Multicolor Flow Cytometry Detects Monoclonal Plasma Cells In the Vast Majority of Marrows From Patients with Plasma Cell Disorders Shown to Have Genetic Abnormalities by Fluorescence In Situ Hybridization (FISH)

Abstract Abstract 1896 Flow cytometric analysis (FCM) is now considered an essential tool for the identification of malignant hemopoietic and lymphoid cells, and it is particularly useful in evaluating plasma cell clonality to differentiate normal from neoplastic plasma cells (PC). Neoplastic PC typically show loss of CD19 and aberrant expression of markers such as CD56 and CD117, but the demonstration of an expansion of PC containing a single immunoglobulin light chain (monotypic or clonal expansion) is critical for determining the nature of a plasma cell population. Genetic abnormalities are becoming essential markers for prognosis of plasma cell disorders (PCD). Because of the relatively low proliferation or low representation of PC in the sample, FISH rather than conventional cytogenetics is used routinely or the detection of karyotypic abnormalities in PCD and the use of FISH in plasma cell-enriched preparations has improved the sensitivity of detection of genetic abnormalities in these disorders. We wished to determine how often FCM would detect monoclonal cells in marrows with genetic abnormalities observed by FISH. For this purpose, we analyzed marrow samples that were either non-enriched or enriched for PC using immunomagnetic beads for cell separation. From February 2009 until present, we studied marrows sent to our laboratory for diagnostic purposes from 158 patients who either had a clinical or laboratory abnormality suggestive of or consistent with PCD, or their marrows demonstrated immunophenotypic abnormalities of PC. The FCM consisted of a simultaneous determination of surface CD45, CD19, CD38, CD56 and intracytoplasmic kappa and lambda immunoglobulin (Ig) light chains in a single tube, using a Canto II cytometer (BD). Between 105 and 3×105 cells were analyzed in each case. Clonal expansions were detected based on the Ig light chain expression in cells selected as normal or abnormal plasma cells by their surface phenotype. FISH analysis was performed using Abbott probes for CEP 7, CEP 9, CCND1/IgH, RB1, and p53 on both enriched and non-enriched interphase cells. FISH samples were processed according to standard procedures and 200 cells were analyzed for each probe. Plasma cell enrichment was performed by incubating marrow cells with CD138-coated immunomagnetic beads, and isolating them on Miltenyi separation columns using an auto MACS Pro Separator. Of the 158 samples studied, 52 underwent plasma cell enrichment. Enrichment was performed on samples with less than 8% PC as assessed by FCM. The fraction of PC in enriched samples was approximately 10 times greater than those in non-enriched samples. FISH abnormalities were detected in enriched samples from cases that contained as low as 0.04% PC prior to enrichment. The majority of the enriched samples that showed FISH abnormalities exhibited multiple FISH alterations, the most common being additional copies of CEP 7, CEP 9 and CCND1 DNA sequences, and deletion of RB1 and p53. Of the 158 samples, 46 (29%) were found to be polyclonal by FCM and normal by FISH. These included 8 samples enriched for PC. Of the 69 non-enriched samples only 33 (48%) demonstrated FISH abnormalities. Of these abnormal samples, 32 (97%) were monoclonal by FCM. Among the 43 samples that underwent plasma cell enrichment, 37 (86%) demonstrated FISH abnormalities. Of these enriched abnormal samples, 34 (92%) were shown to be monoclonal by FCM. Our results showed that, confirming previous observations, targeted FISH analysis using plasma cell enrichment increases the detection frequency of abnormalities in PCD, which can be found in the majority of cases. This study also showed that FCM detected monoclonality in the vast majority of samples of PCD that demonstrated FISH abnormalities, whether or not they were enriched for PC, validating the clinical utility of FCM in the routine diagnostic evaluation of patients with PCD. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of the cytogenetic aberration pattern in amyloid light chain amyloidosis as compared with monoclonal gammopathy of undetermined significance reveals common pathways of karyotypic instability

Blood ◽

10.1182/blood-2007-11-122101 ◽

2008 ◽

Vol 111 (9) ◽

pp. 4700-4705 ◽

Cited By ~ 65

Author(s):

Tilmann Bochtler ◽

Ute Hegenbart ◽

Friedrich W. Cremer ◽

Christiane Heiss ◽

Axel Benner ◽

...

