Dual JAK2/Aurora kinase A inhibition prevents human skin graft rejection by allo-inactivation and ILC2-mediated tissue repair.

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100 significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells. We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

Update on B-cell lymphoproliferative disorders of the gastrointestinal tract.

The gastrointestinal (GI) tract is home to a significant portion of the immune system, which interacts daily with the antigenic milieu of its contents. Therefore, the presence of white blood cells within the walls of the GI tract upon histologic examination is a familiar sight on GI biopsies-both in health and disease. The GI tract is the most common site of extranodal lymphomas, most of which are B-cell neoplasms. Here, we review common and uncommon B-cell neoplasms of the GI tract - extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), mantle cell lymphoma, duodenal-type follicular lymphoma, diffuse large B-cell lymphoma, plasmablastic lymphoma, EBV-positive mucocutaneous ulcer, and post-transplant lymphoproliferative disorders - with special focus on literature published during the past five years. Along with the other articles in this edition of Seminars in Diagnostic Pathology, it is the authors' hope that this review proves to be a useful resource in the workup of the array of hematopoietic processes that can involve the GI tract.

Analysis of Differentially Expressed MicroRNAs and Circulating Tumor Cells as Predictive Biomarkers of Platinum Chemoresistance in Primary Ovarian Carcinomas: A Prospective Study.

LESSON LEARNED: Circulating tumor cells, microRNA markers, or other biomarkers merit examination as part of correlative scientific analyses in prospective clinical trials. BACKGROUND: Platinum chemotherapy resistance occurs in approximately 25% of patients with ovarian carcinoma; however, no biomarkers of ovarian carcinoma chemoresistance have been validated. We performed a prospective trial designed to identify tumor-based predictive biomarkers of platinum resistance. METHODS: Tumor specimens were collected from 29 women with newly diagnosed histopathologically proven primary ovarian carcinoma. Of these, 23 women had specimens accessible for assessment and outcome data available regarding chemosensitive versus chemoresistance status via review of the medical record. Tumor slices were stained with antibodies against two microRNAs (miRNAs 29b and 199a) differentially expressed in chemoresistant ovarian cancer cell lines. Additionally, blood samples obtained at the time of diagnosis were analyzed for the presence of circulating tumor cells (CTCs). RESULTS: The average age of the patients was 64 years, and 82.6% had high-grade epithelial carcinomas. The baseline median CA-125 was 464 (range 32-2,782). No statistically significant differences were observed in miR29b or 199a expression in platinum-resistant/refractory versus platinum-sensitive tumors. Furthermore, the presence of CTCs was not found to be statistically significantly predictive of eventual platinum resistance. CONCLUSION: Our analysis showed no differences in miR29b and 199a expression, and differences in baseline CTCs in women with newly diagnosed ovarian tumors were not statistically significant.

Chronic liver injury alters driver mutation profiles in hepatocellular carcinoma in mice.

Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures. In addition to established human HCC genes and pathways, we identified several injury-associated candidates that represent promising loci for further study. Among them, we found that FIGN is overexpressed in human HCC and promotes hepatocyte invasion. We also validated Gli2's oncogenic potential in vivo, providing direct evidence that Hedgehog signaling can drive liver tumorigenesis in the context of chronic injury. Finally, we show that a subset of injury-associated candidate genes identifies two distinct classes of human HCCs. Further analysis of these two subclasses revealed significant trends among common molecular classification schemes of HCC. The genes and mechanisms identified here provide functional insights into the origin of HCC in a chronic liver damage environment. CONCLUSION: A chronically damaged liver microenvironment influences the genetic mechanisms that drive hepatocarcinogenesis. (Hepatology 2018;67:924-939).

Minimal residual disease prior to allogeneic hematopoietic cell transplantation in acute myeloid leukemia: a meta-analysis.

