B Lymphoblastic Leukemia/lymphoma Research Articles

An unusual case of a de novo high-grade B cell neoplasm with MYC and BCL2 rearrangements, strong TdT expression, and BRAF V600E mutation

Abstract Introduction/Objective Aggressive de novo B cell neoplasms can rarely exhibit overlapping features of B- lymphoblastic leukemia/lymphoma (B-ALL) and high-grade B cell lymphoma (HGBL) making their definitive classification challenging. The distinction between mature and immature B cell neoplasms is critical as treatment approaches differ. We report a unique case of an aggressive B cell neoplasm that not only posed diagnostic difficulties but was also found to harbor a BRAF V600E mutation, an unusual finding in such cases. Methods/Case Report A 33-year-old man without any significant past medical history presented for evaluation of a large tonsil mass and diffuse lymphadenopathy. The tonsil was excised, and histologic examination showed a diffuse atypical infiltrate composed of medium to large monomorphic lymphoid cells with high-grade cytology. By immunohistochemistry, the atypical cells were positive for CD19, CD79a, CD22, PAX5, CD10 (strong), CD45 (strong), BCL2, MYC, and TdT (strong). Ki67 showed a high proliferation index (80%). The atypical cells were negative for CD20, CD34, CD1a, CD30, Cyclin D1, T cell markers, myeloid markers, HHV8, and EBER. Flow cytometry detected a CD10 positive B cell population that was negative for CD20, CD34, and surface light chain expression. Fluorescence in-situ hybridization (FISH) detected BCL2 and MYC rearrangements. Staging marrow showed diffuse involvement by a neoplastic infiltrate with similar features to those observed in the tonsil. Chromosome analysis showed a complex karyotype. The patient was treated with two cycles of da-EPOCH-R. A neck lymph node biopsy two months later showed persistent disease. Next-generation sequencing (NGS) on this sample demonstrated a BRAF V600E mutation at 31% allele frequency. A mutation-specific BRAF-VE1 immunostain was diffusely positive. Results (if a Case Study enter NA) NA Conclusion This was a diagnostically challenging case of a de novo B-cell lineage neoplasm with high-grade features, MYC and BCL2 rearrangements, and strong diffuse TdT expression. There is limited literature on mutational profiles and prognostic data for such cases, and whether they are best classified (and treated) as HGBL or B-ALL remains challenging and controversial. In our case, we also discovered an unexpected finding of a BRAF V600E mutation, which has not been reported in similar cases in the existing literature and could serve as a potential therapeutic target. Additional reporting and genetic profiling of such cases may help further elucidate their biology and guide treatment protocols.

Automated Machine Learning-Based Diagnosis and Molecular Characterization of Acute Leukemias using Flow Cytometry Data

Abstract Current flow cytometric analysis of blood and bone marrow samples for the diagnosis of acute leukemias relies heavily on manual intervention in both the processing and analysis steps, introducing significant subjectivity into the resulting diagnosis and increasing diagnostic turn-around time. Additionally, concurrent molecular characterization of these samples via cytogenetics and targeted sequencing panels can take multiple days, thereby delaying patient diagnosis and treatment. Attention-based multi-instance learning models are machine learning models that can make accurate predictions and generate interpretable insights regarding the classification of a sample from multiple events/cells; while these models have been developed for anatomic pathology applications, they have yet to be applied to flow cytometry data. By utilizing 1,820 flow cytometry samples from 2019-2022 at Brigham and Women’s Hospital, we developed attention-based multi-instance machine learning models for automated diagnosis of acute leukemia, including differentiation of acute myeloid leukemia (AML) from B-lymphoblastic leukemia/lymphoma (B-ALL). Additionally, using concurrent cytogenetic and targeted sequencing data from 674 acute leukemia samples, machine learning models for prediction of molecular aberrancies from flow cytometry data were developed. Machine learning models were created using the TabNet deep learning architecture and VIME self-supervised training algorithm, which are state-of-the-art approaches towards machine learning from tabular data including flow cytometry data. Attention-based multi-instance models demonstrated strong performance for the automated diagnosis of acute leukemia versus non-leukemia samples (AUROC 0.869), as well as the separation of AML from B-ALL samples (AUROC 0.971). These models also accurately predicted cytogenetic aberrancies among AML samples, including t(15;17);PML::RARA (AUROC 1.00), as well as mutations including NPM1 (AUROC 0.725). These models do not require any manual intervention including compensation or gating, and additionally provide quantitative scores for the relative importance of different flow cytometry events and markers for the diagnosis of a particular sample. These importance scores can be integrated into flow cytometry analysis software for visualization and interpretation by hematopathologists. In this study, we have demonstrated the capability of machine learning models to provide automated diagnoses of acute leukemia, as well as accurately predict cytogenetic and molecular aberrancies in blood and bone marrow samples using flow cytometry data. This automated workflow can significantly decrease diagnostic turn-around time and ultimately improve patient outcomes.

