Bone marrow findings post allogeneic transplant for myeloproliferative neoplasms and chronic myelomonocytic leukemia with increased fibrosis.

BACKGROUND: Allogeneic hematopoietic stem cell transplant for myeloid neoplasms with increased fibrosis is uncommon; morphologic features posttransplant can be concerning for persistent disease. METHODS: In this retrospective study, we identified 22 patients transplanted for myeloproliferative neoplasms or chronic myelomonocytic leukemia with fibrosis at our institution, and reviewed slides from pretransplant and posttransplant bone marrow biopsies. Clinical features and results of molecular, chimerism, and cytogenetic studies were retrieved from the medical record. RESULTS: Pretransplant bone marrow biopsies commonly exhibited hypercellularity, atypical megakaryocytes, and reticulin fibrosis. At day 100, 36% of biopsies had reticulin grade >MF1 and 33% of those tested had positive molecular studies, with no significant associations between day 100 marrow characteristics and molecular profile or peripheral count recovery times. In the 1 year posttransplant biopsies (n = 12), 7 of 9 had negative molecular studies; of these, none had reticulin grade >MF1, 1 had trichrome 1+, 2 had atypical megakaryocytes, and 1 was hypercellular. CONCLUSIONS: Supporting recent literature, our study indicates that persistent day 100 reticulin fibrosis/collagen deposition does not show an association with day 100 molecular status. Our study additionally provides data for 12 patients with 1 year posttransplant marrow biopsies, with the majority of those lacking either increased fibrosis or molecular evidence of persistent disease.

Advances in Monitoring and Prognostication for Lymphoma by Flow Cytometry.

Flow cytometry (FC) is a well-established method important in the diagnosis and subclassification of lymphoma. In this article, the role of FC in lymphoma prognostication will be explored, and the clinical role for FC minimal/measurable residual disease testing as a monitoring tool for mature lymphoma will be introduced. Potential pitfalls of monitoring for residual/recurrent disease following immunotherapy will be presented.

Reactive Lymphadenopathy in the Pediatric Population with a Focus on Potential Mimics of Lymphoma.

Benign lymphadenopathy is common in the pediatric population and may be clinically striking. As in adults, lymph node evaluation in pediatric patients requires careful morphologic and immunohistochemical assessment and clinical contextualization of the findings. It is important for the pathologist to be familiar with benign and reactive conditions that may mimic malignancy. This review presents non-neoplastic or indolent processes or patterns of lymphoid hyperplasia that may be confused with or raise the differential of lymphoma, with a focus on those more commonly encountered in the pediatric/adolescent population.

Diagnostic Flow Cytometry in the Era of Targeted Therapies: Lessons from Therapeutic Monoclonal Antibodies and Chimeric Antigen Receptor T-cell Adoptive Immunotherapy.

Therapeutic monoclonal antibodies (therapeutic mAb) and adoptive immunotherapy have become increasingly more common in the treatment of hematolymphoid neoplasms, with practical implications for diagnostic flow cytometry. Their use can reduce the sensitivity of flow cytometry for populations of interest owing to downregulation/loss of the target antigen, competition for the target antigen, or lineage switch. Expanded flow panels, marker redundancy, and exhaustive gating strategies can overcome this limitation. Therapeutic mAb have been reported to cause pseudo-light chain restriction, and awareness of this potential artifact is key. Established guidelines do not yet exist for antigen expression by flow cytometry for therapeutic purposes.

Isolated Light Chain-restricted Germinal Centers are Common in Follicular Hyperplasia by Ultrasensitive In Situ Hybridization.