Keyword(s):

Plasma Cell ◽

Light Chain ◽

Monoclonal Gammopathy ◽

Common Mechanism ◽

Fish Analysis ◽

Tree Model ◽

Light Chain Amyloidosis ◽

Plasma Cell Disorders ◽

Karyotypic Instability ◽

Amyloid Light Chain

AbstractChromosomal aberrations (CAs) have emerged as important pathogenetic and prognostic factors in plasma cell disorders. Using interphase fluorescence in situ hybridization (FISH) analysis, we evaluated CAs in a series of 75 patients with amyloid light chain amyloidosis (AL) as compared with 127 patients with monoclonal gammopathy of unknown significance (MGUS). We investigated IgH translocations t(11;14), t(4;14), and t(14;16) as well as gains of 1q21, 11q23, and 19q13 and deletions of 8p21, 13q14, and 17p13, detecting at least one CA in 89% of the patients. Translocation t(11;14) was the most frequent aberration in AL, with 47% versus 26% in MGUS (P = .03), and was strongly associated with the lack of an intact immunoglobulin (P < .001), thus contributing to the frequent light chain subtype in AL. Other frequent aberrations in AL included deletion of 13q14 and gain of 1q21, which were shared by MGUS at comparable frequencies. The progression to multiple myeloma (MM) stage I was paralleled by an increased frequency of gain of 1q21 (P = .001) in both groups. Similar branching patterns were observed in an oncogenetic tree model, indicating a common mechanism of underlying karyotypic instability in these plasma cell disorders.

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Comparison of Immunophenotypic and Genotypic Profiles of Neoplastic Plasma Cells by Use of Flow Cytometric Immunophenotyping and FISH Analysis Following Plasma Cell Enrichment,

Blood ◽

10.1182/blood.v118.21.3925.3925 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3925-3925

Author(s):

Xiaorong Zhao ◽

Ronald Thomason ◽

Xiao-Xiang Zhang

Keyword(s):

Plasma Cell ◽

Plasma Cells ◽

Flow Cytometric Analysis ◽

Fish Analysis ◽

Flow Cytometric Immunophenotyping ◽

Flow Cytometric ◽

Genetic Abnormalities ◽

Cd20 Expression ◽

Cd56 Expression ◽

Igh Rearrangement

Abstract Abstract 3925 According to The European Myeloma Network(1), FISH analysis of plasma cell myeloma (multiple myeloma, or MM) should be performed on samples enriched for plasma cells or an assay that allows for identification and study of plasma cells apart from other cells should be utilized. We enriched 400 bone marrow samples sent to our laboratory from May-September 2010 using CD138 antibody microbeads for plasma cells and then performed FISH utilizing probes for CEP3, CEP7, CEP9, CEP11, IgH breakapart, IgH/CCND1 (11;14), IgH/MAF (14;16), IgH/FGFR3(4;14), Rb1/13q, and TP53/CEP17. The genotypic profiles were then compared to the immunophenotypic profiles of monoclonal plasma cells derived from flow cytometric immunophenotyping for the following antibodies: CD38, CD19, CD56, CD117, CD20, HLA DR, CD45 and CD10. We identified genetic abnormalities in 90.6% (184/203) of the samples with monoclonal plasma cell percentages of 0.1% or more as detected by concurrent flow cytometric analysis. The most common identified genetic abnormality was aneuploidy {55.5% (222/400)}, either as a sole finding or in combination with translocations or gene loss. The aneuploid samples were further found to be hyperdipoid {23.75% (95/400)}, hypodiploid {15.75% (63/400)}, or polypoid {16.00% (64/400)}. IgH rearrangement was the seond most common abnormality and was seen in 30.2% of the 400 cases. The IgH rearrangements and frequencies were as follows: 11.25% (45/400) with t(11;14), 5.5% (22/400) with t(4;14), 3.0% (12/400) with 14;16, and 10.5% (42/100) with IgH translocation with partners unknown. Deletion of chromosome 13q/monosomy 13 was observed in 26% (104/400) of cases. This was an isolated finding in 3/400 (0.75%), observed with aneuploidy in 71/400 (17.75%), and seen in combination with IgH rearrangement in 64/400 (16.0%). There was a strong association between del (13q14) and IgH rearrangement, especially with t(4;14) and t(14;16). Loss of TP53 was observed in 3.8% (15/400) of cases. This was an isolated finding in 0/400 (0%), observed with aneuploidy in 14/400 (3.5%), and seen in combination with IgH rearrangement in 8/400 (2%). We explored the relationship between identified genetic abnormalities and respective immunophenotypic findings for the cases. Our results showed that t(4;14) was significantly associated with expression of CD56 (17/17 positive, 100%) and the absence of CD117 (76.5% negative, 13/17) and CD20 (82.4% negative, 14/17). On the other hand, t(14;16) was only rarely seen in combination with CD56 expression (88.9% negative, 8/9) or CD20 expression (100% negative, 9/9), but was highly associated with CD117 expression (66.7% positive, 6/9). t(11;14) was associated with CD20 expression (21.9% positive, 7/32), CD56 expression (34.4% positive, 11/32), and CD117 expression (37.5% positive, 12/32). Additionally, our results found that both CD56 and CD117 expression correlate with aneuploidy. Disclosures: No relevant conflicts of interest to declare.