Highly sensitive single tube B-lymphoblastic leukemia/lymphoma minimal/measurable residual disease test robust to surface antigen directed therapy.

Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both. We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments. The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10-5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1). The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy.

Blastoid B-Cell Neoplasms: Diagnostic Challenges and Solutions.

Blastoid B-cell neoplasms mainly include B-lymphoblastic leukemia/lymphoma (B-ALL), blastoid mantle cell lymphoma, and high-grade B-cell lymphoma with blastoid morphologic features (blastoid HGBL). Distinguishing blastoid HGBL from B-ALL can be challenging and we previously developed six-point flow cytometry-focused and three-point immunohistochemistry-focused scoring systems to aid in differential diagnosis. However, the six-point scoring system was derived from bone marrow cases and occasional cases may have a misleading score using either system. In this study, we assessed 121 cases of blastoid-HGBL (37 BM and 84 extramedullary) to validate the six-point scoring system in all tissue types and to further compare the two scoring systems. Compared with 47 B-ALL cases enriched for CD34-negative neoplasm, the 121 blastoid-HGBL cases showed distinctive pathologic features. The six-point scoring system showed a sensitivity of 100%. A comparison of the two scoring systems in blastoid HGBL (n = 64) and B-ALL (n = 37) showed a concordance score rate of 88%. Thirteen cases showed misleading scores, including five HGBL and eight B-ALL, and the diagnosis was further validated by gene transcriptome profiling. Twelve of thirteen cases had discordant scores between the two scoring systems. Simultaneous employment of both scoring systems improved the accuracy of classification of blastoid B-cell neoplasms to 99%. In conclusion, the previously defined six-point scoring system showed an excellent performance regardless of the tissue origin. Using both scoring systems together improves the accuracy of classification of blastoid B-cell neoplasms. Cases with discordant scores between the two scoring systems were extremely challenging neoplasms and classification required correlation with all available clinical and genetic features.

Circulating CD22+/CD19-/CD24- progenitors and CD22+/CD19+/CD24- mature B cells: Diagnostic pitfalls for minimal residual disease detection in B-lymphoblastic leukemia.

Multiparametric flow cytometry (MFC) has become a powerful tool in minimal residual disease (MRD) detection in B-lymphoblastic leukemia/lymphoma (B-ALL). In the setting of targeted immunotherapy, B-ALL MRD detection often relies on alterative gating strategies, such as the utilization of CD22 and CD24. It is important to depict the full diversity of normal cell populations included in the alternative B-cell gating methods to avoid false-positive results. We describe two CD22-positive non-neoplastic cell populations in the peripheral blood (PB), including one progenitor population of uncertain lineage and one mature B-cell population, which are immunophenotypic mimics of B-ALL. Using MFC, we investigated the prevalence and phenotypic profiles of both CD22-positive populations in 278 blood samples from 52 patients with B-ALL; these were obtained pre- and post-treatment with CD19 and/or CD22 CAR-T therapies. We further assessed whether these two populations in the blood were exclusively associated with B-ALL or recent anticancer therapies, by performing the same analysis on patients diagnosed with other hematological malignancies but in long-term MRD remission. The progenitor population and mature B-cell population were detected at low levels in PB of 61.5% and 44.2% of B-ALL patients, respectively. Both cell types showed distinctive and highly consistent antigen expression patterns that are reliably distinguishable from B-ALL. Furthermore, their presence is not restricted solely to B-ALL or recent therapy. Our findings aid in building a complete immunophenotypic profile of normal cell populations in PB, thereby preventing misdiagnosis of B-ALL MRD and inappropriate management.

Recurrent Novel P2RY8/IGH Translocations in B-Lymphoblastic Leukemia/Lymphoma.