Minimal residual disease prior to allogeneic hematopoietic cell transplantation has been associated with increased risk of relapse and death in patients with acute myeloid leukemia, but detection methodologies and results vary widely. We performed a systematic review and meta-analysis evaluating the prognostic role of minimal residual disease detected by polymerase chain reaction or multiparametric flow cytometry before transplant. We identified 19 articles published between January 2005 and June 2016 and extracted hazard ratios for leukemia-free survival, overall survival, and cumulative incidences of relapse and non-relapse mortality. Pre-transplant minimal residual disease was associated with worse leukemia-free survival (hazard ratio=2.76 [1.90-4.00]), overall survival (hazard ratio=2.36 [1.73-3.22]), and cumulative incidence of relapse (hazard ratio=3.65 [2.53-5.27]), but not non-relapse mortality (hazard ratio=1.12 [0.81-1.55]). These associations held regardless of detection method, conditioning intensity, and patient age. Adverse cytogenetics was not an independent risk factor for death or relapse. There was more heterogeneity among studies using flow cytometry-based than WT1 polymerase chain reaction-based detection (I2=75.1% vs. <0.1% for leukemia-free survival, 67.8% vs. <0.1% for overall survival, and 22.1% vs. <0.1% for cumulative incidence of relapse). These results demonstrate a strong relationship between pre-transplant minimal residual disease and post-transplant relapse and survival. Outcome heterogeneity among studies using flow-based methods may underscore site-specific methodological differences or differences in test performance and interpretation.

History of consolidation is prognostic in acute myeloid leukemia patients undergoing allogeneic hematopoietic cell transplantation in minimal residual disease-negative first complete remission.

BACKGROUND: Prognostic factors among acute myeloid leukemia (AML) patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) in minimal residual disease (MRD)-negative first complete remission (CR1) are unknown. We retrospectively attempted to answer the following question: In AML patients undergoing allo-HCT in MRD-negative CR1, does a history of prior consolidation provide additional prognostic information? METHODS: The inclusion criteria were: (i) Age > 18 years, (ii) AML in CR1 after 1-2 cycles of intensive induction chemotherapy, with or without consolidation, (iii) Allo-HCT between 1/2003 and 4/2016 at our institution, (iv) Available standard-sensitivity 4-color flow cytometry results from a bone marrow aspiration at diagnosis and after completion of all previous chemotherapy within one month prior to HCT, (v) Flow cytometry-based MRD-negative status at the time of HCT. RESULTS: A history of prior consolidation was associated with favorable overall survival (Hazard Ratio [95% Confidence Interval]: 0.59 [0.35-0.99], P = .046), relapse-free survival (0.60 [0.37-0.96], P = .036), and relapse (0.50 [0.27-0.92], P = .025). Analysis of potential sources of bias was unrevealing. CONCLUSIONS: In AML patients undergoing allo-HCT in MRD-negative CR1, a history of prior consolidation was associated with favorable outcomes. If the path to pre-HCT MRD negativity includes consolidation, it may identify patients with improved prognosis following HCT in MRD-negative state. These results warrant validation in larger cohorts.

Myeloablative, but not Reduced-Intensity, Conditioning Overcomes the Negative Effect of Flow-Cytometric Evidence of Leukemia in Acute Myeloid Leukemia.

Stringent complete remission (CR) in acute myeloid leukemia (AML) requires the absence of both morphologic and flow cytometric evidence of disease. We have previously shown that persistent AML detected by flow cytometry (FC+) before reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (alloHCT) was associated with significantly increased relapse, shorter disease-free survival (DFS), and poorer overall survival (OS), independent of morphologic blast count. We evaluated the effect of FC status on outcomes of alloHCT for AML after either myeloablative conditioning (MAC) or RIC regimens in 203 patients (MAC, n = 80, and RIC, n = 123) with no morphologic evidence of persistent AML pretransplant on marrow biopsy. The allografts included 130 umbilical cord blood (UCB) and 73 sibling donors. We performed central review of pretransplant standard sensitivity FC to identify detectable FC+. Twenty-five patients were FC+, including 15 (18.7%) receiving MAC and 10 (8.1%) RIC alloHCT. Among RIC patients FC+ was associated with significantly inferior relapse, DFS, and OS (multiple regression HR, 3.8; 95% CI, 1.7 to 8.7; P < .01 for relapse; HR, 2.9; 95% CI, 1.4 to 5.9; P < .01 for DFS; and HR, 3.4; 95% CI, 1.7 to 7; P < .01 for OS). In contrast, FC+ status was not associated with relapse or decreased OS after MAC. These data suggest that MAC, but not RIC, overcomes the negative effect of pretransplant FC+ after sibling or UCB alloHCT. Therefore, a deeper pretransplant leukemia-free state is preferred for those treated with RIC.