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The Molecular Cytogenetic Analysis of MGUS Provides Additional Evidence for a Novel, 14q-Translocation Independent Pathogenetic Pathway in Clonal Plasma Cell Disorders.

Blood ◽

10.1182/blood.v104.11.3648.3648 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3648-3648

Author(s):

Peter Liebisch ◽

Meike Kirschenlohr ◽

Christiane Wendl ◽

Hartmut Dohner

Keyword(s):

Plasma Cell ◽

Light Chain ◽

Chromosomal Aberrations ◽

Plasma Cells ◽

Diagnostic Procedures ◽

Dna Probe ◽

Genetic Changes ◽

Plasma Cell Disorders ◽

Probe Set ◽

Genetic Events

Abstract Introduction: There is growing evidence for two almost mutually exclusive pathways in the pathogenesis of multiple myeloma (MM): the first one is characterized by the activation of oncogenes through one of five "primary" IgH translocations, while the second one seems to be designated by the presence of various chromosomal extra copies ("hyperdiploidy") in the absence of a primary 14q-translocation. However, the pivotal chromosomal gains in hyperdiploid tumors and the crucial genetic events in the transition of MGUS to overt MM are unknown. Aims: i) to evaluate the incidences of chromosomal aberrations using a myeloma-specific DNA probe in pts. with MGUS ii) to compare the FISH data from the present MGUS series with our MM FISH data previously determined on a large series of tumors with the goal to distinguish primary aberrations (already present in MGUS and therefore potentially involved in the process of immortalization) and secondary genetic changes (not or infrequently detectable in MGUS but common in MM, therefore potentially participating in the process of MGUS-MM transition). Material and Methods: Bone marrow aspirates from 50 pts. diagnosed with MGUS were obtained during routine diagnostic procedures. In the majority of cases, a positive selection of plasma cells using immunomagnetic beads (CD 138) was performed. FISH was combined with immunocytology using light-chain fluorescence antibodies for precise detection of plasma cells carrying the respective clonal light chain (kappa or lambda). At least 50 to 100 plasma cells were evaluated for each probe. The DNA probe set comprised probes mapping to chromosome bands 1p22, 1q21, 6q21, 8p11, 9q34, 11q25, 13q14, 14q32, 17p13, and 22q11. Results: The most frequent chromosomal aberrations in our MGUS series were (in order of decreasing prevalence; + denotes gains, mostly trisomies, - denotes losses, mostly deletions): t(14q32)/46%, +9q/35%, +11q/27%, +1q/25%, and 13q-/19%. The incidences of all other aberrations were lower than 8%. Comparing the incidences of aberrations in MM and MGUS, the greatest difference was found for 13q- (43% vs. 19%), while it was <15% for all other abnormalities. Of note, 22q- was present in 13% of cases with MM, while no pt. with MGUS exhibited this abnormality. Conclusions: i) The high incidence of +9q and +11q in MGUS suggests these chromosomal extra copies as early genetic events and provides further evidence for a novel, 14q-translocation independent pathogenetic pathway in clonal plasma cell disorders. ii) Our data indicate that 13q- and 22q- could be relevant for the transition of MGUS to MM. iii) +1q, previously proposed as a marker of advanced MM, is already detectable in 25% of pts. with MGUS.

