Use Of Plasma Cell Immunophenotype As Prognostic Markers In Patients With Systemic AL Amyloidosis

Abstract The prognosis of patients with systemic AL amyloidosis is mainly driven by advanced organ dysfunction, mainly heart involvement. The contribution of the underlying plasma cell clone to prognosis has not been much studied – lately the serum free light chain load has been shown to impact survival. Detailed analysis of plasma cell immunophenotype by multiparameter flow cytometry has been reported to be prognostic in myeloma as well as MGUS. We report that the plasma cell phenotype may allow us to refine prognostic assessment in patients with systemic AL amyloidosis. 48 serial patients with biopsy proven systemic AL amyloidosis had bone marrow as part of a prospective bone marrow study. Flow cytometry was done using previously published protocols (Paiva et al Blood 2010) using fluorescent labelled monoclonal antibodies to the following markers (maximum eight in one tube): CD138, CD38, CD56, CD117, CD28, CD20, CD27, CD19, CD81 and kappa and lambda light chains. Organ involvement, survival and haematological response were analysed in the context of plasma cell phenotypes. Abnormal plasma cell phenotype was defined as cells expressing CD38+CD138+CD19- and further markers were analysed on this population. Cardiac, renal and liver involvement in the cohort was 69%, 75% and 29% respectively. The median follow up was 7 months (range 0.7-18.7months). Results Cardiac involvement was significantly higher in patients co-expressing CD56 (spearman’s correlation coefficient = 0.342 and p=0.021) and a non-significantly greater in those with CD27 expression. 77% had achieved a haematological response on an intention to treat analysis with 65% achieving a VGPR or better. Patients with plasma cells lacking expression of CD27 or CD81 achieved a significantly higher rate of VGPR or better compared to those expressing these markers, (spearman’s correlation coefficient = -.565 and -0.394, p=0.001 and p=0.021 respectively) (Table 1). Median OS was not reached for the whole cohort and the 2 year OS in patients with >5% normal plasma cells (defined as CD38+CD138+CD19+ plasma cells) was 74% compared to 45% in those with <5% normal plasma cells (median OS not reached (NR) vs 4.3 months respectively). Patients with cardiac involvement had a median OS of 5.2 months compared to not reached (NR) for those without cardiac involvement (p=0.017). Within the former group, those with >5% normal plasma cells had a better median OS compared to those with <5% normal plasma cells (NR vs 3.7 months, p=0.564). The median OS was not reached for those achieving a clonal response vs. 3.2 months for non responders (p<0.000). Of the patients who achieved VGPR, those with >5% normal plasma cells appear to have a better OS than those with <5% normal plasma cells with a 2 year survival of 100% and 76%, respectively. Patients with abnormal plasma cell clone expressing CD27 phenotype had a significantly worse OS than those without CD27 (2.6 months vs median OS NR, p=0.010, figure 1). Similarly, expression of CD56 by the abnormal clones had a significantly worse OS, than CD56- plasma cells (4.3 months vs Median OS NR, p=0.052). Patients with abnormal clones expressing CD27+CD56+ had an OS of 1.5 months compared to median OS not reached in the CD27+CD56- group (p=0.003). Median OS was not reached in either CD27-CD56- or CD27-CD56+ patients, but the former group appear to have a better outcome with a 2 year survival of 90% and 67% respectively (figure 2). On a multivariate analysis, expression of CD27 and CD56 on abnormal plasma cells remained significant prognostic factors when analysed along with different variables of cardiac involvement including Mayo staging. Conclusion In summary, presence of >5% normal plasma cells is associated with better OS. Expression of CD56 correlated with cardiac involvement and of CD27 with a poorer haematological response. Expressions of CD27 or CD56 on CD38+CD138+CD19- plasma cells appear to be independent markers of poorer prognosis. Patients expressing both having even worse outcomes with a median OS <2 months. In summary, defining the plasma cell immunophenotype may help to refine the current staging of AL amyloidosis and response to treatment. This observation requires further validation in larger studies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Prognostic Significance of Phenotypically ‘Normal’ Plasma Cells in Chemotherapy Treated AL Patients with Underlying MGUS and Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2073.2073 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2073-2073

Author(s):

Sajitha Sachchithanantham ◽

Ruth M de Tute ◽

Anna Baginska ◽

Christopher Parrish ◽

Shameem Mahmood ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Al Amyloidosis ◽

