Flow cytometric immunophenotypic features of acute myeloid leukemia with mast cell differentiation.

OBJECTIVES: Acute myeloid leukemia (AML) with mast cell (MC) differentiation was recently described as an aggressive subgroup of AML cases. The objectives of this study were to assess the flow cytometric immunophenotypic features of AML-MC cases. METHODS: We characterized the immunophenotypic features of 21 AML-MC cases by flow cytometry and compared them to 20 reactive/regenerating bone marrow specimens. RESULTS: The number of MCs detected by flow cytometry in AML-MC cases ranged from 0.4% to 21.1%, with a median of 3.5%, significantly higher than that of normal/reactive bone marrow (BM) (median, 0.01%; range, 0.000%-0.396%; P < .0001). Immunophenotypically, MCs in AML-MC cases demonstrated immaturity, differing from MCs in normal/reactive BMs, including dimmer CD45 (100% vs 0%), lower side scatter (100% vs 0%), more frequent CD34 (81% vs 20%), and CD123 (100% vs 10%) positivity, and more frequent uniform/increased CD38 expression (95% vs 20%) (all P ≤ .0001). CD2 (0/5) and CD25 (2/6, 1 uniform and 1 partial) were assessed in a subset of cases. The myeloblasts in AML-MC were typically CD34+CD117+HLA-DR+ with unusually frequent expression of CD56 (57%, all partial) and CD25 (63%, mostly partial), increased CD117 (62%), and decreased CD38 (86%). The MC percentage determined by flow cytometry correlated well with MCs detected by tryptase immunohistochemistry (r = 0.76, P < .001). CONCLUSIONS: The MCs in AML-MC cases are characterized by dim CD45, low side scatter, CD34 and CD123 positivity, and uniform and increased CD38 expression. Flow cytometry is an excellent tool for identifying AML-MC cases.

Blast phase of chronic myeloid leukemia presenting as early T-cell precursor acute lymphoblastic leukemia.

OBJECTIVES: The blasts in most cases of chronic myeloid leukemia blast phase (CML-BP) have a myeloid or precursor-B immunophenotype, with only a small subset having T-cell or natural killer-cell lineage. Patients with CML-BP having early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) are extremely rare. METHODS: We report the clinicopathologic, immunophenotypic, and molecular genetic features and outcome of 3 patients with CML-BP who had ETP-ALL, with a review of the literature. RESULTS: Only patient 1 had a history of chronic myeloid leukemia chronic phase. Fluorescence in situ hybridization revealed BCR::ABL1 rearrangement in cells with round nuclei (blasts) and cells with segmented nuclei (neutrophils) in cases 2 and 3, supporting a diagnosis of CML-BP rather than de novo Ph+ ETP-ALL. The blasts were positive for cytoplasmic CD3, CD7, CD33, and CD117; were negative for CD1a and CD8; and had dim CD5 expression in 2 cases. Next-generation sequencing showed a TET2 mutation in case 1 and BCOR, RUNX1, and STAG2 mutations in case 3. All patients received chemotherapy and tyrosine kinase inhibitors. Patients 2 and 3 died 33 days and 39 days, respectively, after diagnosis. Patient 1 received stem cell transplantation and was alive 14 months after blast phase. CONCLUSIONS: Patients with CML-BP may have ETP-ALL. These patients usually have an aggressive clinical course, requiring intensive therapy, and may benefit from stem cell transplantation.

Beyond surgery: the proper role and delivery of radiation therapy in the local-regional management of BIA-ALCL.

For localized breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), surgical resection is crucial; however, radiation therapy (RT) can be utilized as local-regional therapy if surgery is incomplete or not recommended. We present the case of a woman with BIA-ALCL who received systemic therapy and consolidation RT.

Utility of KIT Mutations in Myeloid Neoplasms Without Documented Systemic Mastocytosis to Detect Hidden Mast Cells in Bone Marrow.

