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.

Cytoplasmic Lipid Droplets Predict Worse Prognosis in Diffuse Large B-Cell Lymphoma: Next-Generation Sequencing Deciphering Lipogenic Genes.

Burkitt lymphoma is characterized by high cell turnover and numerous cytoplasmic vacuoles that are demonstrated to be lipid droplets (LDs) decorated by adipophilin. By contrast, cytoplasmic vacuoles are variably observed in diffuse large B-cell lymphoma (DLBCL) and less well characterized. In this study, we first validated in DLBCL that cytoplasmic vacuoles are indeed LDs by Oil-red-O stain, Bodipy fluorescent stain, and electron microscopy. Second, in a cohort of DLBCL patients (n=52) we showed that LDs in effusional lymphoma cells were associated with a poorer prognosis (P=0.029, log-rank test) and higher International Prognostic Index (IPI) score (94% vs. 66%, P=0.026) than those without. Moreover, using adipophilin as a surrogate marker for LDs, we found in another cohort of biopsy specimen (n=85) that expression of adipophilin by lymphoma cells predicted a poorer prognosis (P=0.007, log-rank test) and higher IPI score (63% vs. 30%, P=0.005). In addition, whole exome sequencing of effusional DLBCL cells showed LD-positive DLBCL shared genetic features with the MCD (MYD88 and CD79B mutations) subtype and highlighted OSBPL10 and CUBN as the most frequently mutated genes involved in lipogenesis. Whole transcriptome analysis by comparing effusional DLBCL cells with versus without LDs showed upregulation of EHHADH, SLC1A1, CD96, INPP4B, and RNF183 relevant for lymphoma lipogenesis and upregulation of epithelial-mesenchymal transition and KRAS signaling pathways. Higher expression of EHHADH and CD96 were validated in LD-positive clinical samples and LD-rich cell lines than LD-poor cells along with the known lipogenic gene, FASN. Our findings highlight the roles of LDs and adipophilin expression in DLBCL, suggest that these markers may predict prognosis and show that lipogenic genes may be potential therapeutic targets.

Radiotherapy in the treatment of primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder.

BACKGROUND: Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSM-LPD) is an increasingly recognized entity with heterogeneous management strategies that may include radiotherapy. OBJECTIVE: Our aim was to characterize treatment options for PCSM-LPD, with a focus on the role of radiotherapy. METHODS: This is a retrospective review of 46 patients seen in the Cutaneous Lymphoma Program at the University of Texas MD Anderson Cancer Center, with a clinicopathologic review consistent with PCSM-LPD. All patients were biopsied and underwent observation, topical/intralesional steroids, and/or radiotherapy. Patients were confirmed to have residual disease prior to radiotherapy. RESULTS: All patients achieved a complete response (CR). Sixteen patients (35%) received focal radiotherapy, with a CR in 15 (94%). The CR rate following ultra-low-dose radiotherapy (4 Gy in 1-2 fractions) was 92%. There was no grade 3 toxicity after radiotherapy. Thirty patients were managed without radiotherapy, with excision and observation or steroids. CONCLUSION: Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder has excellent outcomes, and management strategies may include observation following biopsy, steroids, or radiation. Ultra-low-dose radiotherapy results in excellent outcomes with limited toxicity and is effective for persistent lesions after steroidal therapy.

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.

Fifth Edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues: Mature T-Cell, NK-Cell, and Stroma-Derived Neoplasms of Lymphoid Tissues.

This review focuses on mature T cells, natural killer (NK) cells, and stroma-derived neoplasms in the fifth edition of the World Health Organization classification of hematolymphoid tumors, including changes from the revised fourth edition. Overall, information has expanded, primarily due to advancements in genomic understanding. The updated classification adopts a hierarchical format. The updated classification relies on a multidisciplinary approach, incorporating insights from a diverse group of pathologists, clinicians, and geneticists. Indolent NK-cell lymphoproliferative disorder of the gastrointestinal tract, Epstein-Barr virus-positive nodal T- and NK-cell lymphoma, and several stroma-derived neoplasms of lymphoid tissues have been newly introduced or included. The review also provides guidance on how the fifth edition of the World Health Organization classification of hematolymphoid tumors can be applied in routine clinical practice.

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.

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.

From the archives of MD Anderson Cancer Center. Mesothelial/monocytic incidental cardiac excrescence with a review of the literature.