Term Survival ◽

Female Ratio ◽

Bone Marrow Trephine ◽

Normal Plasma ◽

The Impact

Abstract Background: Systemic AL amyloidosis is a rare complication of plasma cell dyscrasias. Much progress has occurred in treatment of AL amyloidosis but long term survival remains limited with advanced organ involvement, in particular, cardiac dysfunction determining outcomes. However, controlling the underlying plasma cell clone with chemotherapy or ASCT is the key to improving outcomes. Yet the role of plasma cell clones in determining prognosis remains to be fully explored and understood. The plasma cell burden in patient with AL amyloidosis is generally lower than that of multiple myeloma but reported degree of plasma cell infiltration has varied. A large study from the Mayo group reported markedly poor outcomes for patients with AL amyloidosis who have >10% BMPCs, even in the absence of symptomatic myeloma (Kourelis et al, JCO 2013). However, apart from just the number of BMPC, the composition appears to be of importance. Multiparameter flow cytometry (MFC) can identify proportion of normal and clonal plasma cells. Patients with >5% normal BMPC (defined as cells expressing CD38+CD138+CD19+) at diagnosis had a better prognosis (Paiva et al. Blood 2011). MFC underestimates the total proportion of BMPCs due to sample dilution effect. We report the impact of normal' plasma cells, as determined by MFC, on the outcome of AL patients in context of the total plasma cell burden as determined by standard morphological techniques in 104 patients with biopsy proven systemic AL amyloidosis, who had both bone marrow trephine and MFC performed at presentation between 2005-2013 assessed at UK National amyloidosis centre and St James's University Hospital. Methods: The bone marrow trephine biopsy (BMTB) plasma cell burden was estimated by morphology supplemented by CD138 immunohistochemistry as required. Patients with >10% CD138+ cells were classified as having AL-multiple myeloma (AL-MM) and those with <10% CD138+ cells as having AL-MGUS. Six or eight colour MFC was used to assess the proportion of CD38+CD138+ plasma cells expressing CD19 in the bone marrow aspirate samples. Results: The median age was 64.8 years (range: 38.5-83.3) with a male-female ratio of 1.7:1. 58 (56%) had cardiac involvement. All patients had treatment and the longest follow up was 8.3 years with 52 patients alive at the time of analysis. BMTB was inadequate for 3 patients, of the remaining 101 patients, 59 (57%) had >10% CD138+ PCs on trephine (classed as AL-MM) and 42 (40%) had <10% (classed as AL-MGUS). All patients had MFC and the median number of normal' PCs was 4.07% (range 0-72.57%). The median neoplastic PC% was 95.9 (range : 9.8-95.3). All patients had CD19 negative PC demonstrable, of which 61 (58%) had CD56 expression thereby confirming the diagnostic utility of MFC in this setting. ROC analysis gave 10% as the most significant cut-off for normal PC. 31 (30%) patients had greater than 10% normal' PCs; 22 (52%) and 8(14%) with underlying AL-MGUS and AL-MM respectively (p<0.001). There was a statistically significant negative correlation between the BMTB PC% and the normal' PC quantity on MFC (Spearman correlation -0.419, p=0.000). The median overall survival (OS) for the whole cohort was 26 months and that for those with AL-MGUS was 37.9months and AL-MM 18.1 months (p=0.140). Those patients with >10% normal PCs had a significantly superior survival (53.4months) compared to those with <10% normal PCs (16 months) (p = 0.019) on MFC (Figure 1). When outcome was assessed according to overall BM burden and MFC it was clear that the presence of >10% normal PCs conferred a favourable outcome regardless of the BM burden (Figure 2, p=0.075). Outcomes were similar for AL-MM and AL-MGUS in those patients with >10% normal PC (p=0.824) and those with <10% (p=0.755). Conclusion: In this study we have confirmed the value of MFC in patients with AL amyloid. Abnormal PC populations are demonstrable in all patients confirming the utility of the assay for diagnostic purposes. This is particularly relevant for those patients with low BM burden. Similarly the presence / absence of normal plasma cells by MFC had a significant effect on outcome which was demonstrable in patients with both AL-MGUS and AL-MM. MFC should be included in the diagnostic workup of all patients with AL. Further studies are required to determine how this additional prognostic data can be incorporated into existing prognostic models. Figure 1: Figure 1:. Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

Download Full-text

AL Amyloidosis and Concomitant Myeloma: Time to Reconsider Assumptions

Blood ◽

10.1182/blood.v116.21.4044.4044 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4044-4044

Author(s):

Wesley Witteles ◽

Ronald Witteles ◽

Michaela Liedtke ◽

Sally Arai ◽

Richard Lafayette ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Plasma Cell ◽

Research Funding ◽

Plasma Cells ◽

Renal Involvement ◽

World Health ◽

Bone Lesions ◽

Al Amyloidosis ◽

Bone Marrow Biopsies

Abstract Abstract 4044 Background: Conventionally, multiple myeloma is believed to coexist in approximately 10% of AL amyloidosis patients. However, it is unclear whether this figure is too low based on current World Health Organization criteria. These criteria, mainly created to differentiate myeloma from monoclonal gammopathy of undetermined significance, include the presence of ≥ 10% plasma cells on a bone marrow biopsy or aspirate as being diagnostic of myeloma. Aims: To define the frequency and relevance of a concomitant diagnosis of myeloma in patients with AL amyloidosis. Methods: Records from consecutive patients with biopsy-proven AL amyloidosis treated at the Stanford University Amyloid Center were reviewed. Plasma cell percentages were determined by manual counts from bone marrow aspirate smears and by CD138 immunohistochemistry (IHC) performed on bone marrow core biopsies. Results: A total of 41 patients (median age 61 years, 32% female) were evaluated. The median number of organs involved with amyloidosis was 2 (range 1–4), with 28 patients (68%) having cardiac involvement, 22 patients (54%) having renal involvement, 15 patients (37%) having gastrointestinal involvement, 12 patients (29%) having soft tissue involvement, and 10 patients (24%) having nervous system involvement. All patients had bone marrow biopsies and aspirates performed at the time of amyloid diagnosis, with most undergoing both manual counts of plasma cells from aspirates and IHC from core biopsies. Based on conventional criteria, manual aspirate counts defined 15/28 (54%) patients as having myeloma, and IHC defined 26/31 (84%) patients as having myeloma (p=0.01). Only nine patients had a detectable serum paraprotein on immunofixation (median 1.1 g/dl, range 0.4–2.6). 81% of patients had an elevated serum free light chain (85% lambda), with a median level of 37.3 mg/dl (range 8.6–256 mg/dl). Compared to the frequency of elevated plasma cells, the prevalence of anemia (29%), hypercalcemia (14%), impaired kidney function (21%), and lytic lesions (7%) was low. After a median follow-up of 13 months (range 1–127 months), the one-year overall survival (74% vs. 58%) and three-year overall survival (50% vs. 50%) was not significantly different between patients with ≥10% plasma cells and patients with <10% plasma cells (p=NS). Discussion: As defined by bone marrow plasma cell involvement, a strikingly high percentage (84%) of AL amyloidosis patients would be considered to have concurrent myeloma. This figure is much higher than has been traditionally quoted in the literature, likely due to the utilization of newer methods of counting plasma cells. There was a low prevalence of myeloma-associated end-organ effects (hypercalcemia, anemia, renal insufficiency, lytic bone lesions), and a myeloma diagnosis had no impact on survival. Conclusion: In this cohort of AL amyloid patients, concomitant myeloma was present in the vast majority of patients using modern diagnostic techniques. The significance of this diagnosis appears to be minimal – calling into question whether the diagnostic criteria for myeloma should be redefined in this population. Disclosures: Witteles: Celgene: Research Funding. Liedtke:Celgene: Lecture fee, Research Funding. Schrier:Celgene: Research Funding.