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Minimal Residual Plasma Cell Content of Autulogous Grafts, Chemosensitivity to Induction or High Dose Therapy and Pre/Post Transplant PET Negativity Predict Long Term Disease Free Survival in Multiple Myeloma.

Blood ◽

10.1182/blood.v120.21.3024.3024 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3024-3024

Author(s):

Sule Mine Bakanay ◽

Klara Dalva ◽

Berna Elif Koksoy ◽

Erol Ayyildiz ◽

Muhit Ozcan ◽

...

Keyword(s):

Multiple Myeloma ◽

Multivariate Analysis ◽

Plasma Cell ◽

Light Chain ◽

Pet Imaging ◽

Plasma Cells ◽

Whole Body ◽

High Dose ◽

Aberrant Expression ◽

Number Of Patients

Abstract Abstract 3024 Introduction: High dose melphalan supported by autologous hematopoietic cell transplantation has been shown to prolong survival and decrease relapse rates compared to conventional chemotherapies in elligible patients with multiple myeloma (MM) but unfortunately relapses remain a problem. Various factors have been shown to affect the relapse after transplantation. Malignant plasma cells (PCs) have been documented to contaminate blood stem cell harvest products. Conflicting results have been reported regarding the levels of PC contamination in apheresis products from patients with MM undergoing transplantation (Tx) and their impact on relapse and survival. Aim of this study was to analyze post induction residual myeloma by flow cytometric detection of the graft and by whole body PET imaging. Method: Standard panel was set up with CD138FITC/CD38PE/CD45ECD/CD56PC5. CD45negCD56pos plasma cells were identified as abnormal plasma cells. If any aberrant expression [such as CD20(loss) CD27(loss) CD28 (gain), CD33(loss), CD34 (gain), CD81(loss), CD117(gain)] is detected at diagnosis, the corresponding antibody was also added to the panel. Univariate and multivariate analysis were performed by SPSS.16. Results: Apheresis products from 118 patients (female/male=53/65; median age at diagnosis= 55 (35–69)) were tested for the presence of abnormal PCs and the number of normal PCs. The number of patients in MM subtypes IgG/IgA/Light chain/non-secretory were 70/20/26/2. International scoring system I/II/III:50/38/30. Most patients (n=108) had received one or two lines of induction and 52 had bortezomib based therapies before transplantation. In univariate analysis age (> 55; ≤ 55), ISS (ISS1 vs ISS2 + ISS3), beta2 mcg (>3.5 and ≤ 3.5) and MM Subtype (light chain vs others) were significantly associated with response to Tx. Presence of abnormal PCs in the harvests did not significantly affect response to Tx or OS. However, both the presence of abnormal PCs and the amount of normal PCs > 7.6 ×105/kg was associated with shorter PFS. Fifty-five of the patients had pre and post-Tx PET-imaging done. On the other hand, post-Tx PET positivity negatively affected the PFS. Post-Tx PET positivity was also significantly associated with relapse at 12 months after transplantation. Pre and post-Tx disease status significantly influenced relapse after transplantation. In multivariate analysis, age, beta2 mcg and light chain myeloma continued to be significantly associated with response to transplantation. Abnormal PC contamination and the amount of normal PCs> 7.6 ×105/kg were not associated with pre-Tx PET positivity. Conclusions: Age, ISS, beta2 mcg, light chain disease are predictors of response to transplant. Response to the treatments before mobilization, response to Tx, post Tx PET positivity, graft plasma cell (abnormal or normal) content, serum LDH are predictors of PFS. Response (≥VGPR) during mobilization correlates with less abnormal plasma cells in the apheresis product. PC contamination of the graft does not prevent response to transplant. However, presence of abnormal PC in the apheresis product as well as PET positivity predicts shorter DFS compared to lack of residual disease by flow or PET imaging (11 vs 18 months). Disclosures: No relevant conflicts of interest to declare.

