Bone Marrow Biopsy Specimens Research Articles

#2672 Cryoglobulin-Induced membranoproliferative glomerulonephritis in a HEV-infected kidney transplant recipient: outcomes post ribavirin failure

Abstract Background and Aims Hepatitis E is a zoonosis caused by hepatitis E virus (HEV), which was first discovered 40 years ago. It is the most prevalent hepatitis virus worldwide. Most hepatitis E cases are self-limiting acute hepatitis, but the virus has been recognized to cause chronic hepatitis. Following the first case report of chronic hepatitis E (CHE) in a transplant recipient, CHE has recently been identified as associated with chronic liver damage induced in immunocompromised patients such as transplant recipients. CHE can induce extra-hepatic manifestations, such as immune-mediated thrombocytopenia, neurological symptoms, and kidney injuries. Few cases of glomerulonephritis (GN) and cryoglobulinemia in transplant patients have been related with CHE, with a good progression thanks to viral control with ribavirin introduction. There have been no reported cases in the literature of good progress in renal function in renal transplant patients despite failure of treatment with Ribavirin. Method We report here what is to our knowledge, the first documented case of HEV-induced de novo cryoglobulinemic membranoproliferative glomerulonephritis in a renal transplant patient with a ten-year history of CHE infection unsuccessfully treated with ribavirin. Results A 69-year-old man underwent a kidney transplant for diabetes type 1. 7 years after transplantation, he was diagnosed with a chronic genotype 3.1H-HEV infection. He was treated by Ribavirin, but it did not allow to eradicate HEV but a biochemical response. At 18 years post transplant, he presented with asthenia, abdominal pains and increased liver enzyme levels: liver tests were disrupted with cytolysis and cholestasis. He presented also a nephritic syndrome with high blood pressure, oedemas, increased serum creatinine level, proteinuria with hypoalbuminemia and de novo microscopic hematuria. Biological analysis revealed presence of type 2 cryoglobulinemia with monoclonal IgM Kappa (2285 mg/L), and polyclonal immunoglobulins association. Rheumatoid factor was increased (573 UI/ml) and C4 level was reduced (0.04 g/L). The concentration of HEV RNA in the serum was 750 000 UI/mL. Viral serologies were performed for HBV, HCV, HAV, HIV, EBV, CMV. Hematologic diseases were ruled out by analyzing a bone marrow biopsy specimen. Histologic analysis of a kidney biopsy specimen revealed diffuse endocapillary proliferation, diffuse mesangial proliferation and focal glomerular basal membrane duplication. One arteriolar section showed a focal lesion of vasculitis (Fig. 1). There was no argument for allograft rejection. Immunofluorescence microscopy revealed mesangial and capillary wall deposits of IgM, IgG, C3 and C1q, polytypic for k and l light chains. We suspected cryoglobulinemic membranoproliferative glomerulonephritis induced by VHE. The initial treatment plan involved 10 sessions of plasmapheresis and steroid pulses (500 mg/d for 3 days), alongside a course of rituximab (1 g/week for 4 weeks). However, the patient experienced intolerance to rituximab, prompting a switch to Obinutuzumab. Subsequently, the anti-HEV therapy was modified, and the patient was placed on a course of Sofosbuvir. Consequently, three months after treatment kidney function kidney improved with return to baseline and cryoglobulinemia became undetectable. However, proteinuria remained impaired and VHE RNA was still present. Conclusion Cryoglobulinemia is rarely associated with glomerular disease in HEV infection, and cases currently reported in the literature showed a favorable outcome with ribavirin. This case is the first to display a chronic HEV infection with cryoglobulin-induced glomerulonephritis with global improvement despite unsuccessful treatment with ribavirin. Although the difficulty of managing immunity in the treatment of cryoglobulinemia by immunodepleting treatment, with the risk of increased viral replication in the absence of an effective antiviral, it seems interesting to offer a first-line treatment of cryoglobulinemia that appears to improve renal function despite continued viral replication. These results need to be confirmed in further studies.

The unnecessary use of short tandem repeat testing on bone marrow samples in patients after 1 year following allogeneic hematopoietic stem cell transplant.