Download Full-text

The Use of Next Generation Gene Sequencing to Measure Minimal Residual Disease in Patients with AL Amyloidosis and Low Plasma Cell Burden: A Feasibility Study

Blood ◽

10.1182/blood-2019-131863 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4353-4353 ◽

Cited By ~ 2

Author(s):

Shayna Sarosiek ◽

Vaishali Sanchorawala ◽

Mariateresa Fulcinti ◽

Allison P. Jacob ◽

Nikhil C. Munshi ◽

...

Keyword(s):

Bone Marrow ◽

Minimal Residual Disease ◽

Plasma Cell ◽

Peripheral Blood ◽

Research Funding ◽

Plasma Cells ◽

Residual Disease ◽

Al Amyloidosis ◽

Next Generation ◽

Minimal Residual

Background: AL amyloidosis is a bone marrow disorder in which clonal plasma cells produce light chains that misfold and deposit in vital organs, such as the kidneys and heart, leading to organ failure and eventual death. Treatment is directed towards the clonal plasma cell population in an effort to halt the production of toxic light chains and recuperate organ function. Pallidini et al. demonstrated that almost 50% of patients with AL amyloidosis who achieved a complete hematologic response to prior therapy had minimal residual disease (MRD) detectable in their bone marrow by multiparametric flow cytometry (MPF).1. Next generation gene sequencing (NGS) has been a successful tool in measuring MRD among patients with multiple myeloma2 though the data regarding its use in AL amyloidosis are limited. AL amyloidosis is a disease with a much smaller plasma cell burden at baseline (typically 5-10%), making the task of isolating an initial clonal sequence even more challenging. We sought to evaluate NGS as a method of isolating a clonal population of plasma cells among patients with systemic AL amyloidosis in a first-ever feasibility study. Methods: Patients were eligible if they had systemic AL amyloidosis and no clinical evidence of concurrent active multiple myeloma. In this study, feasibility was deemed successful if discovery of a clone could be achieved in 3 out of 10 of patients. Approximately five cc's of peripheral blood and bone marrow aspirate were collected from each patient and processed for CD138 selection and DNA isolation/purification. De-identified samples were sent to Adaptive Biotech Inc. (Seattle, WA) for initial clonal identification using the ClonoSEQ immunoglobulin heavy chain (IGH) assay. Genomic DNA was amplified by implementing consensus primers targeting the IGH complete (IGH-VDJH) locus, IGH incomplete (IGH-DJH) locus, immunoglobulin κ locus (IGK) and immunoglobulin l locus (IGL). The amplified product was sequenced and a clone identified based on frequency. After proof of feasibility in the first 10 patients an additional 27 patients had initial clonal identification via the same process mentioned above. Results: In total, 37 patient samples underwent NGS via the ClonoSEQ IGH assay method. The median patient age was 66 years old (range: 44 to 83), 24% of which were female. All 37 patients had measurable disease based on serum electrophoresis and immunofixation and/or serum free light chain assay (Table 1). Four patients had no monoclonal protein detected on SIFE or UIFE and 13 patients had a normal sFLC ratio. Of the 33 patients with monoclonal disease on immunofixation, 12 patients had only a free lambda monoclonal protein and the remaining 21 patients had a clonal heavy chain with an associated light chain. Bone marrow biopsies demonstrated clonal plasmacytosis of 40% or lower. ClonoSEQ IGH assay identified trackable clones in 31 of 37 patients (84%) (see Table 1). Four patients had at least one trackable sequence (range: 1 to 5 sequences) in the peripheral blood and 29 patients had at least one trackable sequence in the bone marrow aspirate (range: 1 to 7 sequences). No correlation was seen between the detection of a clone and standard measures of plasma cell tumor burden (SIFE, SPEP, UIFE, UPEP, and sFLCs). Conclusion: NGS was successful in identifying an initial clone in 29 of 37 patients with systemic AL amyloidosis, four of which were detectable in the peripheral blood. Due to the low clonal burden in patients with AL amyloidosis, it is often difficult to assess disease status, especially post-treatment. These encouraging results may enhance disease monitoring and improve patient care in this rare disease. We are currently tracking MRD in the patients with identifiable clones as they receive systemic treatment, the results of which will be available for presentation in December 2019. REFERENCES 1. Palladini G, Massa M, Basset M, Russo F, Milani P, Foli A, et al. Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response. Abstr 3261. 2016; 2. Ladetto M, Brüggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299-307. Table 1 Disclosures Sarosiek: Acrotech: Research Funding. Sanchorawala:Proclara: Consultancy, Honoraria; Takeda: Research Funding; Caelum: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Prothena: Research Funding; Celgene: Research Funding. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Munshi:Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy.