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Light Chain Shifting: Identification of a Human Plasma Cell Line Actively Undergoing Light Chain Replacement

Blood ◽

10.1182/blood.v93.1.198 ◽

1999 ◽

Vol 93 (1) ◽

pp. 198-207 ◽

Cited By ~ 5

Author(s):

Hirofumi Tachibana ◽

Hirotaka Haruta ◽

Koji Yamada

Keyword(s):

Cell Line ◽

Human Plasma ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

High Rate ◽

Cell Phenotype ◽

Recombination Activating Genes ◽

Λ Light Chain ◽

Rag 1

Abstract We identified an antibody-secreting human B-cell line (HTD8), which actively replaces the production of the original λ light chain with a new λ chain (light chain shifting) at a high rate. Loss of the original rearranged λ light chain occurs by significantly reducing the amount of transcript expressed. Expression of the new λ chain, which replaces the original λ chain, occurs by rearranging new VJ segments on a previously excluded allele. V λ gene usage of these new rearrangements are biased toward Vλ4, Vλ6, and Vλ10 families, which are known to be the least frequently used. In striking contrast to the plasma cell phenotype, recombination activating genes, RAG-1 and RAG-2, were expressed in the HTD8 cells and were shown to be necessary, but insufficient for inducing expression of the new λ chain. These results suggest that human plasma cells have the potential to actively undergo light chain replacement.

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Molecular Profiling of Extramedullary and Medullary Plasmacytomas.

Blood ◽

10.1182/blood.v114.22.1806.1806 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1806-1806 ◽

Cited By ~ 1

Author(s):

Anuj Mahindra ◽

Samir B Amin ◽

Gabriela Motyckova ◽

Aliyah R. Sohani ◽

Kishan Patel ◽

...

Keyword(s):

Gene Expression ◽

Plasma Cell ◽

Research Funding ◽

Plasma Cells ◽

Expression Patterns ◽

Tissue Microarrays ◽

Molecular Characteristics ◽

Fish Analysis ◽

New Therapeutic Approaches ◽

Risk Of Progression

Abstract Abstract 1806 Poster Board I-832 Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH. We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7-87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97-144 months) and for patients with MP was 102 months (95% CI 93-128 months) { p=0.025} MicroRNA (miRNAs) profiling was performed on MP (n=19) and MM samples (n=66). Data was normalized using U6 endogenous control. Three hundred and one miRNAs out of a total 665 were significantly differentially expressed between MP vs MM samples. Gene expression profiling performed on MP will be correlated with the miRNA data to identify genes and transcripts of interest which will be functionally validated. Tissue microarrays were performed on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Of samples analyzed, CD56 positivity was observed in 55% MP and 71% EMP samples (p=0.67). Additional staining for cyclin D1, Bcl 2 and FISH analysis will be reported. Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders. Disclosures Hochberg: Enzon: Consultancy, Speakers Bureau; Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau. Anderson:Millennium: Research Funding. Raje:Celgene, Norvartis, Astrazeneca: Research Funding.

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IDH1 and IDH2 mutations are Not Frequent In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.4992.4992 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4992-4992 ◽

Cited By ~ 1

Author(s):

Christian Langer ◽

Mariam Ibañez ◽

Peter Liebisch ◽

Thorsten Zenz ◽

Stefan Knop ◽

...

Keyword(s):

Multiple Myeloma ◽

High Performance ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Rare Event ◽