To determine whether the information provided by short tandem repeat (STR) testing and bone marrow (BM) biopsy specimens following hematopoietic stem cell transplant (HSCT) provides redundant information, leading to test overutilization, without additional clinical benefit. Cases with synchronous STR and flow cytometric immunophenotyping (FCI) testing, as part of the BM evaluation, were assessed for STR/FCI concordance. Of 1199 cases (410 patients), we found the overall concordance between STR and FCI was 93%, with most cases (1063) classified as STR-/FCI-. Of all discordant cases, 75 (6%) were STR+/FCI-, with only 5 (6.7%) cases best explained as identification of disease relapse. Eight cases were STR-/FCI+, representing relapsed/residual disease. Analysis of cases 1 year or more from transplant (54% of all cases) indicated only 9 (1.5%) were STR+/FCI-, and none uniquely identified relapse. These data suggest that STR analysis performed 1 year or more post-HSCT does not identify unknown cases of relapse. Furthermore, while STR testing is critical for identifying graft failure/rejection within the first year posttransplant, FCI appears superior to STR at detecting late relapses with low-level disease. Therefore, STR testing from patients 1 year or more post-HSCT may be unnecessary, as BM biopsy evaluation is sufficient to identify disease relapse.

Role of BACH1 in multiple myeloma

ABSTRACT Objective Examine Bach1 protein expression in bone marrow biopsy specimens obtained from newly diagnosed multiple myeloma (NDMM) and iron deficiency anemia (IDA) patients. Conduct a thorough analysis to explore the potential connection between Bach1 and the onset as well as treatment response of NDMM. Methods This study investigated Bach1 expression in bone marrow biopsy tissues from NDMM and IDA patients. Immunohistochemical staining and Image-pro Plus software were utilized for quantitatively obtaining the expression level of Bach1 protein. Arrange Bach1 expression levels from high to low, and use its median expression level as the threshold. Samples with Bach1 expression level above the median are categorized as the high-expression group, while those below the median are categorized as the low-expression group. Under this grouping, a detailed discussion was conducted to explore relationship of the Bach1 expression level with the patients’ gender, ISS stage, and survival rate based on the Bortezomib (Btz) therapy. Results Our experiment indicates that the expression level of Bach1 in NDMM patients is significantly higher than in IDA patients. Furthermore, we discovered that patients in the high-expression group exhibit better prognosis compared to those in the low-expression group after Btz-treatment. Bioinformatics analysis further confirms this conclusion. Conclusion By categorizing Bach1 expression level as high and low, our study offers a unique perspective on understanding the relationship between Bach1 and NDMM.

P53 immunohistochemistry as an ancillary tool for rapid assessment of residual disease in TP53-mutated acute myeloid leukemia and myelodysplastic syndromes.

Measurable residual disease flow cytometry (MRD-FC) and molecular studies are the most sensitive methods for detecting residual malignant populations after therapy for TP53-mutated acute myeloid leukemia and myelodysplastic neoplasms (TP53+ AML/MDS). However, their sensitivity is limited in suboptimal aspirates or when the immunophenotype of the neoplastic blasts overlaps with erythroids or normal maturing myeloid cells. In this study, we set out to determine if p53 immunohistochemistry (IHC) correlates with MRD-FC and next-generation sequencing (NGS) in the posttherapy setting and to determine the utility of p53 IHC to detect residual disease in the setting of negative or equivocal MRD-FC. We retrospectively identified 28 pre- and posttherapy bone marrow biopsy specimens from 9 patients with TP53+ AML/MDS and a p53 overexpressor phenotype by IHC (strong 3+ staining at initial diagnosis). Next-generation sequencing and/or MRD-FC results were collected for each specimen. Using a threshold of more than ten 2-3+ cells in any one 400× field, p53 IHC detected residual disease with a sensitivity of 94% and a specificity of 89%. The threshold used in this study showed a high degree of concordance among 6 blinded pathologists (Fleiss κ = 0.97). Our study suggests that p53 IHC can be used as a rapid tool (within 24 hours) to aid in the detection of residual disease that may complement MRD-FC or NGS in cases in which the flow cytometry immunophenotype is equivocal and/or the bone marrow aspirate is suboptimal.

Comprehensive morphologic characterization of bone marrow biopsy findings in a large cohort of patients with VEXAS syndrome: A single-institution longitudinal study of 111 bone marrow samples from 52 patients.

VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context.

Correlation analysis of bone marrow microvessel density and miRNA expression on drug resistance in patients with chronic myelogenous leukemia after tyrosine kinase inhibitor treatment

ABSTRACT Objective: This study analyzed the relationship between bone marrow microvessel density (MVD) and the expression of four miRNAs with chronic myelogenous leukemia (CML) resistance after tyrosine kinase inhibitor (TKI) treatment. Methods: 234 CML patients were divided into resistance and non-resistance groups in terms of the results of the 5-year follow-up. Patients were divided into the Optimum response group and the Warning/Failure group based on TKI response. MVD was determined by immunohistochemistry, and the expression levels of four miRNAs (miR-106a, miR-155, miR-146a, and miR-340) in bone marrow biopsy specimens were examined by qPCR. We evaluated the association of MVD with four miRNAs and them predictive value for CML resistance after TKI treatment. Results: The MVD and the levels of miR-106a, miR-155, and miR-146a were significantly higher while the miR-340 level was lower in the resistance group than the non-resistance group. Besides, MVD had a significant correlation with the levels of miR-340 and miR-155. According to the results of survival analysis, MVD as well as miR-340 and miR-155 levels were observably correlated with 5-year survival of patients without TKI resistance. The results of the ROC curve indicated that the MVD, miR-106a, miR-340, and miR-155 had good predictive accuracy for CML resistance after TKI treatment. As for the results of multivariate analysis, disease stage, risk level (high risk), high MVD, low miR-340 expression, and high miR-155 expression were all independent risk factors for CML resistance. Conclusion: MVD and the expression of miR-340 and miR-155 are closely associated with CML resistance after TKI treatment.