Download Full-text

Melphalan and Dexamethasone Is Less Effective for Patients with Immunoglobulin Light Chain Amyloidosis (AL) with High Bone Marrow Plasmacytosis.

Blood ◽

10.1182/blood.v112.11.1735.1735 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1735-1735 ◽

Cited By ~ 1

Author(s):

Nelson Leung ◽

Heidi D Gunderson ◽

Tow S Tan ◽

Angela Dispenzieri ◽

Morie A Gertz ◽

...

Keyword(s):

Bone Marrow ◽

Plasma Cell ◽

Light Chain ◽

Cardiac Involvement ◽

Plasma Cells ◽

High Plasma ◽

High Dose ◽

Immunoglobulin Light Chain ◽

Hematologic Response ◽

Immunoglobulin Light Chain Amyloidosis

Abstract Introduction: Currently, the most extensively tested treatments for immunoglobulin light chain amyloidosis (AL) are high dose melphalan followed by autologous stem cell transplantation (ASCT) and melphalan and dexamethasone (MDex). A recent randomized controlled trial comparing the two regimens showed MDex is at least equivalent if not superior to ASCT. Most patients with symptomatic systemic AL have a low plasma cell load; however, approximately 30% have bone marrow plasmacytosis greater than 20% but do not meet criteria for multiple myeloma with significant anemia or lytic bone disease. This leads to the speculation that the plasma cell biology (and therefore outcomes) might differ between low and high plasma cell burden AL. We undertook this study to address whether these two standard regimens are equally effective in AL patients with low and high plasma cell burden. Methods: Patients with confirmed AL treated with MDex were recruited for this study. Some of the patients treated were not ASCT candidates while others were eligible but favored MDex. Hematologic response was defined as 50% reduction in serum M-protein or 90% reduction in urine M-protein if one was present, otherwise a 50% reduction in serum free light chain levels was used. Organ involvement was defined according to the Consensus Opinion from the 10th International Symposium on Amyloid and Amyloidosis. Patients were separated into a high and a low marrow plasma cells (PC) group based on their percentage of bone marrow plasma cells. Cutoff for the bone marrow plasmacytosis was calculated using receiver-operator characteristic (ROC) curve. Overall survival (OS) and progression free survival (PFS) were compared using Kaplan-Meier method. OS was also calculated for 342 AL patients treated with ASCT for comparison. Results: Seventy-three patients with AL received MDex between 05/01 and 05/07. Median follow-up was 16.8 months. At the time of the study, 42.5% had died. Calculating for OS and PFS, the best cutoff for bone marrow plasmacytosis was found to be 20%. In our cohort, 28.8% had >20% bone marrow plasmacytosis. Age (64.8 yr vs 61.0 yr, p = 0.61) and sex (81% male vs 63.4% male, p = 0.13) were similarly distributed between patients with high and low PC. Cardiac troponin T (cTnT) was also comparable between the 2 groups (0.03 ng/ml (high) vs 0.06 ng/ml (low), p = 0.41). Cardiac involvement were similar between high and low PC groups (78.9% vs 85.7% respectively, p = 0.49) but renal involvement was less common in the high PC group (70.6% vs 38.1%, p = 0.01). No differences were noted in the gastrointestinal and neurological involvement. Patients with high PC received a median of 4 cycles of MDex vs 5 in the low PC group (p = 0.68). Hematologic response was achieved in 75% of patients with high PC and 54.9% of the low’s (p = 0.11). A significant difference was noted in the PFS and OS between the 2 groups. The PFS and OS were 13.5m and 15.3m, respectively for patients with high PC but neither was reached in those with < 20% plasmacytosis (p=0.02 and p = 0.03 respectively). In the multivariate analysis, hematologic response, cardiac involvement and plasmacytosis >20% were independent predictors of PFS and OS. To determine if plasmacytosis had the same affect on survival of ASCT treated patients, 342 patients were analyzed. No relationship between OS and % of plasmacytosis was found. Conclusion: Our study suggests that ASCT and MDex may not be equivalent for all AL patients. Even though hematologic response rates were similar, patients with plasmacytosis >20% had a worse OS and PFS when treated with MDex. This disparity in survival was not evident in patients treated with ASCT. This effect was independent of cardiac involvement. If confirmed, this could have a significant impact on the choice of therapy for AL patients. The best therapy may be determined by the extent of marrow plasmacytosis, with higher plasma cells favoring high dose therapy.