Gene Mutations ◽

Isocitrate Dehydrogenase 1 ◽

Aberrant Expression ◽

Genetic Abnormalities ◽

Idh1 And Idh2 Mutations

Abstract Abstract 4992 Multiple myeloma (MM) is characterized by frequent and complex genomic abnormalities. However, most genetic abnormalities are already present in the precursor state of MM, the monoclonal gammopathy of undetermined significance (MGUS). Therefore, it is likely that secondary genetic events might contribute to the development from MGUS to symptomatic MM. Mutations in isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) genes have recently been described as relatively frequent molecular lesions in gliomas and in acute myeloid leukemia (AML). However no larger study has so far examined the frequency of IDH1 and IDH2 gene mutations in patients (pts) with MM. Methods: In 188 pts with MM genomic DNA from CD138 sorted plasma cells was used for analyses. Exon 4 of both IDH1 and IDH2 were amplified by PCR and the amplicons were analyzed using a combination of denaturing high-performance liquid chromatography and DNA sequencing. All patients were also characterized by a comprehensive set of FISH probes for the presence of recurring cytogenetic abnormalities. Results: 185 out of 188 samples were evaluable for analyses. One missense mutation in the IDH2 gene (c.G419A) was identified in the cohort of 185 MM pts (0.5%). This mutation was described as the most frequent IDH2 mutation in AML and is predicted to cause an amino acid change from arginine to glutamin at position 140 (p.R140Q). On cytogenetic analysis this patient harbored a translocation t(11;14) resulting in aberrant expression of CCND1. Additionally, in 15 pts (8%) the recently described single nucleotide polymorphism (SNP) in the IDH1 gene (rs11554137) was detected that has been reported as an adverse prognostic factor in cytogenetically normal AML. Summary: Mutations in the IDH1/2 genes are a rare event in MM (0.5%). Further studies are warranted to address the issue if IDH1/2 mutations are restricted to distinct genetic subgroups as for example the group of MM pts with translocation t(11;14). Disclosures: No relevant conflicts of interest to declare.

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Not All Polyps Are Created Equal: Multiple Myeloma in the Gut

Blood ◽

10.1182/blood.v128.22.5602.5602 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5602-5602

Author(s):

Divya Akella ◽

Fnu Aparna ◽

Marijeta Pekez ◽

Nirmala S. Nathan ◽

Hemchand Ramberan ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Correct Diagnosis ◽

Colonic Polyps ◽

Immunohistochemical Analysis ◽

Recurrent Bleeding ◽

Dexamethasone Therapy

Abstract Background: Multiple myeloma is a neoplastic proliferation of plasma cells producing a monoclonal immunoglobulin usually restricted to the bone marrow. Recent literature confirms increased extramedullary involvement of skin, liver and lymph nodes but gastrointestinal multiple myelomas remain rare. Case: We report a case of 57-year-old female with a past medical history of progressive multiple myeloma IgA lambda on elotuzumab, lenalidomide and dexamethasone therapy, who presented with generalized weakness and black stools for approximately one week. Initial laboratory work demonstrated a hemoglobin of 6.7 grams per deciliter and heme positive stools consistent with anemia secondary to presumed gastrointestinal blood losses. Esophagogastroduodenoscopy (EGD) was unremarkable. Colonoscopy revealed 6 colonic polyps scattered throughout the distal transverse, cecal and descending colon which were excised and sent for pathology. Pathology of the polyps showed plasma cell myeloma with anaplastic features. Immunohistochemistry demonstrated cells that were positive for CD-138 and negative for keratin staining, confirming plasma cell origin. Furthermore analysis was positive for lambda light chain, but negative for kappa light chain. The patient was managed with packed red cell transfusion with no further evidence of recurrent bleeding. Conclusion: Gastrointestinal multiple myeloma are rare, but as our case demonstrates, they must be considered in the differential diagnosis of patients with gastrointestinal bleeding, particularly those with multiple myeloma. The endoscopic appearance of multiple myeloma polyps may be similar to other more common conditions, making pathological and immunohistochemical analysis of biopsies essential for making a correct diagnosis. Disclosures No relevant conflicts of interest to declare.

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In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽

10.1182/blood-2019-125952 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3100-3100 ◽

Cited By ~ 2

Author(s):

Ken Flanagan ◽

Muntasir M Majumder ◽

Romika Kumari ◽

Juho Miettinen ◽

Ana Slipicevic ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Plasma Cell ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Al Amyloidosis ◽

Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

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B-Cell Maturation Antigen (BCMA) in Systemic Light-Chain Amyloidosis (AL): Association with Disease Activity and Its Modulation with Gamma-Secretase Inhibition

Blood ◽

10.1182/blood-2019-126988 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4409-4409 ◽

Cited By ~ 2

Author(s):

Amandeep Godara ◽

Ping Zhou ◽

Benjamin Rosenthal ◽

Adin Kugelmass ◽

Denis Toskic ◽

...