Erythroid variant evolving from chronic myeloid leukemia resistant to multiple tyrosine kinase inhibitors: a rare case report

BackgroundChronic myeloid leukemia (CML) is characterized by the presence of BCR::ABL1 fusion gene resulting from a reciprocal translocation, t(9;22)(q34;q11.2), leading to prominent granulocytic proliferation. The majority of patients initially present in chronic phase (CP), which may progress to advanced CML with predominantly granulocytic phenotypes in the absence of proper treatment or response to tyrosine kinase inhibitors (TKIs). We present an exceptionally rare case in which an erythroid variant emerged from a CML patient resistant to multiple TKIs. This variant is characterized by the detection of t(9;22) BCR::ABL1 fusion in erythroid precursors at various maturation stages and the absence of granulocytic progenitor hyperplasia typically seen in classical CML.Case presentationA 33-year-old female with CP-CML had received multiple TKI therapies since her initial diagnosis in 2015. Due to intolerable side effects and inconsistent adherence, she exhibited an inadequate response and developed new-onset pancytopenia. Bone marrow (BM) biopsy specimen revealed a hypercellular marrow with significant erythroid hyperplasia (90% of marrow cellularity) and a reversed myeloid-to-erythroid (M: E) ratio of 1:10. Both erythroid and myeloid cells displayed progressive maturation without dysplasia or excess blasts. Chromosomal analysis identified t(9;22) (q34;q11.2) in 19 out of 20 metaphase cells. BCR::ABL1 fusion transcript (p210 isoform) was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Notably, no additional pathogenic cytogenetic abnormalities or ABL1 kinase domain mutations were detected. Here, we report the first published case of an erythroid variant emerging in a CML patient resistant to multiple TKIs—a distinct entity from the erythroid blast crisis evolving from CML.ConclusionThe erythroid variant of CML is distinguished by the presence of t(9;22) (q34;q11.2) BCR::ABL1 in predominant erythroid precursors at different stages of maturation. In a myeloid neoplasm showing predominant erythroid hyperplasia without typical CML features, it is vital to correlate morphology and t(9;22) BCR::ABL1 cytogenetic testing for accurate diagnosis, and to prevent confusion with PEL transformation in CML.

Late transplant-associated thrombotic microangiopathy verified in bone marrow biopsy specimens is associated with chronic GVHD and viral infections.

To describe late transplant-associated thrombotic microangiopathy (TA-TMA) as chronic endothelial complication in bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (HSCT). BM specimens along with conventional diagnostic parameters were assessed in 14 single-institutional patients with late TA-TMA (more than 100 days after HCST), including 11 late with history of early TA-TMA, 10 with early TA-TMA (within 100 days), and 12 non TA-TMA patients. Three non-HSCT patients served as control. The time points of BM biopsy were +1086, +798, +396, and +363 days after HSCT, respectively. Late TA-TMA patients showed an increase of CD34+ and von Willebrand Factor (VWF)+ microvascular endothelial cells with atypical VWF+ conglomerates forming thickened VWF+ plaque sinus in the BM compared to patients without late TA-TMA and non-HSCT. Severe chronic (p = .002), steroid-refractory GVHD (p = .007) and reactivation of HHV6 (p = .002), EBV (p = .003), and adenovirus (p = .005) were pronounced in late TA-TMA. Overall and relapse-free survival were shorter in late TA-TMA than in patients without late TA-TMA (5-year OS and RFS: 78.6% vs. 90.2%, 71.4% vs. 86.4%, respectively). Chronic allo-immune microangiopathy in BM associated with chronic, steroid-refractory GVHD and/or viral infections are key findings of late, high-risk TA-TMA, which deserves clinical attention.

The Role of FDG- PET/CT in Detecting Bone Marrow Involvement in Childhood Solid Tumors.