Download Full-text

Detection and Characterization of Plasma Cell and B Cell Clones in Patients with Systemic Light Chain Amyloidosis Using Flow Cytometry

Blood ◽

10.1182/blood.v124.21.2068.2068 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2068-2068

Author(s):

Stefan Schönland ◽

Ute Hegenbart ◽

Christoph Kimmich ◽

Katarina Lisenko ◽

Dirk Hose ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Cell Count ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

Cell Clone ◽

Flow Analysis ◽

Al Amyloidosis ◽

Cell Clones

Abstract Introduction: AL amyloidosis is a rare and life-threatening protein-deposition disorder caused by a small B cell (mostly plasma cell) clone which produces amyloidogenic light chains. The goal of therapy is to target this clone and halt the uncontrolled release of free light chain, which might subsequently lead to improvement of organ function. In routine diagnostic some of these B cell clones are missed as they might be extremely small. However, specific treatment can only be applied if the clone is well characterized. Hardly any data on the characteristics of these cells using flow cytometry have been reported. (e.g. Paiva et al., Blood 2011). Study design: We performed a retrospective analysis of consecutive patients who were referred to our amyloidosis center (March to July 2014) and have been thoroughly studied (immunhistology of amyloid, free light chain assay, immunofixation, bone marrow diagnostic: cytology, flow cytometry and interphase-FISH cytogenetics (iFISH)). Patients and Methods: Twenty-two patients were included (all untreated, 21 AL patients, one pt with monoclonal gammopathy of renal significance (MGRS)). Plasma cells were detected by their co-expression of CD38 and CD138 antigens. Differentiation between malignant and normal plasma cells was achieved by analysis of aberrant CD45 and CD19 expressions and proof of intracellular light chain restriction (see Figure 1). To evaluate potential targets for an antibody-based immunotherapy, we stained CD20, CD22, CD30, CD52 and CS-1 on these plasma cells. Overall, positivity was defined as >20% expression of the antigen. iFISH was done after CD138 selection as previously described (Bochtler et al., Blood 2011). Results: Main characteristics and results are shown in Table 1. Median dFLC was 304 mg/l, three patients had a dFLC of less than 50 mg/l. Median plasma cell count in cytology was 10%, 3 patients had less than 5%. Median plasma cell count by flow was 3.8%, three patients had less than 1%. Correlation between dFLC, plasma cell count in cytology and flow was low (FLC vs. flow: spearman=0.25, p=0.26; FLC vs. cytology: spearman=0.49, p=0.02; flow vs. cytology, spearman=0.36, p=0.1). Detection of the amyloidogenic clone by flow was possible in all but one patient (95%). In this patient we were not able to show a light chain restriction although we detected a relevant aberrant plasma cell clone (CD45low, CD19low). In one patient we found a B cell lymphoma as underlying disease for MGRS type IgG lambda (CD19+, CD20+, lambda+, CD5-, CD22+, FMC7-, CD23-, CD25+, CD103-, CD38+ typical for marginal zone lymphoma). In all 21 patients the light chain restriction demonstrated by flow was confirmed by immunofixation, FLC, and immunohistology of the amyloid. All patients analyzed for the expression of CS-1 were positive. 25% were also positive for CD20 and none was positive for CD22, CD30 and CD52. Detection of the plasma cell clone by iFISH was possible in all 21 patients (see Table 1). Conclusion: Flow cytometric analysis of the bone marrow is a very sensitive method to detect and characterize the amyloidogenic clone in AL amyloidosis. B cell lymphomas can easily be distinguished from pure plasma cell clones. Secondly, flow provides useful information to specify immune-chemotherapy in AL amyloidosis and related disorders. Table 1: Patients (n=22) Characteristics and Results Age in yrs (median / range) 67 (41 – 77) Sex: female / male 9 / 13 Type of light chain: kappa / lambda 4 / 18 Median dFLC in mg/l (range) 304 (22 - 6621) Median % of plasma cells in BM cytology (range) 10 (0 – 68) Underlying disease leading to AL amyloidosis“MG” / MM III / B-NHL 20 / 1 / 1 Median % of PC by flow (range) 3.8 (0.2 - 34) Detection of the amyloidogenic clone by flow 21 / 22 Flow analysis of clonal plasma cells (% of pts)CD20+ / CD22+ / CD30+ / CD52+ / CD56+ / CS-1+ 25 / 0 / 0 / 0 / 75 / 100 Detection of a clone by iFISH 21/21 % of pts with t(11;14) / Gain of 1q21 / Hyperdiploidy / High-risk cytogenetic (del 17p13, t(4;14)) 52 / 10 / 14 / 10 Figure 1: Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Figure 1:. Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Disclosures Schönland: Janssen: Honoraria; Celgene: Honoraria. Hegenbart:Janssen: Honoraria; Celgene: Honoraria. Hose:Novartis: Research Funding. Hundemer:Celgene: Honoraria, Research Funding.

Download Full-text

Macroglossia – Not Always AL Amyloidosis

Blood ◽

10.1182/blood.v116.21.5007.5007 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5007-5007 ◽

Cited By ~ 1

Author(s):

Andrew J Cowan ◽

Martha Skinner ◽

J. Mark Sloan ◽

John L Berk ◽

Carl J O'Hara ◽

...