Ultrasensitive bright-field RNA in situ hybridization (BRISH) can be used to detect lower levels of light chain expression than immunohistochemical stains or conventional colorimetric RNA in situ hybridization. In this study, we retrospectively reviewed 77 lymph node specimens with follicular hyperplasia and kappa/lambda BRISH performed as part of the diagnostic evaluation. Thirty-two of the specimens had ≥1 germinal center(s) (GC) showing light chain restriction (14 specimens with lambda-restricted GC, 9 with kappa-restricted GC, and 9 with separate kappa-restricted or lambda-restricted GC). In all but 1 specimen, the light chain-restricted GC represented a minority of the total GC (average: 10%, range: 0.2% to 60%). There was no significant difference in age, sex, type of biopsy (core vs. excision), number of GCs, proportion of cases with a light chain-restricted B-cell population by flow cytometry, or proportion of cases with a positive IgH gene rearrangement study between the specimens with and without restricted GCs. In our cohort of follicular hyperplasia cases, BRISH identified light chain-restricted GC more frequently than flow cytometry identified a monotypic B-cell population. Our findings highlight the potential for overinterpretation of light chain restriction in limited samplings such as fine needle aspiration cell blocks or core needle sampling and reinforce that interpretation of BRISH staining needs to occur in the context of the morphologic features including tissue architecture and results of additional immunohistochemical stains.

An interactive e-learning module on peripheral blood smear analysis is an effective option for teaching pathology trainees.

OBJECTIVES: This study compares the effectiveness of an interactive e-learning module with a traditional text-based method for teaching peripheral blood smear analysis. METHODS: Pathology trainees at Accreditation Council for Graduate Medical Education residency programs were asked to participate. Participants completed a multiple-choice test on peripheral blood smear findings. Trainees were randomized into completing an e-learning module or a PDF reading exercise with the same educational content. Respondents rated their experience and completed a postintervention test composed of the same questions. RESULTS: In total, 28 participants completed the study; 21 improved their score in the posttest (mean, 21.6 correct answers) compared with the pretest (19.8; P < .001). This improvement was seen in both the PDF (n = 19) and interactive (n = 9) groups, with no difference in performance between the 2 groups. Trainees with less clinical hematopathology experience showed a trend of having the largest performance improvement. Most participants completed the exercise within 1 hour, rated the exercise as easy to navigate, were engaged, and reported learning new information about peripheral blood smear analysis. All participants indicated that they would likely complete a similar exercise in the future. CONCLUSIONS: This study suggests that e-learning is an effective tool for hematopathology education and equivalent to traditional narrative-based methods. This module could easily be incorporated into a curriculum.

Work-up of Patients with Decreased Hemoglobin A2 Identified by Capillary Zone Electrophoresis: A North American Institutional Experience.

OBJECTIVE: Isolated low hemoglobin A2 (HbA2) is rarely encountered in our clinical practice using capillary zone electrophoresis. The study goal was to characterize the work-up at our institution of patients with low HbA2. METHODS: Patients with low HbA2 and a control cohort with normal capillary zone electrophoresis were identified and relevant information extracted from the medical record. RESULTS: Of 44 patients with isolated decreased HbA2, 28 (64%) had corresponding complete blood count/ferritin values. Compared to control patients, patients with low HbA2 were more likely to have iron deficiency and demonstrated a more microcytic, hypochromic blood picture. However, 46% (13/28) of patients with low HbA2 and ferritin for evaluation did not have iron deficiency. Only 2 patients had genetic testing. CONCLUSION: This study redemonstrates the association between low HbA2 and iron deficiency and reinforces the need for iron indices to interpret capillary zone electrophoresis results. Our study population showed incomplete or absent iron studies in most cases.

Geographic Variability of Nodular Lymphocyte-Predominant Hodgkin Lymphoma.

OBJECTIVES: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) differs from classic Hodgkin lymphoma (CHL) in terms of clinicopathologic features, including Epstein-Barr virus (EBV) association. CHL geographic variability is well known, with higher frequencies of mixed-cellularity subtype and EBV positivity in low/middle-income countries (LMICs), but there are few well-characterized series of NLPHL from LMICs. METHODS: We detail clinicopathologic findings of 21 NLPHL cases received in consultation from Kenya and summarize reports of NLPHL with EBV testing published since 2000. RESULTS: Median age of consultation cases was 36 years, and male/female ratio was 3.2. All cases involved peripheral lymph nodes and showed at least some B-cell-rich nodular immunoarchitecture, with prominent extranodular lymphocyte-predominant (LP) cells and T-cell-rich variant patterns most commonly seen. LP cells expressed pan-B-cell markers, including strong OCT2; lacked CD30 and CD15 expression in most cases; and were in a background of expanded/disrupted follicular dendritic cell meshworks and increased T-follicular helper cells. LP cells were EBV negative in 18 cases. Historical cases showed a low rate of EBV positivity with no significant difference between LMICs and high-income countries. CONCLUSIONS: Unlike CHL, NLPHL shows few geographic differences in terms of clinicopathologic features and EBV association. These findings have implications for diagnosis, prognostication, and treatment of patients with NLPHL in LMICs.