BACKGROUND: KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM. Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM. METHODS: We searched NGS result in our institution with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST). RESULTS: Bone marrow biopsies were assessed with immunohistochemical stains for CD117 and mast cell tryptase (n = 49). Most patients had acute myeloid leukemia (AML, n = 38) or chronic myelomonocytic leukemia (CMML, n = 6). Immunohistochemical stains for CD117 and tryptase were performed in all 49 patients. A total of 4 patients (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the WHO criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (∼ 50%) except one patient (1%). CONCLUSION: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations fulfilled the diagnostic criteria for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies is recommended to identify underrecognized SM when KIT mutations are found by molecular assays.

Expression of CD6 in Aggressive NK/T-cell Neoplasms and Assessment as a Potential Therapeutic Target: A Bone Marrow Pathology Group Study.

BACKGROUND: Aggressive NK/T-Cell neoplasms are rare hematological malignancies characterized by the abnormal proliferation of NK or NK-like T (NK/T) cells. CD6 is a transmembrane signal transducing receptor involved in lymphocyte activation and differentiation. This study aimed to investigate the CD6 expression in these malignancies and explore the potential of targeting CD6 in these diseases. MATERIALS AND METHODS: We conducted a retrospective study with totally 41 cases to investigate the expression of CD6 by immunohistochemistry, including aggressive NK-cell leukemia/lymphoma (ANKLL: N = 10) and extranodal NK/T-cell lymphoma (ENKTL: N = 31). A novel ANKLL model was applied for proof-of-concept functional studies of a CD6 antibody-drug-conjugate (CD6-ADC) both in vitro and in animal trial. RESULTS: CD6 was expressed in 68.3% (28/41) of cases (70% (7/10) of ANKLL and 67.7% (21/31) of ENKTL). The median overall survival (OS) for ANKLL and ENTKL cases was 1 and 12 months, respectively, with no significant difference in OS based on CD6 expression (p > 0.05, Kaplan-Meier with log-rank test). In vitro exposure of the CCANKL cell line, derived from an ANKL patient, to an anti-CD6ADC resulted in dose dependent induction of apoptosis. Furthermore, CCANKL engraftment in NSG mice could be blocked by treatment with the anti-CD6 ADC. CONCLUSION: To date, this is the first report to explore the expression of CD6 in ANKLL and ENKTL and confirms its expression in the majority of cases. The in vitro and in vivo data support further investigation of CD6 as a potential therapeutic target in these aggressive NK/T-cell malignancies.

Optical genome mapping improves the accuracy of classification, risk stratification, and personalized treatment strategies for patients with acute myeloid leukemia.

Cytogenomic characterization is crucial for the classification and risk stratification of acute myeloid leukemia (AML), thereby facilitating therapeutic decision-making. We examined the clinical utility of optical genome mapping (OGM) in 159 AML patients (103 newly diagnosed and 56 refractory/relapsed), all of whom also underwent chromosomal banding analysis (CBA), fluorescence in situ hybridization, and targeted next-generation sequencing. OGM detected nearly all clinically relevant cytogenetic abnormalities that SCG identified with >99% sensitivity, provided the clonal burden was above 20%. OGM identified additional cytogenomic aberrations and/or provided information on fusion genes in 77 (48%) patients, including eight patients with normal karyotypes and four with failed karyotyping. The most common additional alterations identified by OGM included chromoanagenesis (n = 23), KMT2A partial tandem duplication (n = 11), rearrangements involving MECOM (n = 7), NUP98 (n = 2), KMT2A (n = 2), JAK2 (n = 2), and other gene fusions in 17 patients, with 10 showing novel fusion gene partners. OGM also pinpointed fusion genes in 17 (11%) patients where chromosomal rearrangements were concurrently detected by OGM and CBA. Overall, 24 (15%) aberrations were identified exclusively by OGM and had the potential to alter AML classification, risk stratification, and/or clinical trial eligibility. OGM emerges as a powerful tool for identifying fusion genes and detecting subtle or cryptic cytogenomic aberrations that may otherwise remain undetectable by CBA.

Morphology, immunophenotype, and suggested diagnostic criteria of TCL1 family-negative T-prolymphocytic leukemia.