Translocations involving the immunoglobulin heavy chain (IGH) locus are common abnormalities in B-lymphoblastic leukemia/lymphoma (B-ALL) and multiple myeloma. These rearrangements result in a juxtaposition of IGH enhancers to the vicinity of oncogenes, such as MYC and CRLF2, leading to the upregulation of oncogenes. Here, we identified recurrent novel P2RY8/IGH translocations in three B-ALL patients by transcriptome sequencing. Noncoding exon 1 of P2RY8 was translocated to different sites of the IGH gene, resulting in transcripts of P2RY8/IGHM, P2RY8/IGHV, and P2RY8/IGHD. However, a high expression level of truncated P2RY8 was observed in the patients compared with healthy donors, which might be related to the aggressive clinical course and inferior outcome. In summary, we described recurrent novel P2RY8/IGH translocations with high expression levels of P2RY8, which may contribute to the guidelines for clinical diagnosis and treatment.

Synchronous plasma cell neoplasm and B lymphoblastic leukemia/lymphoma at initial presentation: first report of an unusual association with a good outcome.

A synchronous diagnosis of a plasma cell neoplasm (PCN) and a non-plasma cell hematologic malignancy is very rare. We report what we believe is the first instance of a synchronous PCN and B lymphoblastic leukemia/lymphoma (B-ALL) diagnosed at initial presentation. The patient underwent laboratory evaluation for an underlying plasma cell neoplasm, including immunology studies, bone marrow biopsy, and flow cytometry immunophenotyping. Serum lambda free light chain and serum IgG were elevated, with an IgG lambda M-protein identified by serum protein electrophoresis and immunofixation. The clinical working diagnosis was plasma cell myeloma. Bone marrow biopsy was positive for a composite PCN and B-ALL. The patient received treatment with VDT-PACE chemotherapy followed by autologous stem cell transplant and maintenance therapy with bortezomib/daratumumab and is in complete remission for both diseases 3.5years after diagnosis. This case not only adds to the known repertoire of hematologic neoplasms that can occur in association to a PCN, but also demonstrates that patients presenting with this rare combination of hematopoietic neoplasms can be effectively treated simultaneously with excellent responses. Additional research is warranted to understand the pathophysiology, to identify potential prognostic factors, and to develop specific therapeutic plans for these patients.

Abnormal B-lymphoblasts in myelodysplastic syndromes and myeloproliferative neoplasms other than chronic myeloid leukemia.

Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN). We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL). We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL. These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation.

Development of B-lymphoblastic leukemia in patients treated for multiple myeloma – A case series of 14 patients

Abstract Development of B-lymphoblastic leukemia/lymphoma (BALL) is uncommon in patients with plasma cell neoplasm (PCN). Currently, little is known about risk factors for and characteristics of BALL arising in patients who have undergone treatment for PCN. We reviewed the clinicopathologic features of 14 patients with PCN who developed subsequent BALL and assessed the clonal relationship between these two neoplasms in 5 of the 14 patients. Clinical and laboratory data were extracted from the electronic medical record in accordance with an institutional review board-approved protocol. For the IgH clonality studies DNA was extracted from formalin fixed paraffin embedded tissue and analyzed utilizing the LymphoTrack® IGH FR2/3 Assay -- S5/PGM™ reagents from Invivoscribe Technologies. Next-generation sequencing of libraries was performed on a ThermoFisher Scientific Ion Torrent S5™ sequencer. The LymphoTrack® Software - S5/PGM™ Version 2.4.5 was used to analyze the IGH rearrangement sequences and relative proportion of the sequences was determined as a percentage of total sequencing reads. All PCN patients in our cohort received an autologous stem cell transplant (auto-SCT) with subsequent lenalidomide maintenance therapy. The mean time from the start of lenalidomide treatment after auto-SCT to BALL development was 62 months. None of the BALL cases were Philadelphia chromosome-positive. There was increased incidence of TP53 mutations and deletions (36%). IgH clones were identified in all but one sample. Comparison of the IgH clones from the original PCN and subsequent BALL revealed distinct clones in all 5 patients tested. In 2 of the 5 patients, the original PCN clone was also identified along with the distinct BALL clone. Our study reveals that BALL cases arising in patients with treated PCN are not clonally related to the original disease, and may represent therapy-related malignancy associated to prior treatment including lenalidomide maintenance therapy. More studies are needed to establish the incidence and understand the pathogenesis of this possible therapy-related complication.