Indolent B-Lineage Precursor Populations Identified by Flow Cytometry and Immunohistochemistry in Benign Lymph Nodes.

OBJECTIVES: In this retrospective study, we report a series of benign lymph nodes showing small populations of normal B-cell precursors characterized by flow cytometry and immunohistochemistry. METHODS: Ten cases identified during clinical flow cytometry practice were retrospectively reanalyzed with particular attention to hematogone categorization and enumeration. Immunohistochemical staining was performed on five excisional lymph node biopsy specimens to characterize the morphologic correlate. RESULTS: Populations of hematogones ranging from 0.13% to 1.86% (median, 0.51%) of all viable leukocytes were demonstrated in 10 benign lymph node samples from eight different patients ranging in age from 17 to 45 years (median, 37.5). These hematogones showed a characteristic immunophenotype (CD19+/CD10+) and maturational pattern by flow cytometry, with progression from stage 1 (median, 0.03%) to stage 2 (median, 0.19%) to stage 3 (median, 0.26%) seen in all cases. Immunohistochemical staining on five excisional biopsy specimens demonstrated a distinct perisinusoidal distribution of CD10+/CD20+ cells with a subset of TdT+ cells, providing a morphologic correlate. CONCLUSIONS: To our knowledge, this is the first study to characterize distinct hematogone populations within benign lymph nodes by both flow cytometry and immunohistochemistry. Recognizing these normal B-cell precursor populations is important to avoid their miscategorization as a CD10+ B-cell neoplasm.

Variant Acute Promyelocytic Leukemia Presenting Without Auer Rods Highlights the Need for Correlation with Cytogenetic Data in Leukemia Diagnosis.

Variant acute promyelocytic leukemia (vAPL) is a rare leukemia characterized by rearrangement between RARα and a non-PML partner gene. This type of leukemia can be difficult to recognize by histomorphologic evaluation, particularly in patients with few or no Auer rods, and by flow cytometry, but it can be identified by distinct cytogenetic features. Herein, we report on a patient with vAPL with t(11;17)(q23;q21) who presented an initial diagnostic challenge. Detailed flow cytometry findings are presented for this rare entity. Our case study also presents novel treatment (chemotherapy in combination with venetoclax) chosen based on mechanistic data from preclinical studies.

Self-regressing oral CD30-positive, EBV-negative, T-cell lymphoproliferative lesions. A poorly understood process highlighted by ominous clinicopathologic features and indolent behavior.

OBJECTIVE: Intraoral, primary, CD30-positive (CD30+) T-cell lymphoproliferative disorders (TLPDs) are uncommon, and their clinicopathologic presentation and management can vary and may be challenging. Herein, we present a retrospective study of 4 examples of self-regressing primary CD30+ TLPD affecting the gingiva. STUDY DESIGN: Archived files were retrospectively reviewed for oral CD30+ TLPDs featuring (1) proper immunohistochemical documentation, (2) Epstein-Barr virus negativity, (3) adequate follow-up information corroborating regression, and (4) no history of hematopoietic malignancy or related-mucocutaneous disease. RESULTS: Three women and 1 man (age range, 55-82 years; mean, 68.3 years) presented with rapidly growing gingival ulcers. Microscopic evaluation revealed diffuse infiltration by sheets of large, atypical cells admixed with lymphocytes and eosinophils, showing angiocentric distribution, focal neurotropism, and muscle infiltration. The lesional cells consistently stained for CD3 and CD30 and were variably immunoreactive against CD2, CD4, CD5, CD7, and CD8, but were negative for ALK1 and EBV-encoded small RNA. TCR-γ gene rearrangement studies revealed a monoclonal T-cell population in 1 case. All lesions showed complete regression 2 to 8 weeks postoperatively (mean follow-up, 4.5 weeks). CONCLUSIONS: Notwithstanding their alarming clinicopathologic appearance, there are CD30+ TLPDs confined to the oral cavity that have an indolent course. However, clinical staging is essential to exclude aggressive systemic malignancy.