Parenchymal infiltration and lymphoma-associated membranoproliferative pattern of glomerular injury: an unusual presentation of mantle cell lymphoma.

Lymphomatous processes have been shown to involve the kidney by direct and paraneoplastic mechanisms. Direct injury can manifest by effacement of typical parenchymal architecture by the lymphomatous infiltrate, and indirect, paraneoplastic mechanisms have been associated with a variety of glomerular lesions. Mantle cell lymphoma (MCL) has rarely been reported to be associated with both direct infiltration and/ or paraneoplastic glomerular lesions. We describe a patient with rapidly progressive glomerulonephritis whose renal biopsy showed effacement of the renal parenchyma by MCL and a membranoproliferative pattern of glomerular injury. The patient's bone marrow was also involved by MCL, and serology revealed small M-spikes and a positive rheumatoid factor. The clinicopathologic findings were consistent with a membranoproliferative pattern of glomerular injury secondary to MCL with infiltrative destruction of renal parenchyma. This case is unusual in that MCL was diagnosed on renal biopsy, that there was a two-pronged mechanism of renal injury, and that there were two separate monoclonal immunoglobulins elaborated by the lymphoma that could be associated with the glomerular injury. Although it is uncommon to make an initial diagnosis of lymphoma from a renal biopsy, it should be recognized that patients with lymphoma might develop clinically significant renal sequelae secondary to both direct and indirect mechanisms of lymphoma-mediated nephropathy.

Enhancing hematopathology peripheral blood smear education through asynchronous video material: A pilot report.

This study explores the effectiveness of asynchronous video material as a supplementary educational tool for trainees in hematopathology. Traditional pedagogical methods often rely heavily on faculty involvement, potentially limiting the breadth of information trainees receive due to constraints in faculty time and the variety of cases covered in a limited time interval/rotation. Asynchronous video-based learning presents a potential solution to these challenges. This concept has been utilized effectively in various fields of medical education. In this study, we describe our implementation of an educational program utilizing asynchronous video material to supplement traditional learning methods for peripheral blood smear interpretation for learners on a hematopathology clerkship. Following a pre-test/post-test assessment with 13 trainees, we analyzed the correlation between video viewing percentage and changes in test scores. The results indicate an improvement in test scores following exposure to video content, supporting the positive impact of asynchronous video material on hematopathology education. Trainees had positive feedback regarding this new educational tool. This study suggests that such self-directed learning could enhance traditional teaching methods, ensuring broader and more consistent coverage of hematopathology concepts.

Application and pitfalls of immunophenotyping in challenging plasma cell neoplasms: a case series.

Multiple myeloma (MM) is an incurable malignant plasma cell neoplasm, representing the second most common hematopoietic cancer. As plasma cell neoplasms are clonal and often secrete a monoclonal protein (M-spike), laboratory diagnosis is usually straightforward, especially when ancillary studies such as immunohistochemistry, flow cytometry, and protein electrophoresis are available in addition to microscopic examination. Despite the repertoire of diagnostic tools, rare cases pose diagnostic dilemmas, especially when reagent antibodies do not react as expected, extent of disease is patchy, or when disease occurs in unique age groups. In this retrospective study, we report a series of challenging diagnostic cases, discussing aberrant findings and comparing them to more classic counterparts. Twelve cases collected during routine clinical sign-out were reanalyzed and include examples of MGUS, classic multiple myeloma, t(11;14) rearranged myeloma, minimal residual disease, AA and AL amyloidosis, truncated light chain, non-secretory and non-producer myeloma, biphenotypic myeloma, oligoclonal expansion after bone marrow transplant, and plasma cell leukemia in a young adult. This cohort showcases the diversity of atypical presentations of plasma cell neoplasms, and we highlight standardized approaches to workup to avoid diagnostic pitfalls.

