimens (59.5% by CC and 92.1% by FISH). In addition, as the only technique capable of detecting copy neutral loss of heterozygosity (CN-LOH) and chromothripsis, CMA appears to be the most powerful tool in risk stratification as it successfully re-stratified 9 (25.7%) and 12 (34.3%) specimens from standard risk (determined by CC and FISH, respectively) to high risk. Based on the encouraging data presented by our study and others, we conclude that implementation of CMA with PC enrichment is of great value in routine clinical workup in achieving a more complete genetic profile of patients with PCN.

Development of Anti-CD32b Antibodies with Enhanced Fc Function for the Treatment of B and Plasma Cell Malignancies.

The sole inhibitory Fcγ receptor CD32b (FcγRIIb) is expressed throughout B and plasma cell development and on their malignant counterparts. CD32b expression on malignant B cells is known to provide a mechanism of resistance to rituximab that can be ameliorated with a CD32b-blocking antibody. CD32b, therefore, represents an attractive tumor antigen for targeting with a monoclonal antibody (mAb). To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. Their complementarity-determining regions (CDR) bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcγRIIIa on immune effector cells. The NVS32b mAbs selectively target CD32b+ malignant cells and healthy B cells but not myeloid cells. They mediate potent killing of opsonized CD32b+ cells via antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) as well as complement-dependent cytotoxicity (CDC). In addition, NVS32b CDRs block the CD32b Fc-binding domain, thereby minimizing CD32b-mediated resistance to therapeutic mAbs including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b+ xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor and enhancement of dendritic cell maturation in response to immune complexes. Finally, the activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed using samples from patients with B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. The findings indicate the promising potential of NVS32b mAbs as a single agent or in combination with other mAb therapeutics for patients with CD32b+ malignant cells.

Patients with Plasma Cell Disorders Have High EBV DNA in Peripheral Blood than the General Population.

Epstein-Barr virus (EBV) is involved in the development of a wide range of B cell lympho-proliferative disorders. Its association with plasma cell disorders (PCD) however is not clear, especially in immunocompetent patients. To explore any relationship, 39 patients of suspected PCD with positive M-band on electrophoresis and 50 healthy controls were enrolled. EBV DNA in peripheral blood was quantified using quantitative Real Time Polymerase Chain Reaction (qPCR). Of 39 patients, 15 (38.5%) had EBV DNA compared to 8/50 (16%) controls (p = 0.0008). The mean viral copy number was found to be significantly high in patients compared to controls (1.8 × 105; range = 2.6 × 103-7.6 × 105 copies/ml and 1.7 × 104; range = 7.0 × 102-6.1 × 104 copies/ml respectively; p = 0.003). This is the first study, which characterizes the frequency of EBV in circulation in patients of PCD. The significance of increased prevalence of circulating EBV and a higher viral load in our immunocompetent patients however, needs further evaluation.

IgA plasma cell neoplasms are characterized by poorer long-term survival and increased genomic complexity compared to IgG neoplasms.

The characteristics of the IgA plasma cell neoplasms are not clearly reported in the literature. The main goal of this study is to examine IgA plasma cell neoplasms (PCN) and to compare them to IgG lesions. After at least 5 years from the identification of an M protein, 98 cases were selected, the presentation and clinical evolution of 45 IgA neoplasms were compared to 43 cases of IgG gammopathies. The classification at presentation as monoclonal gammopathy of undermined significance (MGUS)-22 of 45 IgA and 20 of 43 IgG (49 vs 46%), plasma cell myeloma (PCM)-22 of 45 IgA and 22 of 43 IgG (49 vs 51%) and smoldering PCM (SPCM)-1 each (2% for both) was essentially identical. No solitary plasmacytomas were identified. At presentation, IgA patients were younger (66.5 ± 11.3 vs. 69.2 ± 10.7 years), less likely to have bone lesions (12/45 vs 18/43, p < 0.14) or immunoparesis (51% vs. 63%), differences statistically insignificant. Cases with normal fluorescence in-situ hybridization (FISH) results, 27% for IgA vs 61% for IgG (p < 0.037) were statistically different. The IgA patients had worse survival (80 vs 108 months median IgA vs IgG, p < 0.013), difference not detectable in the first 5 years, but substantial after 10. In conclusion, poorer long-term survival and increased genomic complexity by FISH are characteristics of IgA PCNs.