Impact of initial chemotherapy regimen on outcomes for patients with double-expressor lymphoma: A multi-center analysis.

Diffuse large B-cell lymphoma featuring overexpression of MYC and B-Cell Lymphoma 2 (double expressor lymphoma, DEL) is associated with poor outcomes. Existing evidence suggesting improved outcomes for DEL with the use of more intensive regimens than R-CHOP is restricted to younger patients and based on limited evidence from low patient numbers. We retrospectively evaluated the impact of intensive frontline regimens versus R-CHOP in a multicenter analysis across 7 academic medical centers in the United States. We collected 90 cases of DEL, 46 out of 90 patients (51%) received R-CHOP and 44/90 (49%) received an intensive regimen, which was predominantly DA-EPOCH-R. Treatment cohorts were evenly balanced for demographics and disease characteristics, though the intensive group had a higher lactate dehydrogenase (LDH, 326 vs. 230 U/L p = 0.06) and presence of B-symptoms (50% vs. 22%, p = 0.01) compared to the R-CHOP cohort. There was no difference in PFS (median 53 vs. 38 months, p = 0.49) or overall survival (67 vs. not reached months, p = 0.14) between the R-CHOP and intensive therapy cohorts, respectively. On multivariate analysis, intensive therapy was associated with a hazard ratio of 2.35 (95% CI 0.74-7.41), though this was not statistically significant. Additionally, a subgroup analysis of intermediate high-risk lymphoma defined by IPI ≥3 did not identify a difference in survival outcomes between regimens. We conclude that in our multi-center cohort there is no evidence supporting the use of intensive regimens over R-CHOP, suggesting that R-CHOP remains the standard of care for treating DEL.

Entrustable Professional Activities in Hematopathology Pathology Fellowship Training: Consensus Design and Proposal.

Hematopathology fellowship education has grown in complexity as patient-centered treatment plans have come to depend on integration of clinical, morphologic, immunophenotypic, molecular, and cytogenetic variables. This complexity is in competition with the need for timely hematopathology care with stewardship of patient, laboratory, and societal resources. Accreditation Council for Graduate Medical Education Milestones provide a guidance document for hematopathology training, but fellows and their educators are in need of a simple framework that allows assessment and feedback of growth toward independent hematopathology practice. Entrustable professional activities provide one such framework, and herein, we provide proposed Hematopathology Fellowship Entrustable Professional Activities based on review of pertinent guidelines and literature, with multiple rounds of expert and stakeholder input utilizing a modified mini-Delphi approach. Ten core entrustable professional activities deemed essential for graduating hematopathology fellows were developed together with skills and knowledge statements, example scenarios, and corresponding Accreditation Council for Graduate Medical Education Milestones. Application of these entrustable professional activities in program design, fellow evaluation, and decisions regarding level of supervision is discussed with consideration of benefits and barriers to implementation. These entrustable professional activities may be used by hematopathology fellowship directors and faculty to provide fellows with timely constructive feedback, determine entrustment decisions, provide the Clinical Competency Committee with granular data to support Milestone evaluations, and provide insight into areas of potential improvement in fellowship training. Fellows will benefit from a clear roadmap to independent hematopathology practice with concrete and timely feedback.

Normal gastrointestinal tract inflammatory cells and review of select benign hematolymphoid proliferations.