Mesothelial/monocytic incidental cardiac excrescence (MICE) is a rare benign lesion composed of monocytes and mesothelial cells that is most often encountered during cardiothoracic surgery. We describe a case in a 71-year-old man with known aortic valve stenosis who presented with gradual onset dyspnea over a few weeks, made worse with minimal exertion. A transesophageal echocardiogram revealed severe aortic stenosis and mild pericardial effusion. The patient underwent aortic valve replacement, coronary artery bypass, and amputation of the left atrial appendage. Histological examination of a 0.8 cm blood clot received along with the atrial appendage showed an aggregation of bland cells with features of monocytes associated with small strands and nodules of mesothelial cells, fat cells, fibrin and a minute fragment of bone. Immunohistochemical analysis showed that the monocytic cells were positive for CD4 and CD68 (strong) and negative for calretinin and keratin. By contrast, the mesothelial cells were positive for calretinin and keratin and negative for all other markers. In sum, the morphologic and immunohistochemical findings support the diagnosis of MICE. Based on our review of the literature, about 60 cases of MICE have been reported previously which we have tabulated. We also discuss the differential diagnosis.

From the archives of MD Anderson Cancer Center: Monomorphic epitheliotropic intestinal T-cell lymphoma: A case with an unusual immunophenotype and discussion of differential diagnosis.

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare and aggressive T-cell neoplasm associated with poor survival. We report a case of MEITL that presented as an ulcerated mass in the jejunum with perforation. Microscopic examination showed that the neoplasm involved the full thickness of the intestinal wall, extended into the mesentery, and was composed of monomorphic, small to medium-size cells. Immunohistochemical analysis showed that the neoplastic cells were positive for T-cell receptor (TCR) delta, CD3, CD7, CD8 (small subset), BCL-2 and TIA-1, and negative for TCR beta, CD4, CD5, CD10, CD20, CD30, CD34, CD56, CD57, CD99, ALK, cyclin D1, granzyme B, MUM1/IRF4, and TdT. The Ki-67 proliferation index was approximately 50 %. In situ hybridization for Epstein-Barr virus-encoded RNA (EBER ISH) was negative. Next-generation sequencing (NGS) analysis showed mutations involving SETD2 and STAT5B. The patient was treated with aggressive chemotherapy and consolidative autologous stem cell transplant and had clinical remission, but relapsed after about one year. Retreatment led to another one-year interval of clinical remission, but at last follow up the patient has relapsed disease involving the ileum and colon. We also discuss the differential diagnosis of MEITL.

The Spectrum of Non-neoplastic Changes Associated With Breast Implants: Histopathology, Imaging, and Clinical Significance.

Breast implant-associated anaplastic large cell lymphoma has been recognized as a distinct entity in the World Health Organization classification of hematolymphoid neoplasms. These neoplasms are causally related to textured implants that were used worldwide until recently. Consequently, there is an increased demand for processing periprosthetic capsules, adding new challenges for surgeons, clinicians, and pathologists. In the literature, the focus has been on breast implant-associated anaplastic large cell lymphoma; however, benign complications related to the placement of breast implants occur in up to 20% to 30% of patients. Imaging studies are helpful in assessing patients with breast implants for evidence of implant rupture, changes in tissues surrounding the implants, or regional lymphadenopathy related to breast implants, but pathologic examination is often required. In this review, we couple our experience with a review of the literature to describe a range of benign lesions associated with breast implants that can be associated with different clinical presentations or pathogenesis and that may require different diagnostic approaches. We illustrate the spectrum of the most common of these benign disorders, highlighting their clinical, imaging, gross, and microscopic features. Finally, we propose a systematic approach for the diagnosis and handling of breast implant specimens in general.

TP53 mutation is frequent in mantle cell lymphoma with EZH2 expression and have dismal outcome when both are present.

Enhancer of zeste homolog 2 (EZH2) expression is found in about 40% of mantle cell lymphoma (MCL) patients, which is associated with aggressive histology, high Ki-67 proliferation rate, p53 mutant pattern and inferior overall survival (OS). We conducted 11-gene (ATM, BIRC3, CCND1, KMT2C, KMT2D, NOTCH1, NOTCH2, RB1, TP53, TRAF2 and UBR5) next generation sequencing panel to shed more light on MCL with EZH2 expression (EZH2+ MCL). EZH2+ MCL more frequently harbor TP53 mutation compared to EZH2(-) MCL (41.2% vs. 19.1%, respectively, p = 0.045). TP53 mutation and EZH2 expression demonstrated overlapping features including aggressive histology, high Ki-67 proliferation rate and p53 mutant pattern by immunohistochemistry. Comparative analysis disclosed that EZH2 expression correlates with high Ki-67 proliferation rate irrespective of TP53 mutation. Aggressive histology is associated with EZH2 expression or TP53 mutation, possibly via independent mechanisms. p53 mutant pattern is due to TP53 mutation. MCL patients with EZH2 expression or TP53 mutation show inferior outcome and when both are present, patients have dismal outcome.