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

Plasma Cell Dyscrasia ◽

Al Amyloidosis ◽

Amyloid Deposits ◽

Immuno Electron Microscopy ◽

Congo Red Staining

Abstract Abstract 5007 Introduction: Amyloidosis is characterized by extracellular deposition of abnormal insoluble fibrillar proteins. The two most frequent systemic amyloidoses are the light-chain (AL amyloidosis) and familial transthyretin (ATTR) forms. Clinical presentations often vary between the two types. Macroglossia is viewed as pathognomic of AL amyloidosis, and has not previously been described in patients with hereditary TTR amyloidosis. Here, we describe two cases of systemic amyloidosis with macroglossia in which immuno-electron microscopy diagnosed ATTR in one and AL in the other. Case Presentations: A 61 year old woman presented initially to her general internist with weight loss, difficulty swallowing, and tongue numbness. Her clinical exam revealed macroglossia and peripheral neuropathy. Tongue and axillary lymph node biopsies demonstrated amyloid deposits by Congo red staining. There was no evidence of renal, cardiac or other vital organ involvement. She had no evidence of a plasma cell dyscrasia with negative serum and urine immunofixation electrophoresis, normal serum free light chain concentration and ratio as well as polytypic plasma cells in the bone marrow. Immuno-electron microscopy using gold-labeled antibodies was performed on the tongue biopsy. The fibrils were immunoreactive with anti-TTR but not anti-kappa, anti-lambda, or anti-AA antibodies. DNA sequencing identified a known amyloidogenic T60A TTR mutation in exon 3 of chromosome 18, confirming the diagnosis of ATTR with amyloidotic polyneuropathy and macroglossia. The second case involved a 59 year old man with renal insufficiency. He complained of fatigue, weight loss, and tongue swelling. Physical examination was significant for macroglossia and submandibular gland enlargement. Tongue biopsy demonstrated amyloid deposits by Congo red staining. As in the previous case, markers of plasma cell dyscrasia with clonal plasma cells in the bone marrow, blood, and urine were absent. Immuno-electron microscopy of the tongue biopsy documented antibody reactivity to lambda light chain and not TTR, kappa light chain or AA proteins, confirming the diagnosis of AL amyloidosis. He subsequently underwent treatment with high dose intravenous melphalan followed by stem cell transplantation achieving a good clinical response sustained for 2 years to date. Discussion: While macroglossia is thought to be pathognomonic of AL amyloidosis, we report a case of macroglossia with fibrillar ATTR amyloid deposits diagnosed by immuno-electron microscopy. This is contrasted with a clinical presentation consistent with AL in which routine laboratory testing failed to identify evidence of a plasma cell dyscrasia. In both cases, electron microscopy demonstrated immunoreactivity for the fibrils of a single pathogenic protein. The first case was confirmed by DNA sequencing, and the second had a typical response to anti-plasma cell chemotherapy, in spite of the lack of identifiable markers of disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Exome Sequencing To Define A Genetic Signature Of Plasma Cells In Systemic AL Amyloidosis

Blood ◽

10.1182/blood.v122.21.3098.3098 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3098-3098 ◽

Cited By ~ 2

Author(s):

Brian A Walker ◽

Dorota Rowczienio ◽

Eileen M Boyle ◽

Christopher P Wardell ◽

Sajitha Sachchithanantham ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Plasma Cell ◽

Copy Number ◽

Monoclonal Gammopathy ◽

Plasma Cells ◽

Chromosomal Abnormalities ◽

Light Chains ◽

Al Amyloidosis ◽

Plasma Cell Disorders

Abstract Systemic amyloid light chain amyloidosis (AL) is characterized by the deposition of immunoglobulin light chains as amyloid fibrils in different organs, where they form toxic protein aggregates. Most AL patients have relatively low levels of circulating free light chains and bone marrow plasmacytosis. The underlying disease is a plasma cell disorder, likely a monoclonal gammopathy, but limited data are available on the biology of the plasma cell clone underlying AL and existing studies have concentrated on chromosomal abnormalities. Many of the chromosomal abnormalities identified in AL are also seen in other plasma cell disorders, such as monoclonal gammopathy of undetermined significance (MGUS) and myeloma. These abnormalities include translocations involving the IGH locus, gains of 1q and deletions of 13q and 17p. Fluorescence in situhybridization studies have identified the translocation t(11;14) to be more frequent in AL and hyperdiploidy to be rare. The causal link between genetic changes in plasma cells and light chain instability remains unknown and progression to symptomatic myeloma is rare. We report the initial findings of the first exome sequencing to define the plasma cell signature in AL and compared this to MGUS and myeloma. CD138+ cells were selected using either EasySep (Stem Cell Technologies) or MACSort (Miltenyi) from the bone marrow of 18 AL patients and 5 MGUS patients. DNA was extracted from the CD138+ cells using the AllPrep kit (Qiagen). Non-involved DNA was isolated from peripheral white blood cells using the Flexigene kit (Qiagen). 200 ng DNA was subjected to exome sequencing using NEBNext kit (NEB) and SureSelect Human All Exon kit v5 and sequenced using 76-bp paired end reads. Fastq files were aligned to the reference genome using BWA and Stampy aligners. BAM files were recalibrated using the GATK and deduplicated using Picard. Paired tumour/normal BAMs were realigned together using the GATK indel realigner and SNVs were called using Mutect. Copy number data were estimated using the R package ExomeCNV. The median depth across all samples was 42x with 97% of the exome covered at 1x and 72% covered at 20x. Exome data to determine the cytogenetic groups of AL samples identified 42% hyperdiploid and 21% with t(11;14). The AL samples with t(11;14) did not contain any other copy number abnormalities. Exome sequencing on samples from patients with MGUS and myeloma was also performed to compare the genetic makeup and mutation spectrum of these well characterised plasma cell neoplasias with AL samples. MGUS samples had a median of 30 acquired nonsynonymous variants (range 24-189) and AL amyloidosis samples had a median of 17 acquired nonsynonymous variants (range 4-44). The AL samples had four recurrent mutations in PCMTD1 (n=3; L267F, P266S and M187I), C21orf33 (n=2; E72K), NLRP12 (n=2; L1018P, W959* ) and NRAS (n=2; Q61R, Q61H). In this small dataset, only 5 genes were mutated in both the MGUS and AL samples (DNMBP, FRG1, HIST1H1B, KRTAP4-11 and MCCC1). In order to assess the similarity (or differences) of plasma cells in AL to malignant plasma cells in general, we compared them to a random sampling of 20 multiple myeloma samples which had also been exome sequenced (median number of acquired nonsynonymous variants = 39 vs. 17 in AL samples). This revealed that the AL contained 21 mutated genes in common with the myeloma cohort, including DIS3 and NRAS. There were two DIS3 mutations in one AL sample at c.379D>E (p.D479E) and c.1999A>T (p.M667L), both of which were in the Ribonuclease II/R catalytic domain. Data on correlation of gene mutations and organ involvement in AL amyloidosis will be presented. We conclude that exome sequencing identifies a genetic signature of AL amyloidosis which is similar to other plasma cell disorders. This not only includes copy number abnormalities and translocations but also a similar number of nonsynonymous mutations to MGUS and fewer than the advanced myeloma samples. Study of further samples is in progress. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Significance Of Abnormal Plasma Cell Clone In Bone Marrow Of Primary Systemic Light Chain Amyloidosis Patients