Assessing genome-wide copy number aberrations and copy-neutral loss-of-heterozygosity as best practice: An evidence-based review from the Cancer Genomics Consortium working group for plasma cell disorders.

BACKGROUND: Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal gammopathy of undetermined significance, smoldering myeloma, plasma cell myeloma, and plasma cell leukemia. Molecular subtypes have been defined by recurrent cytogenetic abnormalities and somatic mutations that are prognostic and predictive. Karyotype and fluorescence in situ hybridization (FISH) have historically been used to guide management; however, new technologies and markers raise the need to reassess current testing algorithms. METHODS: We convened a panel of representatives from international clinical laboratories to capture current state-of-the-art testing from published reports and to put forward recommendations for cytogenomic testing of plasma cell neoplasms. We reviewed 65 papers applying FISH, chromosomal microarray (CMA), next-generation sequencing, and gene expression profiling for plasma cell neoplasm diagnosis and prognosis. We also performed a survey of our peers to capture current laboratory practice employed outside our working group. RESULTS: Plasma cell enrichment is widely used prior to FISH testing, most commonly by magnetic bead selection. A variety of strategies for direct, short- and long-term cell culture are employed to ensure clonal representation for karyotyping. Testing of clinically-informative 1p/1q, del(13q) and del(17p) are common using karyotype, FISH and, increasingly, CMA testing. FISH for a variety of clinically-informative balanced IGH rearrangements is prevalent. Literature review found that CMA analysis can detect abnormalities in 85-100% of patients with PCNs; more specifically, in 5-53% (median 14%) of cases otherwise normal by FISH and cytogenetics. CMA results in plasma cell neoplasms are usually complex, with alteration counts ranging from 1 to 74 (median 10-20), primarily affecting loci not covered by FISH testing. Emerging biomarkers include structural alterations of MYC as well as somatic mutations of KRAS, NRAS, BRAF, and TP53. Together, these may be measured in a comprehensive manner by a combination of newer technologies including CMA and next-generation sequencing (NGS). Our survey suggests most laboratories have, or are soon to have, clinical CMA platforms, with a desire to move to NGS assays in the future. CONCLUSION: We present an overview of current practices in plasma cell neoplasm testing as well as an algorithm for integrated FISH and CMA testing to guide treatment of this disease.

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.

Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders.

We have developed an automated assay to enumerate and characterize circulating multiple myeloma cells (CMMC) from peripheral blood of patients with plasma cell disorders. CMMC show expression of genes characteristic of myeloma and fluorescence in situ hybridisation results on CMMC correlated well with bone marrow results. We enumerated CMMC from over 1000 patient samples including separate cohorts of newly diagnosed multiple myeloma and high/intermediate risk smouldering multiple myeloma (SMM) with clinical follow-up data. In newly diagnosed myeloma patient samples, CMMC counts correlated with other clinical measures of disease burden, including the percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (P < 0·001). Patients with CMMC counts ≥100 at remission showed reduced survival relative to patients with CMMC counts <100. Patients with undetectable CMMC in remission showed further overall survival benefits. In the SMM cohort, there was a trend toward higher CMMC in patients with higher-risk myeloma precursor states. Significantly higher CMMC counts were observed between intermediate/high risk SMM patients that progressed versus those without progression (P = 0·031). CMMC allow a non-invasive means of monitoring tumour biology and may have use as a prognostic test for patients with plasma cell disorders.

Detection of complex genomic signatures associated with risk in plasma cell disorders.

Plasma cell disorders (PCD) range from benign to highly malignant disease. The ability to detect risk-stratifying aberrations based on cytogenetic and molecular genetic assays plays an increasing role in therapeutic decision making. In this study, 58 patients were chosen for screening by comparative genomic hybridisation microarray (aCGH) to identify the new high-risk prognostic markers of chromothripsis and chromoanasynthesis. All patients had an unequivocal clinical diagnosis of a plasma cell disorder (plasma cell myeloma (PCM)(n = 51) or monoclonal gammopathy of undetermined significance (MGUS)(n = 7)) and an abnormal FISH result. There were a total of 17 complex genomic events identified across 9 patient samples, which were selected for further investigation by high definition single nucleotide polymorphism (HD-SNP) microarray. Each event was analysed and characterised for chromothripsis, chromoanasynthesis or a complex step-wise chromosomal event. We describe an effective method to identify the new high-risk prognostic markers of chromothripsis and chromoanasynthesis in plasma cell disorders.