Fifth Edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues: Acute Lymphoblastic Leukemias, Mixed-Phenotype Acute Leukemias, Myeloid/Lymphoid Neoplasms With Eosinophilia, Dendritic/Histiocytic Neoplasms, and Genetic Tumor Syndromes.

This manuscript represents a review of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia/lymphoblastic lymphoma), acute leukemias of ambiguous lineage, mixed-phenotype acute leukemias, myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangements, histiocytic and dendritic neoplasms, and genetic tumor syndromes of the 5th edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The diagnostic, clinicopathologic, cytogenetic, and molecular genetic features are discussed. The differences in comparison to the 4th revised edition of the World Health Organization classification of hematolymphoid neoplasms are highlighted.

Multicolor flow cytometric immunophenotyping is highly sensitive and specific in identifying aberrant mast cells in the diagnostic workup of systemic mastocytosis.

OBJECTIVES: Flow cytometric immunophenotyping (FCI) is a fast and sensitive method for characterizing hematolymphoid neoplasms. It is not widely used in the workup of systemic mastocytosis (SM), in part because of the technical challenges and in part because the utility of FCI in assessing mast cells is not well understood. The objectives of this study were to assess the diagnostic utility of FCI in establishing a diagnosis of SM and distinguishing SM from nonneoplastic mast cells and to examine the immunophenotypic findings among SM subtypes. METHODS: We performed FCI on bone marrow samples suspicious for SM using a panel consisting of CD2, CD25, CD30, CD45, CD117, and HLA-DR. RESULTS: The cohort included 88 SM cases: 67 without an associated hematologic neoplasm (AHN) (PureSM) and 21 with an AHN (SM-AHN). We also assessed 40 normal/reactive controls. Overall, FCI was adequate for interpretation in 87 of 88 (99%) cases and detected at least 1 immunophenotypic aberrancy in 100% of SM cases. CD2, CD25, and CD30 were positive in 78%, 98%, and 90% of SM cases vs 0%, 13%, and 13% of cases with normal/reactive mast cells (P < .0001 for all). Two or 3 abnormalities were observed in 92% of SM cases but not in normal/reactive mast cells. Among SM cases, SM-AHN showed statistically significant less CD2 (38% vs 91%, P < .0001) and less co-expression of all 3 aberrant markers (CD2, CD25, and CD30 positive in 38% vs 86% of cases; P < .0001) than PureSM. Immunohistochemical analysis showed consistently weaker or focal expression of CD2, CD25, and CD30 than FCI, with CD2 and CD30 being falsely negative in 40% and 50% cases, respectively. A KIT D816V mutation was detected in 67% of PureSM cases and 76% of SM-AHN cases. CONCLUSIONS: Flow cytometric immunophenotyping is a quick, sensitive, high-yield tool for evaluating the immunophenotype of mast cells. An abnormal FCI finding should prompt careful histologic evaluation and sensitive KIT D816V mutation testing to address the possibility of SM. CD2, CD25, and CD30 are important markers for the detection of immunophenotypic aberrancy of mast cells, and their frequencies of aberrancy differ across SM subtypes.

Potential genetic mechanisms driving B/myeloid conversion in patients with follicular lymphoma and Langerhans cell neoplasms.

Transformation of follicular lymphoma (FL) to a Langerhans cell (LC) neoplasm is extremely uncommon. The shared IGH::BCL2 rearrangement is a robust finding in most transformed tumors underscoring that the cell of origin is perhaps a pre-B cell harboring IGH::BCL2 with the propensity to undergo further genetic alterations in the germinal centers of lymph nodes: does IGH::BCL2 in pre-B cells set off a plasticity cell state? Do FL and LC neoplasms develop separately through a common progenitor or via a multistep process of transdifferentiation or dedifferentiation/redifferentiation? Here, we review the literature and relevant cases presented in the Society for Hematopathology/European Association of Haematopathology 2021 Workshop to better understand this rare and complex phenomenon. We discuss clinical data, clonal relationship, and the mutational profile of these tumors and review proposed mechanisms of B/myeloid conversion based on in vitro and in vivo models.