Blood ◽

10.1182/blood.v122.21.5342.5342 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5342-5342

Author(s):

Yang Hu ◽

Mangju Wang ◽

Yan Chen ◽

Xue Chen ◽

Fang Fang ◽

...

Keyword(s):

Bone Marrow ◽

Plasma Cell ◽

Light Chain ◽

Plasma Cells ◽

Cell Clone ◽

Morphological Examination ◽

Light Chain Restriction ◽

Significant Difference ◽

Bone Marrow Plasma ◽

Λ Light Chain

Abstract In this study we analyzed the immunophenotype characteristics of the plasma cells and evaluated the significance of the abnormal plasma cell clone in bone marrow in primary systemic light chain amyloidosis (AL) patients. Fresh bone marrow samples were collected from 74 cases of plasma cell disease (PCD), including 51 cases of AL, 21 cases of multiple myeloma (MM), 2 cases of Waldenström's macroglobulinemia (WM). All patients diagnosed according to WHO 2008 diagnostic criteria. The diagnosis of AL was confirmed by the presence of monoclonal immunoglobulin or free light chain in blood or urine, and/or amyloidosis in fat tissues or biopsies by Congo red staining. Ten healthy donors were also collected as controls. Their clinical characteristics and immunophenotype of bone marrow cells were compared and analyzed. The immunophenotype were analyzed with a panel of antibodies including CD45, CD38, CD138, CD117, CD56, CD19, CD20, Igκ, Igλ, CD7, CD22, CD3, CD34 and CD27 by flow cytometry (FCM). The results were for statistical processing. The prominent feature of AL patients was multi-organ and multi-system involvement. Kidney was the major organ involvement (82.4%), followed by cardiovascular system (58.8%); MM mainly had the clinical manifestations of bone lesions (85.7%) and renal involvement (47.6%). The serum immunoglobulin of AL mainly manifested as λ light chain (74.5%), while the majority of MM manifested as κ (61.9%). In the 51 patients of AL, the ratio of plasma cellsin bone marrow was mean 3.87% (0.17∼9.34%) by FCM, and 4.47% (0∼14.5%) by morphological examination. In MM, the ratio of plasma cells was mean 13.17% (1.30∼48.91%) by FCM and 33.55% (3.0∼81.5%) by morphological examination. The plasma cells proportion between AL and MM had significant difference (P< 0.05). The κ or λ light chain restriction can be used for the detection of abnormal plasma cell clones in AL patients. The κ/λ ratio>4.0 or <0.5 can be used as the criteria to identify light chain restriction in plasma cells in AL patients. The 31/51 cases of AL could detected abnormal plasma cell clone that used κ/λ light chain restriction and were mainly expressed λ light chain (24/31, 77.4%). The 21 cases of MM had light chain restriction, mainly expressed κ light chain (13/21, 61.9%) (P<0.05). In CD45/SSC scattergram, the position of abnormal plasma cells of AL patients varied in a wider range. According to the features of CD38+/CD138+ as the basic markers for plasma cells, abnormal plasma cells were CD45 negative or weak positive in AL patients, similar to the CD45 level distribution in malignant plasma cells in MM. In WM, the proliferated cells were plasmacytoid lymphocytes with CD45 weakly or strong positive. FCM can identify abnormal plasma cell clone in bone marrow of AL patients. In 51cases of AL, 78.4% of bone marrow plasma cells were CD56+, 68.6% were CD117+, and 88.2% were CD19-. In 21 of MM, 66.7% were CD56+, 38.1% were CD117+, and 90.4% were CD19-. These results manifested significant difference compared with those of normal plasma cells (P< 0.05). In 2 cases of WM, these plasmacytoid lymphocytes were CD19+ and CD56-, CD117-.The ratios of CD56+, CD117+, CD19-, and CD45-/dim in bone marrow plasma cells were significantly higher in AL patients than in WM patients and healthy individuals (P<0.05), but were similar to those in MM patients (P>0.05). The main difference between AL and MM was the larger size of plasma cell group in MM (P＜0.05). In summary, according to light chain restricted expression and abnormal immunephenotype by FCM analysis we can determine abnormal plasma cell clone in bone marrow of AL patients and the abnormal plasma cells clone can be used as an important diagnostic marker of AL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Biological properties of bone marrow plasma cells influence their recovery in aspirate specimens: impact on classification of plasma cell disorders and potential bias to evaluation of treatment response

Annals of Hematology ◽

10.1007/s00277-020-04249-2 ◽

2020 ◽

Vol 99 (11) ◽

pp. 2599-2609

Author(s):