To compare the results of 18F-Fluorodeoxy positron emission tomography/computed tomography (18 F-FDG-PET/CT) and bone marrow biopsy (BMB) procedures in the initial evaluation of bone marrow involvement (BMI) in pediatric solid tumors. We conducted a retrospective analysis of newly diagnosed pediatric cases with lymphoma, neuroblastoma, Ewing sarcoma, rhabdomyosarcoma. Each case underwent both PET-CT imaging and BMB. Presence of tumor infiltration in BMB specimens and/or positive FDG-PET/CT findings indicate as BMI were regarded as true positive results. Sixty-four patients were included in the study. BMI was detected in 23/64 (36%) patients, FDG-PET/CT imaging and BMB results were concordant in 54/64 patients. In 9/64 patients the finding was FDG-PET/CT (+), BMB (-) indicating a false negative BMB result. In only 1/64 patients FDG- PET/CT (-), BMB (+), indicating a false negative FDG-PET/CT result. In the whole patient group, the sensitivity, specificity, positive predictive value and negative predictive value of PET/CT and BMB in detecting bone marrow involvement were 95.6%, 100%, 100% and 97.6% and 60.8 %, 100%, 100% and 82%, respectively. PET/CT has a high sensitivity and specificity for the assessing marrow involvement in pediatric solid tumors. We believe that PET/CT imaging should be performed as the first step in diagnostic staging, and BMB may not be necessary in every patient, only in patients with suspicious PET/CT results for bone marrow involvement. Additionally, for a more precise determination of bone marrow involvement, it is reasonable to perform BMB from FDG-retaining areas, using PET/CT as a guide tool.

Phase II Study Assessing Safety and Preliminary Efficacy of High Dose Intravenous Ascorbic Acid in Patients with TET2 Mutant Clonal Cytopenias

Background: Clonal cytopenia(s) of undetermined significance ( CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm ( MN)-associated somatic mutations ( MT) in hematopoietic stem cells. Patients ( PTs) with TET2MT CCUS have a high probability of progression to MN. To date, no FDA-approved therapies exist for CCUS, and PTs often have similar cytopenias/transfusion needs as those with myelodysplastic syndromes ( MDS). TET2 is an ascorbic acid (AA)-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine ( 5mC) to 5-hydroxymethylcytosine ( 5hmC), providing rationale to study high-dose IV (HI)-AA in TET2MT CCUS. Here we report the final results of this pilot trial. Methods: This is an investigator-initiated, single-institutional, phase II trial assessing the safety and efficacy of HI-AA in PTs with TET2MT CCUS (NCT03418038). PTs≥ 18 years with ≥1 TET2MT with or without additional somatic MTs, without prior treatment, and with any of the following laboratory criteria: (1) hemoglobin ( Hb) ≤10g/dL, (2) absolute neutrophil count ( ANC) ≤1(10^9/L), (3) platelet count ( PLT)≤100 (10^9/L) were eligible. HI-AA (1g/kg, maximum 100g) was given 3 times weekly for 12 weeks. The primary endpoint was hematologic response rates determined by MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement ( HI)- erythropoietic ( HI-E), platelets ( HI-P), neutrophils ( HI-N). Secondary endpoints include safety and adverse events ( AEs), graded by NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction ( VAF), in vitro colony formation and differentiation, global and sequence-specific DNA methylation/hydroxymethylation, and quantitative 5hmC estimated by IHC in bone marrow biopsy specimens. Results: Ten patients were enrolled, with a median age of 71.4 (range: 65, 79) years, 8 (80%) males. The median number of mutations was 3, with 9 (90%) having co-mutations. All PTs but one [-Y] had normal karyotype. Baseline Hb levels were normal in all but one who was red blood transfusion dependent. Five (50%) PTs were thrombocytopenic, and 5 (50%) PTs had ANC<1 (10^9/L). ( Table). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (40%) and polydipsia (40%, 3 Grade 1, 1 Grade 2). One (10%) PT had constipation and headaches (both Grade 1), and 1 (10%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs' or deaths were reported. The median follow-up duration was 16.8 months (range: 9.4, 24.1). Overall, no PTs met the criteria for HI. There were no significant differences with regards to median Hb, PLT, and ANC values at baseline, week 20, and 1-year (median Hb: 13.3, 12.5, and 12.7 g/dL, p=0.93; PLT: 97 vs. 120 vs. 92.5X10^9/L, p>0.99, and ANC: 0.9 vs. 1.2 vs. 1.3X10^9/L, p=0.99). By CTCAE grading criteria for cytopenias, 3 (30%) PTs had an improvement in severity of cytopenias, while 2 had worsening. One (10%) PT had an improvement in PLT (baseline vs. week 20: 62 vs 192X10^9/L) but was morphologically deemed to meet criteria for CMML-1 at week 20. Four (40%) PTs met criteria for disease progression (PT_4, 7,8,10); CMML-1- 2 and MDS-2. The median time to progression was 5 months (95% CI: 4.9, unreached). Two PTs had mutational clonal evolution, with 1 (PT_8) acquiring a RUNX1 (VAF: 20%) and 1 (PT_9) acquiring 2 CBL mutations (both VAF:4%) at one-year follow-up. There were no significant differences regarding the clinical characteristics or the TET2 variants between PTs who had stable disease vs. progression. There were no significant differences in TET2 VAF at baseline, week 20, and 1-year (median: 39% vs. 43% vs. 42%, p=0.54). We profiled changes in 5-mC and 5-hmC (Ilumina EpicArray) in PTs pre- and post-IV AA treatment and did not observe global changes. However, we were able to highlight site-specific changes in differentially methylated regions, largely resulting in hypomethylation at enhancer sites and affecting actively transcribed states in PTs with an improvement in cytopenia(s) severity by CTACE criteria ( Figure). Conclusion: We report the final safety and efficacy analysis of HI-AA in PTs with TET2 MT CCUS. The trial data suggest that HI-AA is safe and well tolerated. Although there were no significant responses by IWG MDS criteria, alleviation in severity of cytopenias in a subset of PTs (n=3) along with correlative epigenetic changes in enhancer regions, needs further exploration.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Clinical Utility and Reimbursement of Next-Generation Sequencing–Based Testing for Myeloid Malignancies