Early Life Stress Is Associated with Alterations in Lymphocyte Subsets Independent of Increased Inflammation in Adolescents.

Early life stress (ELS) is linked to an elevated risk of poor health and early mortality, with emerging evidence pointing to the pivotal role of the immune system in long-term health outcomes. While recent research has focused on the impact of ELS on inflammation, this study examined the impact of ELS on immune function, including CMV seropositivity, inflammatory cytokines, and lymphocyte cell subsets in an adolescent cohort. This study used data from the Early Life Stress and Cardiometabolic Health in Adolescence Study (N = 191, aged 12 to 21 years, N = 95 exposed to ELS). We employed multiple regression to investigate the association between ELS, characterized by early institutional care, cytomegalovirus (CMV) seropositivity (determined by chemiluminescent immunoassay), inflammation (CRP, IL-6, and TNF-a determined by ELISA), and twenty-one immune cell subsets characterized by flow cytometry (sixteen T cell subsets and five B cell subsets). Results reveal a significant association between ELS and lymphocytes that was independent of the association between ELS and inflammation: ELS was associated with increased effector memory helper T cells, effector memory cytotoxic T cells, senescent T cells, senescent B cells, and IgD- memory B cells compared to non-adopted youth. ELS was also associated with reduced percentages of helper T cells and naive cytotoxic T cells. Exploratory analyses found that the association between ELS and fewer helper T cells and increased cytotoxic T cells remained even in cytomegalovirus (CMV) seronegative youth. These findings suggest that ELS is associated with cell subsets that are linked to early mortality risk in older populations and markers of replicative senescence, separate from inflammation, in adolescents.

Digital and manual interfollicular Ki-67 are associated with a progression-free survival in patients with low-grade follicular lymphoma.

OBJECTIVES: Novel histopathologic prognostic factors are needed to identify patients with follicular lymphoma (FL) at risk of inferior outcomes. Our primary objective was to evaluate the Ki-67 proliferative index in follicular and interfollicular areas in tissue biopsy specimens from patients with newly diagnosed FL and correlate with clinical outcomes. Our secondary objective was to correlate PD-L1 and LAG-3 with clinical outcomes. METHODS: Seventy cases of low-grade FL from the University of Minnesota were evaluated with Ki-67 immunohistochemical stain. Ki-67 expression as a continuous variable was interpreted digitally and manually in follicular and interfollicular areas. Progression-free survival (PFS) and overall survival (OS) were analyzed by Cox regression, and hazard ratios (HRs) per 10-point increase in Ki-67 were calculated. RESULTS: Progression-free survival at 4 years was 28% (95% CI, 19%-41%). Interfollicular, but not follicular, Ki-67 was associated with PFS by manual (HR, 1.33; P = .01) and digital (HR, 1.38; P = .02) analysis. Digital and manual Ki-67 were only moderately correlated but demonstrated similar effects on PFS. At 4 years, OS was 90% with no association with follicular or interfollicular Ki-67 proliferation. CONCLUSIONS: Higher interfollicular Ki-67 by either digital or manual analysis is associated with a poorer PFS in patients with low-grade FL. These results suggest further validation of this marker is warranted to improve pathologic risk stratification at FL diagnosis. PD-L1 and LAG-3 were not associated with PFS or OS.

Perigestational dietary folic acid deficiency protects against medulloblastoma formation in a mouse model of nevoid basal cell carcinoma syndrome.