Svitlana Demyanets ◽

Alexandra Kaider ◽

Ingrid Simonitsch-Klupp ◽

Günther Bayer ◽

Almira Subasic ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cell ◽

Plasma Cells ◽

Cell Phenotype ◽

Special Focus ◽

Plasma Cell Disorders ◽

Bone Marrow Plasma ◽

The Impact

Abstract Methods to estimate bone marrow plasma cells (BMPC) basically include histopathology, cytomorphology, and flow cytometry. The present study compares the outcomes of these methods with special focus on the impact of BMPC-specific characteristics on their recovery by either method. Laboratory reports of diagnostic samples from 238 consecutive patients with suspected or known plasma cell disease were retrospectively analyzed. The median (IQR) proportion of BMPC was 30.0% (15.0–70.0%) by histological review (hBMPC), 7.0% (2.0–16.0%) by smear review (sBMPC), and 3.0% (0.8–10.0%) by flow cytometry (fBMPC). The disparity of results between core biopsy and aspirate smear was enhanced in case of poor quality of the smear, increased BM fiber content, higher grade cell atypia, expression of CD56 (all P < 0.0001), the number of cytogenetic aberrations (P = 0.0002), and abnormalities of the MYC gene (P = 0.0002). Conversely, expression of CD19 and a non-clonal plasma cell phenotype were associated with a lower difference between hBMPC and sBMPC (both P < 0.0001). The disparity between the percentages of sBMPC and fBMPC was associated with the quality of the smear (P = 0.0007) and expression of CD56 (P < 0.0001). Our results suggest that the recovery of BMPC in aspirate specimens not only is a matter of sampling quality but also depends on biological cell properties. Aspiration failure due to malignant type features of BMPC may lead to misclassification of plasma cell disorders and represent a bias for the detection of minimal residual disease after therapy.

Download Full-text

Constitutively High Expression of CD137L on Primary MM Cells Could be Inhibited By Chemotherapy but Re-Emerged after Desease Relapse

Blood ◽

10.1182/blood.v124.21.5198.5198 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5198-5198

Author(s):

Chengcheng Fu ◽

Hui Liu ◽

Ling Ma ◽

Shuang Yan ◽

Juan Wang ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Lines ◽

Plasma Cell ◽

Plasma Cells ◽

De Novo ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

High Expression ◽

Normal Plasma

Abstract Objective: Multiple myeloma (MM) is a kind of malignant monoclonal plasma cell disorder. CD137L molecules are important member of TNFsuperfamily. Under physiological conditions, CD137L express on the surface of various active APCs and participate in keeping the balance of immune system. Under pathological conditions, CD137L could express on the surface of various tumor cells. For example, in hematologic malignancies, about 35% of AML with FAB type M4 or M5, and most CLL, B-cell lymphoma cells express CD137L. CD137L molecules play an important role in the maturation process of B cells. Active CD137L can promote proliferation, differentiation and immune response of B cells. But when B cells transform to plasma cells, the majority of B antigens will be lost, such as CD19, CD20, HLA-DR. CD137L are also lost in the process of transformation. Our previous studies showed that there is no expression of CD137L and CD137 molecular on normal plasma cells. But CD137L were highly expressed on MM cell lines U-266, KMS-11, My-5, LP-1 and 8266. CD137L signaling promoted U266 cell proliferation remarkably, which could be blocked by Bortizomib. CD137L signaling also pushed MM cells in the G1 phase to enter the S phase. To understand the expression of CD137L and CD137 on MM primary cells，We examined the bone marrow specimens of MM patients, then analyzed the relationship between the expression of CD137L and the clinical characteristics of patients. Methods: Flow cytometry was used to detect the CD137L and CD137 expression. Markers of normal plasma cell were CD45+CD38+CD138+CD19+CD56-, Markers of abnormal plasma cell were CD45-/CD45lowCD38++CD138+CD19-CD56+. bone marrow specimens from 127 cases of MM patients treated in the First Affiliated Hospital of Soochow University from 2012 to 2013 and the normal control from 10 volunteers were collected with the consent of patients and volunteers. 45 patients were newly diagnosed, 72 patients were checked after treatment, 10 patients were relapsed and refractory; 68 patients were males, 59 patients were females; The median age was 58(36-73); 3 pts were D-S staging I, 14 pts II, 59 pts III; 25 pts were ISS staging I, 65 pts II, 35 pts III. Results: Normal plasma cells didn’t express CD137L and CD137 molecular .CD137L molecular but not CD137 were expressed on primary MM cells in all 45 de novo patients. The median level was 46.7 (3.6–96.7)%, significantly higher than that on normal plasma cells (P <0.05). A retrospective analysis had been made to find no correlation between CD137L expression and patients’ age, gender, type, DS stage, ISS stage , LDH, creatinine, serum calcium, AKP, M protein and extramedullary plasmacytoma. The CD137L expression of MM cells from 12 de novo patients treated with PAD were decreased gradually with the increase of treatment courses and improvement of efficacy. The expression of CD137L on MM cells in 79 patients grouped in CR (n = 12), PR (n = 51), recurrent (n = 16) were 4.5 (0–18)%，10 (−056)% (P<0.05), 30.5 (5.3–95)% (P = 0.00) respectively. Conclusions: CD137L molecular without CD137 were constitutively highly expressed on MM cell lines and primary MM cells, but hardly on normal plasma cells. There was no relationship between the expression of CD137L with patients’ clinical and biological characteristics. But our data showed that constitutively high expression of CD137L molecular on primary MM cells could be inhibited by chemotherapy but re-emerged after desease relapse. Disclosures No relevant conflicts of interest to declare.

Download Full-text