Next-generation sequencing is becoming increasingly important for the diagnosis, risk stratification, and management of patients with established or suspected myeloid malignancies. These tests are being incorporated into clinical practice guidelines and many genetic alterations now constitute disease classification criteria. However, the reimbursement for these tests is uncertain. This study analyzed the clinical impact, ordering practices, prior authorization, and reimbursement outcomes of 505 samples from 477 patients sequenced with a 50-gene myeloid next-generation sequencing panel or a 15-gene myeloproliferative neoplasm subpanel. Overall, 98% (496 of 505) of tests provided clinically useful data. Eighty-nine percent of test results, including negative findings, informed or clarified potential diagnoses, 94% of results informed potential prognoses, and 19% of tests identified a potential therapeutic target. Sequencing results helped risk-stratify patients whose bone marrow biopsy specimens were inconclusive for dysplasia, monitor genetic evolution associated with disease progression, and delineate patients with mutation-defined diagnoses. Despite the clinical value, prior authorization from commercial payors or managed government payors was approved for less than half (45%) of requests. Only 51% of all cases were reimbursed, with lack of medical necessity frequently cited as a reason for denial. This study demonstrates the existence of a substantial gap between clinical utility and payor policies on test reimbursement.

Efficacy and Safety of BCMA-Specific CAR T Cell-Based Therapy in Relapsed/Refractory Multiple Myeloma Patients with Extramedullary Disease

Background Extramedullary disease (EMD) is an aggressive subentity of multiple myeloma (MM), characterized by the ability of a subclone to grow independent of the bone marrow microenvironment, resulting in a high-risk state associated with increased proliferation, evasion of immune surveillance and treatment resistance. Relapsed/refractory multiple myeloma (R/R MM) patients with EMD have unfavorable prognoses and no effective treatment options. B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown promising efficacy in R/R MM; however, data on patients with EMD remain limited. We aimed to assess the efficacy and safety of CAR T-cell therapy in R/R MM patients with EMD. Methods We conducted a retrospective multi-institutional study of 55 R/R MM patients confirmed with EMD at referral. The definition of EMD included (1) soft tissue masses in extraosseous locations resulting from hematogenous spread (EM-E) and (2) bone-related plasmacytomas that extend via disruption of cortical bones into contiguous soft tissues (EM-B). The overall response, long-term outcomes, and safety were assessed. We also characterized differential response between medullary and extramedullary disease, analyzed unique attributes of compartmental toxicity, and explored patterns of relapse in the setting of EMD. Gene expression profiling was performed using nCounter Human CAR-T Characterization Panel (NanoString, #XT-CSO-HCART1-12) on 40 biopsy specimens from patients enrolled in this study, including EMD and bone marrow biopsy specimens at pretreatment or/and at relapse. Results were analyzed based on outcome (no durable response [progression within 6 months, NDR] vs durable response [progression beyond 6 months, DR]). Multiplex immunofluorescence (mIF) was performed for detection of populations and phenotyping of infiltrated CD8 + T cells (Panel A: CD8/PD-1/LAG-3/TIM-3) and macrophages (Panel B: CD68/CD86/CD163) with EMD biopsies. Results The infusion resulted in an overall response rate of 90.9% (95% confidence interval [CI], 80.4-96.1) in medullary disease and 70.9% (95% CI, 57.9-81.2) in EMD ( P = 0.008). Discrepant outcomes between medullary and extramedullary response were observed, with suboptimal and delayed overall response and shortened duration of response achieved in EMD. With a median follow-up of 27.3 months, the median progression-free survival was 8.7 months (95% CI, 3.7-18.8), and the median overall survival was 16.0 months (95% CI, 13.5-20.1). Landmark analysis was conducted to compare OS in patients who progressed versus remained progression-free at the 6-month landmark time point. We demonstrated that progression before 6 months post-infusion is strongly associated with an increased risk of death (HR = 11.15 [95% CI, 3.49 to 35.60]; P < 0.001). Local cytokine release syndrome (CRS) was depicted in 21.8% patients and was moderately associated with the occurrence of systemic CRS (r = 0.322; P = 0.017). 78.2% patients experienced post-infusion progression in EMD, and BCMA + progression constituted the main pattern of EMD progression. At the time of EMD progression, peripheral CAR + cells were detectable, and 50% patients had ongoing documented B cell aplasia. We next interrogated NanoString gene expression data of tumor microenvironment (TME) genes for possible association with clinical response and toxicity. Pretreatment TME enriched for immune checkpoint, and type I interferon signaling were associated negatively with clinical outcomes. We found that the infiltration of type 2 macrophages (CD68 +CD163 +) in pretreatment TME is associated with the density of activated or exhausted CD8 + T cell ( PD-1 +LAG-3 +/-), and is associated with no durable response in EMD. We interrogated TME dynamic changes of patients developing EMD progression between cohorts of DR and NDR. An increase in immune counter-regulatory marker and lower diversity of T-cell receptor (TCR) repertoire, was observed at relapse in cohort of NDR. Discussion Our study shows that anti-BCMA CAR-T therapy has provided apparent therapeutic advantage over the existing drugs in response rate and long-term survival. Compared with medullary disease, BCMA-specific CAR T cell-based therapy showed limited and discrepant efficacy in EMD, perhaps due to the the properties of EMD-specific microenvironment.