Keyword(s):

Cell Surface ◽

Disease Activity ◽

Board Of Directors ◽

Plasma Cell ◽

Light Chain ◽

Research Funding ◽

San Diego ◽

Plasma Cells ◽

Advisory Committees ◽

Nsg Mice

Introduction: Systemic light-chain (AL) amyloidosis results from clonal plasma cells that secrete toxic fibril-forming free light chains. Therapies directed at the plasma cell clone form the backbone of its management. Identification of cell-surface receptors on the clonal cells can provide targets for therapy. BCMA is one such cell-surface glycoprotein; it is principally expressed on plasma cells and supports their long-term survival (J Exp Med. 2004;199:91-98). Anti-BCMA immunotherapies are currently being studied in multiple myeloma (N Engl J Med. 2019;380:1726-1737). Membrane-bound BCMA (mBCMA) is also shed as a soluble form, sBCMA, due to γ-secretase activity that can be inhibited by a small molecule (GSI, LY-411575) (Nat Commun. 2015;6:7333; J Immunol. 2017;198(8):3081-3088). We report on mBCMA on the clonal plasma cells of AL patients and its modulation by GSI in vitro, and on sBCMA in the blood of AL patients and of mice xenografted with an AL cell line, demonstrating its correlations in vivo with free light chain (FLC) levels and plasma cell tumor burden. Methods: We analyzed mBCMA and sBCMA levels in marrow aspirate and peripheral blood samples from AL patients under an IRB approved protocol. We isolated mononuclear cells (MNC) from patient marrow aspirates with anti-CD138 microbeads (Miltenyi Biotec, Auburn, CA), and used the CD138-selected cells in culture with LY-411575 (Sigma Aldrich, St Louis, MO). We analyzed mBCMA expression by flow cytometry using APC conjugated anti-CD269 (BCMA) antibody (Biolegend, San Diego, CA, USA) and CD138 expression by PE-conjugated anti-CD138 antibody (Biolegend, San Diego, CA, USA), along with appropriate isotype controls. We injected 107 ALMC-1 reporter cells in the flanks of NOD scid gamma (NSG) mice to create a xenograft model of AL clonal plasma cell disease (Jackson Laboratories, Bar Harbor, ME). sBCMA in patients and mice and FLC in mice were measured by ELISA (R&D Systems, Minneapolis, MN; Bethyl lab Montgomery, TX respectively). Pearson and Spearman correlation analysis was used to examine associations of sBCMA and clinical disease parameters. Paired t-test was applied to compare BCMA expression before and after treatment with GSI. Results: Marrow and blood were obtained from 20 AL patients, 8 newly diagnosed, 4 with progression of disease, and 8 after treatment with >VGPR. Their median age was 65 years (range, 48-77) and 50% were female. Median plasma cells in the marrow aspirates and involved FLC levels were 5% (1-20%) and 33 mg/L (6.6-2220mg/L) respectively. Median mBCMA expression on CD138+ marrow MNC and sBCMA levels in plasma were 39% (4-83) and 28.5 ng/ml (6.6-100.3) respectively (Figure 1A-B). sBCMA levels correlated with bone marrow plasma cell percentage and iFLC (both p<0.001, Figure 1C-D). In culture with LY-411575, the percentages of CD138 cells positive for mBCMA increased from 85% to 100% with ALMC-1 cells and from 36% to 68% (p < 0.01) with patient CD138-selected cells while the sBCMA levels in culture supernatant decreased by over 50%. In NSG mice with ALMC-1 reporter cell xenografts, medians of luciferin-based bioluminescence FLUX (photons/s), λ FLC and sBCMA were 3.9x1010 (2.02x109-1.2x1011), 949.1 mg/L (868.8-23629.2), and 3.8 ng/ml (0.9-23.6) respectively. sBCMA levels correlated with FLC (Pearson r= 0.99, p<0.0001) and with FLUX (Pearson r=0.61, p=0.07). Conclusions: BCMA is expressed on AL plasma cells and sBCMA is detected in the blood of all AL patients. In this light chain disease, sBCMA may be useful as a marker of disease activity even in patients with low FLC. Furthermore, expression of mBCMA can be manipulated by treatment with a GSI, an approach which may be useful therapeutically in AL. These results provide the basis for applying anti-BCMA immunotherapies in clinical trials in relapsed refractory AL patients. Disclosures Comenzo: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Unum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Caelum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Prothena Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Myself: Patents & Royalties: Patent 9593332, Pending 20170008966.

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