OBJECTIVES: We sought to investigate the morphologic and immunophenotypic characteristics of TCL1 family-negative T-cell prolymphocytic leukemia (T-PLL). METHODS: Twenty cases of TCL1 family-negative T-PLL were studied. RESULTS: The doubling time of leukemic cells ranged from less than 2 days to more than 5 years, with a median of 5.5 months. Leukemic cells were small to medium-sized, with round to irregular nuclei, variably condensed chromatin, and small amounts of agranular cytoplasm. A visible nucleolus was identified in 11 (55%) cases. Cytoplasmic blebs/protrusions were identified in all cases, but their occurrence was highly variable from case to case. Bone marrow biopsy showed an interstitial pattern in 90% of cases and a diffuse pattern in the remaining 10% of cases. Flow cytometric immunophenotypic analysis showed that the leukemic cells in all cases were CD4 positive; 3 (15%) also showed concurrent CD8 expression. All cases were positive for CD2 and CD5. Surface CD3 and CD7 were positive in 19 of 20 (95%) cases, and all CD3-positive cases expressed the T-cell receptor αß. Compared with prototypic T-PLL cases, these 2 groups shared many immunophenotypic findings, except CD8 and CD26, both of which were more commonly expressed in prototypic T-PLL cases. CONCLUSIONS: TCL1 family-negative T-PLL cases have morphologic and immunophenotypic features that are similar to prototypic T-PLL. They are characterized by neoplastic proliferation of small to medium-sized mature T cells with CD4-positive T-cell receptor αß phenotype. Tumor cells frequently maintain pan-T antigen expression. Recognizing these morphologic and immunophenotypic features will aid in accurately diagnosing this rare subset of T-PLL.

Advancing Diagnostic Accuracy and Quality of Patient Care Through the Implementation of a Flow Cytometry Quality Assurance Program.

CONTEXT.­: Flow cytometry immunophenotypic analysis plays an important role in the diagnosis, classification, and disease monitoring of hematologic neoplasms. The interpretation of flow cytometry testing can be challenging. OBJECTIVE.­: To explore ways to improve diagnostic accuracy and in turn enhance the quality of patient care. DESIGN.­: A flow cytometry quality assurance (QA) program was developed. Cases from various complex flow cytometry panels were randomly selected and cross-reviewed. The outcomes of the QA review were categorized into 3 groups: complete agreement, minor discrepancy, and major discrepancy. Each discrepancy underwent a process of documentation, discussion, and resolution. Here we summarize our 3 years of experience with this program. RESULTS.­: In total, 6166 cases were evaluated; 6028 cases (97.7%) showed complete concordance, 120 cases (2.0%) showed minor discrepancies, and 18 cases (0.3%) showed major discrepancies. Among the top 5 panels evaluated, the panel evaluating mature T-cell abnormalities showed the highest rate of discrepancy, whereas the panel for evaluation of myelodysplastic syndromes showed the lowest discrepancy rate. When analyzing the trends of concordance and discrepancy over time, we observed a statistically significant decrease in discrepancy rate over time, from 4% at the beginning of the 6-month period to 1.5% in the final 6-month period. CONCLUSIONS.­: The overall concordance rate was 97.7%. The remaining 2.3% of cases showed discrepancies that required a correction, underscoring the value and necessity of having a QA program. The overall discrepancy rates exhibited a gradual decline over time, indicative of the positive impact of the QA program on enhancing diagnostic competency and accuracy over time.

Long-term results of the sequential combination of cladribine and rituximab in Hairy cell leukemia.