Neoplastic Monocyte-Derived Fibrocytes Induce Bone Marrow Fibrosis in Patients with Primary Myelofibrosis

Progressive bone marrow (BM) fibrosis is a defining feature of primary myelofibrosis (PMF). The extent of BM fibrosis has been closely associated with the clinical manifestations of advanced disease, including anemia and thrombocytopenia, rendering it an important prognostic indicator. Mesenchymal stromal cells (MSCs) have long been regarded as the exclusive inducer of BM fibrosis. However, using xenograft and transgenic animal models, we and other investigators demonstrated that neoplastic monocyte-derived fibrocytes independently contribute to the induction of BM fibrosis in PMF. Notably, our data revealed that PMF fibrocytes but not MSCs, carry pro-fibrotic signaling pathways that are absent in healthy individuals' fibrocytes or MSCs. Despite these findings, the precise contribution of BM fibrocytes and MSCs to the induction of BM fibrosis in patients with PMF remains elusive. To determine which of two subsets of collagen-producing cells are the main contributors to the induction of BM fibrosis in PMF, we analyzed BM biopsies of patients with PMF and assessed whether their distribution spatially correlated with areas of fibrosis. We analyzed formalin-fixed paraffin-embedded BM trephine biopsies obtained from previously untreated patients with PMF (n = 85) and age-matched healthy donors (n = 6). Using a 7-plexed fluorescence immunohistochemistry (IHC) in conjunction with hematoxylin/eosin, Masson's trichrome, and Gomorri's silver stains we analyzed those BM biopsy specimens. To ensure comprehensive assessment, we imaged serial whole-tissue sections from each patient at 400x resolution, meticulously aligning and carefully correcting for bone trabeculae and any artifacts. To quantitate the spatial distribution of individual cells, collagen fibers, and reticulin fibers, we developed a set of analytical algorithms based on a U-Net neural network in Visiopharm software. Our analysis revealed a 6-fold higher density of fibrocytes, defined as CD45 +/CD68 +/procollagen type I + cells, in the BM of patients with PMF ( P=0.0003) compared to normal controls. By contrast, the number of MSCs, defined as CD45 -/CD90 +/CD105 + cells, in the PMF BM was not different from the number of MSCs in normal BM. After dividing the area of each BM biopsy into a 100 μm regular hexagonal grid, the observed spatial distribution of fibrocytes strongly and positively correlated with the areas covered by reticulin and collagen fibers ( P<0.0001 in both). Furthermore, spatial analysis showed that increased densities of fibrocytes were associated with an increased number of fibrotic cellular zones in the BM tissue of PMF patients. To investigate the clonality of PMF BM fibrocytes, we conducted DNA in situ hybridization (ISH) analysis, employing two sets of locus-specific probe pairs. The DNA-ISH analysis provided valuable insights, revealing that fibrocytes obtained from PMF patients with trisomy 9 or 20q deletion carried the same corresponding cytogenetic abnormalities, indicating that PMF fibrocytes indeed originate from the neoplastic PMF clone. Because increased peripheral blood monocyte counts, commonly detected in patients with PMF, are associated with a poor prognosis, we sought to determine the potential correlation between BM fibrocyte and circulating monocyte counts. As expected, we found a significant positive correlation between BM fibrocyte counts and the percentage of circulating monocytes ( P=0.001). Moreover, we also found that fibrocyte numbers correlated with the levels of the plasma cytokines pentraxin 3, known to induce the differentiation of monocytes into fibrocytes, and matrix metalloproteinase 2, as assessed by using magnetic immunoassay, suggesting that neoplastic fibrocytes induce BM fibrosis in PMF and that fibrocyte quantification could serve as a valuable indicator of disease severity and prognosis. Taken together, our quantitative imaging analysis of BM biopsy tissue from treatment naïve PMF patients revealed that neoplastic fibrocytes, rather than MSCs, are the main contributors to the induction of BM fibrosis in PMF. Our findings underscore the importance of BM fibrocytes in the pathogenesis of PMF and warrant further investigation to identify novel therapeutic agents that will either inhibit fibrocyte differentiation or block their pro-fibrotic function.