PAX5 P80R mutated acute leukemia followed by genetically-related histiocytic proliferation with clonal IGH gene rearrangements

Abstract Introduction/Objective PAX5 is a transcription factor critical for B-cell development and its genetic alterations are common in B lymphoblastic leukemia/lymphoma (B-ALL). We report a case of PAX5 P80R mutated acute leukemia with predominantly monocytic immunophenotype followed by a genetically-related histiocytic proliferation. Methods/Case Report A 27-year-old male presented with pancytopenia, epistaxis, and blurry vision. Bone marrow exam showed 95% blasts with nuclear indentations, occasional prominent nucleoli and basophilic cytoplasm. Blasts were positive for bright CD33, HLA-DR, CD14, bright CD64, partial CD123 and partial CD19. In addition, a minute population of B lymphoblasts positive for CD19, CD20, and dim TdT was seen. The AML FISH panel showed markedly aneuploid population with all probes demonstrating 3 to 5 signals. PAX5 P80R and KRAS G13D mutations were detected by NGS. Post induction bone marrow showed no evidence of acute leukemia with normal cytogenetics and FISH results. Subsequent two bone marrow exams performed due to progressive cytopenias demonstrated a prominent histiocytic proliferation with sheets of mature appearing histiocytes with abundant cytoplasm, oval to folded nuclei and occasional hemophagocytosis. This population was positive for CD163, CD14, CD68R, CD68, cyclin D1 with variable OCT2 expression and low proliferative activity. PAX5 P80R and KRAS G13D were detected. AML FISH panel showed aneuploidy in histiocytic appearing cells and IgH gene rearrangement studies by PCR showed prominent clonal population. The patient remained pancytopenic and died of disseminated fungal infection. Results (if a Case Study enter NA) NA Conclusion PAX5 P80R mutation has been primarily reported in B-ALL and is exceedingly rare in acute myeloid leukemias. This alteration has been linked to downregulation of B-cell lineage genes affecting B-cell maturation, and not surprisingly a proportion of PAX5 P80R mutated B-ALL cases show a switch to a monocytic lineage. The reported case demonstrates diagnostic caveats including unusual features of acute leukemia at the time of initial diagnosis and subsequent genetically-related histiocytic proliferation.

CD123 Expression Patterns and Potential of IMGN632, a CD123-Targeted Antibody Drug Conjugate, in Acute Lymphoblastic Leukemia

Background: The interleukin (IL)-3 receptor α-chain, CD123, is the primary low-affinity subunit of the IL-3 receptor. CD123 is expressed at various levels in hematologic malignancies and has thus become an attractive target of therapy. While CD123 expression in precursor B-lymphoblastic leukemia/lymphoma (B-ALL) has been reported, only limited data in T-ALL exists. Furthermore, the potential of CD123-targeted therapies in ALL remains largely unexplored. In this study, we examine CD123 expression levels in a large cohort of ALL patients and assess the impact of IMGN632, a conjugate of a CD123-binding antibody with the novel DNA-alkylating IGN payload DGN549, on in vitro viability of patient ALL cells and cell lines.Methods: The study cohort (n=188) included 162 B-ALL and 26 T-ALL patients diagnosed and treated at the University of Texas MD Anderson Cancer Center between 12/2012 and 1/2017. Among B-ALL patients, 59 (36%) were Philadelphia chromosome-positive (Ph+). T-ALL patients were further subtyped into the following immunophenotypic categories: early, early T precursor (ETP), thymic, and mature. CD123 expression was evaluated using multiparameter flow cytometry (MFC). Blasts were defined by lineage markers as well as CD45dim blast gate, and CD123 expression was measured for the percentage of positivity and mean fluorescence intensity (MFI) ratio over controls. An arbitrary cutoff of 20% was used for expressing nominal MFC results. Median follow up duration was 21.1 months. CD123 expression was correlated with clinical, laboratory, and outcomes results. The number of binding sites for IMGN632 on ALL blast and cell lines was quantified by QuantiBRITE method. Cytotoxicity of IMGN632 was evaluated after continuous exposure up to five days by WST-8 assays on cell lines or by MFC on primary ALL patient samples.Results: Patients had a median age of 46 years (range, 16-88 years) and included 117 (62%) men. The median CD123 positivity on ALL blasts was 78% (range, 0.3-100) with a median MFI ratio of 11 (range, 0-203). For categorical analysis purposes, we used a standard cutoff of 20% to define positive CD123 expression. Prevalence of CD123 expression was overall higher in B-ALL compared to T-ALL [145 (90%) vs. 11 (42%); p<0.001]. Expression prevalence was highest in Ph+ B-ALL (57/59; 97%) followed by Ph- B-ALL (88/103; 85%) and T-ALL (11/26; 42%). In Ph- B-ALL, CD123 expression correlated with CD34 expression (p=0.002) and lower bone marrow blast count (p=0.003). In T-ALL, CD123 expression correlated with immunophenotypic subtypes (early) (p=0.006). To test the potential of targeting CD123 in ALL, B-ALL cell lines (n=12) which express CD123 at levels similar to AML patient samples were exposed to titrated IMGN632 levels. IMGN632 IC50 values ranged between 0.6 and 20 pM, and the conjugate cytotoxicity was highly CD123-specific, as masking CD123 with a competing anti-CD123 antibody reduced the potency more than 1000-fold. To test the anti-leukemia activity of IMGN632 on primary B-ALL cells, bone marrow cells from B-ALL patients were exposed to IMGN632 following which blasts and non-malignant cells were enumerated by MFC. Notably, IMGN632 eliminated more than 90% of B-ALL blasts in 6 out of 8 samples from newly-diagnosed as well as relapsed-refractory patients at low pico-molar concentrations. Normal cells were not affected by IMGN632 at 100-fold higher concentrations.Conclusions: CD123 expression is prevalent across ALL subtypes, including 90% of B-ALL and 42% of T-ALL. CD123 expression was associated with CD34 expression in B-ALL and immunophenotype in T-ALL. The CD123 targeted antibody-drug conjugate IMGN632 demonstrates promising activity and safety in pre-clinical models of B-ALL._ [Display omitted] DisclosuresDaver:Incyte Corporation: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy. Zweidler-McKay:ImmunoGen: Employment. Kantarjian:ARIAD: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Kiromics: Research Funding; Pfizer: Research Funding; Angle: Research Funding.