We report on the long-term efficacy and safety of a phase 2 trial of sequential cladribine and rituximab in hairy cell leukemia (HCL). One-hundred and thirty-nine patients were enrolled: 111 in the frontline setting, 18 in first relapse, and 10 with variant HCL (HCLv). A complete response (CR) was achieved in 133 of 137 evaluable participants (97%) with measurable residual disease (MRD) negativity in 102 (77%). MRD status was not associated with significant differences in event-free survival (EFS) or overall survival (OS). With a median follow-up of 7.8 years (range: 0.40-18.8), eight patients have experienced disease relapse (5.8%), 4/111 with newly diagnosed HCL (3·6%) and 4/10 with HCLv (40%) (p = 0.002). The 10-year EFS and OS rates were 86.7% and 91.1%, respectively. Grade 3 adverse events were observed in 28 participants (20·1%), mostly due to infections. Treatment of HCL with sequential cladribine followed by rituximab is associated with excellent efficacy and safety results both in the frontline and relapsed settings.

Integrative immunophenotypic and genetic characterization of acute myeloid leukemia with CBFB rearrangement.

OBJECTIVES: We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. METHODS: Sixty-one cases of AML with CBFB rearrangement were evaluated. RESULTS: The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non-type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. CONCLUSIONS: Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts.

Predictors of clinical outcome in myeloproliferative neoplasm, unclassifiable: A Bone Marrow Pathology Group study.

OBJECTIVES: Myeloproliferative neoplasm, unclassifiable (MPN-U, revised to MPN, not otherwise specified in the fifth edition of the World Health Organization classification) is a heterogeneous category of primary marrow disorders with clinical, morphologic, and/or molecular features that preclude classification as a more specific MPN subtype due to stage at diagnosis, overlapping features between MPN subtypes, or the presence of coexisting disorders. Compared with other MPN subtypes, the contribution of the mutational landscape in MPN-U in conjunction with other clinical and morphologic biomarkers to prognosis has been less well investigated. METHODS: We performed a multicenter, retrospective study of MPN-U (94 cases) to better define the clinicopathologic features, genetic landscape, and clinical outcomes, including subgroups of early-stage, advanced-stage, and coexisting disorders. The Dynamic International Prognostic Scoring System (DIPSS) plus scoring system was applied to assess its relevance to MPN-U prognosis. RESULTS: Multivariate analysis demonstrated bone marrow blast count and DIPSS plus score as statistically significant in predicting overall survival. Univariate analysis identified additional potential poor prognostic markers, including abnormal karyotype and absence of JAK2 mutation. Secondary mutations were frequent in the subset analyzed by next-generation sequencing (26/37 cases, 70.3%) with a borderline association between high molecular risk mutations and overall survival. CONCLUSIONS: This study, as one of the largest of MPN-U studies incorporating both clinicopathologic and molecular data, moves toward identification of biomarkers that better predict prognosis in this heterogeneous category.

Characteristics and outcomes of acute myeloid leukaemia patients with baseline CD7 expression.

Targeted therapy development for acute myeloid leukaemia (AML) requires an understanding of specific expression profiles. We collected flow cytometry data on 901 AML patients and recorded aberrant CD7 expression on leukaemic blasts. 263 (29.2%) had blasts positive for CD7. CD7+ AML was more likely to be adverse risk (64.6% vs. 55.6%, p = 0.0074) and less likely to be favourable risk (15.2% vs. 24.1%, p = 0.0074) by European LeukemiaNet 2022 criteria. Overall survival was inferior (11.9 [95% CI, 9.7-15.9] vs. 19.0 months [95% CI, 16.1-23.0], p = 0.0174). At relapse, 30.4% lost and 19.0% gained CD7, suggesting moderate instability over time.

Diagnostic utility of immunohistochemistry in detection of NPM1 mutations in acute myeloid leukemia with a patchy distribution.

Nucleophosmin 1 (NPM1) mutations occur in approximately one-third cases of adult de novo acute myeloid leukemia (AML). Identification of NPM1 mutations is important for classification, risk stratification, tailored therapy, and monitoring minimal residual disease. Mutational analysis is widely used for detecting NPM1 mutations. Immunochemistry assessing abnormal cytoplasmic localization of NPM1 protein has been used as a surrogate marker for NPM1 mutations. We present a case of AML with mutated NPM1 that was missed by sequencing analysis but detected by immunohistochemistry. This case highlights the value of immunohistochemistry in identifying NPM1 mutations in a subset of AML cases.