Clinical relevance of cyclooxygenase 2 and vascular endothelial growth factor-A expression in classical Hodgkin lymphoma

Abstract Background Classical Hodgkin lymphoma (cHL) is a clonal lymphoid neoplasm derived from B cells. Cyclooxygenase 2 (COX2) and vascular endothelial growth factor-A (VEGF-A) play major roles in angiogenesis and impact cHL prognosis. Aim To measure COX2 and VEGF-A expression in cHL patients and assess their potential association with other laboratory and clinical parameters. Patients and methods Seventy-six cHL bone marrow (BM) biopsy specimens were histopathologically examined and immunohistochemically stained for COX2 and VEGF-A expression. Correlations between COX2 and VEGF-A expression and clinicopathologic factors were evaluated. Results COX2 and VEGF-A were expressed in 67/76 (88.2%) and 48/76 (63.2%) of BM specimens, respectively. VEGF-A was associated with advanced cHL stage (P=0.044) and BM infiltration confirmed by CD30 positivity (P=0.023). A significant association was found between VEGF-A positivity and mediastinal lymphadenopathy (P=0.049), inguinal lymphadenopathy (P=0.046), and pulmonary nodules (P=0.048). COX2 positivity was significantly associated with cervical lymphadenopathy (P=0.011). A positive association was found between expression of both markers (COX2 and VEGF-A) (P=0.001). Coexpression of COX2 and VEGF-A was associated with disease staging (P=0.016), mediastinal lymphadenopathy (P=0.019), and inguinal lymphadenopathy (P=0.044). Conclusion COX2 and VEGF-A, as major players in angiogenesis, are associated with tumor progression in cHL. These findings support targeting both markers as the potential therapeutic approach in cHL.

The clinicopathologic significance of NPM1 mutation and ability to detect mutated NPM1 by immunohistochemistry in non-AML myeloid neoplasms.

NPM1 mutated non-AML myeloid neoplasms (MN; <20% blasts) are characterized by an aggressive clinical course in a few studies. In this retrospective study, we evaluate the clinicopathologic and immunohistochemical features of non-AML MN patients with NPM1 mutations. We assessed NPM1 mutation by targeted next generation sequencing (NGS). Cytoplasmic NPM1 expression was assessed by immunohistochemistry (IHC) on formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. We evaluated 34 non-AML MN patients with NPM1 mutations comprising MDS (22), MPN (3) and MDS/MPN (9). They commonly presented with anemia (88%), thrombocytopenia (58%) and leukopenia (50%). Bone marrow dysplasia was common (79%). The karyotype was often normal (64%). NGS for MN-associated mutations performed in a subset of the patients showed a median of 3 mutations. NPM1 mutations were more often missense (c.859C > T p. L287F; 65%) than frameshift insertion/duplication (35%) with median variant allele frequency (VAF; 9.7%, range 5.1%-49.8%). Mutated NPM1 by IHC showed cytoplasmic positivity in 48% and positivity was associated with higher VAF. The median overall survival (OS) in this cohort was 70 months. Nine patients (26%) progressed to AML. OS in patients who progressed to AML was significantly shorter than the one of patients without progression to AML (OS 20 vs. 128 months, respectively, log rank p = 0.05). NPM1 mutated non-AML MN patients commonly had cytopenias, dysplasia, normal karyotype, mutations in multiple genes, and an unfavorable clinical outcome, including progression to AML. Our data demonstrated that IHC for NPM1 can be a useful supplementary tool to predict NPM1 mutation in some non-AML MN; however, genetic testing cannot be replaced by IHC assessment.