The luminal gastrointestinal tract can be a site of robust immune response in which reactive lymphoproliferative processes can sometimes be difficult to distinguish from lymphoma. In this article, we review gastrointestinal tract normal resident inflammatory cells and common nonneoplastic lymphoproliferative responses with emphasis on their differential and links to lymphoma. Topics that are covered include lymphocytic esophagitis, gastric chronic inflammation, mucosa-associated lymphoid tissue, and ulceration, small intestinal lymphoid hyperplasia, celiac disease, microscopic colitis, inflammatory bowel disease, primary immunodeficiency, graft-versus-host disease, and anti-programmed cell death protein-1 effect. We additionally present the less common differential of histiocytic processes within the gastrointestinal tract. The aim of this paper is to serve as a reference for practicing pathologists facing lymphoid, lymphoplasmacytic, or histiocytic processes in the luminal gastrointestinal tract. We hope to help the practicing pathologist distinguish benign from malignant entities and identify features requiring further workup.

Daratumumab Interference in Flow Cytometry Producing a False Kappa Light Chain Restriction in Plasma Cells.

False kappa light chain restriction on hematogones (normal B-lineage precursors) has been described in patients on the therapeutic anti-CD38 monoclonal antibody daratumumab. In this article, we present a novel case report of pseudo-kappa light chain restriction on lambda-restricted neoplastic plasma cells in a patient with progressive plasma cell myeloma while on daratumumab. Flow cytometric technologists and pathologists need to be aware of this potential diagnostic pitfall.

VS38 Identifies Myeloma Cells With Dim CD38 Expression and Plasma Cells Following Daratumumab Therapy, Which Interferes With CD38 Detection for 4 to 6 Months.

OBJECTIVES: We report our institutional experience using VS38 to evaluate plasma cells by flow cytometry. METHODS: Flow cytometry data were reanalyzed to compare plasma cell percentages between the standard panel and VS38 panel. Natural killer (NK) and plasma cell CD38 median fluorescence intensity (MFI) values were calculated. RESULTS: Our cohort included 63 specimens from 38 patients. Twenty-six had received daratumumab (monoclonal anti-CD38 therapy) between less than 1 month and 17 months prior. For NK and plasma cells, CD38 MFI values were suppressed for 0 to 4 months and started to increase 4 to 6 months after last exposure. There was no significant difference in clonal plasma cell percentage calculated by the VS38 and standard panels; however, identification and quantification using the VS38 panel were easier. CONCLUSIONS: VS38 is a viable alternative to bright CD38 to identify plasma cells and particularly helpful in myeloma cases with dim CD38 and after daratumumab. Daratumumab interference with CD38 identification persists 4 to 6 months after the last exposure.

Comparison of evaluation techniques, including digital image analysis, for MYC protein expression by immunohistochemical stain in aggressive B-cell lymphomas.

Incorporation of an MYC immunohistochemical stain in the workup of large B-cell lymphomas has become common in hematopathology practice. Evaluation of this stain can be difficult because of staining heterogeneity and can have interobserver variability, particularly when performed on the entire tumor sections. We identified 87 cases of aggressive B-cell lymphoma (34 core needle and 53 excisional biopsies) and compared the following methods of MYC immunohistochemical staining evaluation: the original pathologist's interpretation, a systematic retrospective method of evaluation by manual analysis, and a retrospective method of evaluation by digital image analysis (using scanned slides analyzed via the Aperio Nuclear algorithm). Overall, concordance among these methods was around 80% with κ statistics showing good agreement. However, nearly one-third of our cases had a percent MYC positivity in the 30% to 50% range, and for these cases, concordance among the various methods was marginal/poor. This suggests limited utility as a prognostic or predictive marker using 40% as a cutoff value. In our series, core biopsy specimens were poor predictors of MYC gene rearrangement, and there was no association between MYC immunohistochemical stain and MYC gene gain/amplification. Our retrospective digital image analysis showed strong correlation in MYC percent positivity with our retrospective manual review (correlation coefficient of 0.90) and similar concordance to pathologist interpretation as among pathologists, suggesting that digital image analysis is a viable alternative to manual determination of MYC percent positivity. Digital image analysis provides further opportunities for more sophisticated and standardized scoring systems, which may be helpful in future prognostic/predictive studies.