Hereditary nevoid basal cell carcinoma syndrome (NBCCS) is caused by PTCH1 gene mutations that result in diverse neoplasms including medulloblastoma (MB). Epidemiological studies report reduced pediatric brain tumor risks associated with maternal intake of prenatal vitamins containing folic acid (FA) and FA supplements specifically. We hypothesized that low maternal FA intake during the perigestational period would increase MB incidence in a transgenic NBCCS mouse model, which carries an autosomal dominant mutation in the Ptch1 gene. Female wild-type C57BL/6 mice (n = 126) were randomized to 1 of 3 diets with differing FA amounts: 0.3 mg/kg (low), 2.0 mg/kg (control), and 8.0 mg/kg (high) 1 mo prior to mating with Ptch1 (+/-) C57BL/6 males. Females were maintained on the diet until pup weaning; the pups were then aged for tumor development. Compared to the control group, offspring MB incidence was significantly lower in the low FA group (Hazard Ratio = 0.47; 95% confidence interval 0.27-0.80) at 1 yr. No significant difference in incidence was observed between the control and high FA groups. Low maternal perigestational FA levels may decrease MB incidence in mice genetically predisposed to tumor development. Our results could have implications for prenatal FA intake recommendations in the presence of cancer syndromes.

Reporting and Establishment of Reference Intervals for Antiphospholipid Antibody Immunoassays: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

CONTEXT.­: Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. OBJECTIVE.­: To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (anti-ß2GPI) assays. DESIGN.­: Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACL)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. RESULTS.­: The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-ß2GPI (n = 354), and IgM anti-ß2GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-ß2GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. CONCLUSIONS.­: ELISA remains the most used method for detecting aPL antibodies with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable.

Perinatal folate levels do not influence tumor latency or multiplicity in a model of NF1 associated plexiform-like neurofibromas.

OBJECTIVE: In epidemiological and experimental research, high folic acid intake has been demonstrated to accelerate tumor development among populations with genetic and/or molecular susceptibility to cancer. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder predisposing affected individuals to tumorigenesis, including benign plexiform neurofibromas; however, understanding of factors associated with tumor risk in NF1 patients is limited. Therefore, we investigated whether pregestational folic acid intake modified plexiform-like peripheral nerve sheath tumor risk in a transgenic NF1 murine model. RESULTS: We observed no significant differences in overall survival according to folate group. Relative to controls (180 days), median survival did not statistically differ in deficient (174 days, P = 0.56) or supplemented (177 days, P = 0.13) folate groups. Dietary folate intake was positively associated with RBC folate levels at weaning, (P = 0.023, 0.0096, and 0.0006 for deficient vs. control, control vs. supplemented, and deficient vs. supplemented groups, respectively). Dorsal root ganglia (DRG), brachial plexi, and sciatic nerves were assessed according to folate group. Mice in the folate deficient group had significantly more enlarged DRG relative to controls (P = 0.044), but no other groups statistically differed. No significant differences for brachial plexi or sciatic nerve enlargement were observed according to folate status.

Transposon delivery for CRISPR-based loss-of-function screen in mice identifies NF2 as a cooperating gene involved with the canonical WNT signaling molecular class of hepatocellular carcinoma.

Various molecular subclasses of hepatocellular carcinoma (HCC) exists, with many novel cooperating oncogenes and tumor suppressor genes involved in its tumorigenesis. The emerging importance of WNT signaling in HCC has been established. However, the intricate genetic mechanisms involved in this complex signaling pathway remains to be elucidated. Importantly, while some cooperating genes have been identified, there are still many unknown genes associated with catenin beta 1 (CTNNB1)-induced HCC. Mutations in both oncogenes and tumor suppressor genes are required for HCC tumorigenesis. The emergence of the CRISPR/Cas9 system has allowed researchers now to target both alleles efficiently. In this novel study, the Sleeping Beauty transposon system was used as a gene delivery system in vivo to stably integrate an expression cassette that carry pools of gRNAs and overexpress a mutant version of CTNNB1 into the hepatocyte genome. We identified 206 candidate genes that drive HCC tumorigenesis in the context of WNT signaling activation and, neurofibromin 2 (NF2) gene, a known tumor suppressor gene with clinical relevance was validated in this proof-of-principle study.

PCNEO, a New Proficiency Testing Program for Flow Cytometric Analysis of Plasma Cell Neoplasms From the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee.

CONTEXT.­: In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program-PCNEO-to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. OBJECTIVE.­: To report the results from the first 4 years of the PCNEO program. DESIGN.­: Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. RESULTS.­: A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells), or the presence of nonneoplastic plasma cell populations (correctly identified by 91%-96% of participants). CONCLUSIONS.­: Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations, and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.