Bone marrow microenvironment disruption and sustained inflammation with prolonged haematologic toxicity after CAR T-cell therapy.

Mechanisms of prolonged cytopenia (PC) after chimeric antigen receptor (CAR) T-cell therapy, an emerging therapy for relapsed or refractory diffuse large B-cell lymphoma, remain elusive. Haematopoiesis is tightly regulated by the bone marrow (BM) microenvironment, called the 'niche'. To investigate whether alterations in the BM niche cells are associated with PC, we analysed CD271+ stromal cells in BM biopsy specimens and the cytokine profiles of the BM and serum obtained before and on day 28 after CAR T-cell infusion. Imaging analyses of the BM biopsy specimens revealed that CD271+ niche cells were severely impaired after CAR T-cell infusion in patients with PC. Cytokine analyses after CAR T-cell infusion showed that CXC chemokine ligand 12 and stem cell factor, niche factors essential for haematopoietic recovery, were significantly decreased in the BM of patients with PC, suggesting reduced niche cell function. The levels of inflammation-related cytokines on day 28 after CAR T-cell infusion were consistently high in the BM of patients with PC. Thus, we demonstrate for the first time that BM niche disruption and sustained elevation of inflammation-related cytokines in the BM following CAR T-cell infusion are associated with subsequent PC.

Comparison of bone marrow biopsy and fluorodeoxyglucose-positron emission tomography for the assessment of bone marrow infiltration in children with Hodgkin's lymphoma.

To compare bone marrow biopsy (BMB) with [18F]-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (PET/CT) imaging in the demonstration of bone marrow involvement in children with Hodgkin's Lymphoma (HL) and to investigate the effectiveness of PET/CT imaging and thus the necessity for BMB at staging. Pediatric patients with HL, who underwent both bilateral iliac BMB and PET/CT imaging at disease staging were retrospectively analyzed. In determining bone marrow involvement (BMinv), BMB and/or first/follow-up PET/CT imaging were eligible for review. Fifty-six patients were included. BMinv was detected by PET/CT imaging in 6/56 (10.7%), whereas the proportion was 3/56 (5.3%) in BMB specimens. Bone marrow biopsies and PET/CT images were concordant in 53/56 (94.6%) patients with BMB specimens missing three cases of BMinv detected by PET/CT. When diagnostic accuracy was calculated, sensitivity, specificity, positive predictive value and negative predictive values for PET/CT were 100%, 100%, 100%, 100%, respectively, and the same values for BMB were 50%, 100%, 100%, 94.3%, respectively. The results of PET/CT and BMB for staging of pediatric HL patients were compatible, and PET/CT imaging was found to provide high diagnostic performance in determining BMinv. In keeping with earlier research, the current study showed that BMB may not be necessary in every patient at staging, and should be reserved for cases where PET/CT is inconclusive.

DUSP1 Signaling Pathway Regulates Cytarabine Sensitivity in Acute Myeloid Leukemia.

Objectives: Dual specificity phosphatase 1 (DUSP1) is high-expressed in various cancers and plays an important role in the cellular response to agents that damage DNA. We aimed to investigate the expressions and mechanisms of DUSP1 signaling pathway regulating cytarabine (Ara-C) resistance in acute myeloid leukemia (AML). Methods: Immunohistochemistry was performed on bone marrow biopsy specimens from AML and controls to explore the expression of DUSP1. Western blot and Q-PCR were used to detect the protein and mRNA expression levels. MTT assay was used to detect the proliferation of cells. Cell apoptosis was detected by flow cytometry. The immune protein-protein interaction (PPI) network of DUSP1 was analyzed in the platform of Pathway Commons, and immune infiltration analysis was used to study the immune microenvironment of AML. Results: We found that the expression levels of DUSP1 in AML patients exceeded that in controls. Survival analysis in public datasets showed that AML patients with higher levels of DUSP1 had poor clinical outcomes. Further public data analysis indicated that DUSP1 was overexpressed in NRAS mutated AML. DUSP1 knockdown by siRNA could sensitize AML cells to Ara-C treatments. The phosphorylation level of mitogen-activated protein kinase (MAPK) pathway was significantly elevated in DUSP1 down-regulated NRAS G13D mutated AML cells. The PPI analysis showed DUSP1 correlated with immune gene CREB1 and CXCL8 in NRAS mutated AML. We also revealed a correlation between tumor-infiltrating immune cells in RAS mutated AML microenvironment. Conclusion: Our findings suggest that DUSP1 signaling pathways may regulate Ara-C sensitivity in AML.