How I Diagnose Minimal/Measurable Residual Disease in B Lymphoblastic Leukemia/Lymphoma by Flow Cytometry.

Assessment for minimal/measurable residual disease (MRD) is a powerful prognostic factor in B lymphoblastic leukemia/lymphoma (B-LL/L) that is quickly becoming standard of care in assessing patients with B-LL/L posttherapy. MRD can be assessed using methodologies including flow cytometry and molecular genetics, with the former being rapid, relatively inexpensive, and widely applicable in many hematopathology/flow cytometry laboratories. This article presents an approach to MRD detection in B-LL/L by flow cytometry through case presentations with illustration of several potential pitfalls. We review normal maturation patterns, antigens used for assessment, flow panels that can be utilized, considerations to be made during therapy, and clinical impact. The benefits and drawbacks when using the "different from normal" and "leukemia associated phenotype" approaches are considered. Evaluation for MRD in B-LL/L by flow cytometry relies on a knowledge of normal immunophenotypic patterns associated with B-cell maturation in states of rest and marrow regeneration so that one can identify patterns of antigen expression that differentiate abnormal, leukemic populations from regenerating hematogones or B-cell precursors. The nature of therapy can affect normal patterns, a phenomenon especially important to take into consideration given the increased use of targeted therapies in the treatment of B-LL/L. Flow cytometry is widely available in many laboratories and is a cost-effective way to evaluate for B-LL/L MRD. However, panel validation and interpreter education are crucial for accurate assessment.

Relationship between CD45 Expression and Outcomes in B Lymphoblastic Leukemia/Lymphoma

Introduction : B lymphoblastic leukemia/lymphoma (B-ALL) constitutes the most common type of lymphoblastic leukemia, with about two thirds of the cases being of B-cell lineage. It represents a significant cause of cancer related morbidity and mortality in both pediatric and adult populations. B-ALL immunophenotype has been extensively studied and used for diagnosis. Recent data underscores the importance of the CD45 transmembrane phosphatase in the modulation of signal transduction pathways in leukemic cells. In fact, the gene encoding CD45, PTPRC, has been identified as a tumor suppressor that is deleted in 10% of cases of T-ALL. Based on the impact of CD45 on leukemic cell biology, we hypothesized a relationship between CD45 protein expression and clinical parameters. Methods : This is a retrospective, single center analysis of patients diagnosed with B-ALL at Indiana University Simon Comprehensive Cancer Center from December 2003 until December 2018. Patients were followed until July 2020. Variables included patient demographics, and clinical and treatment characteristics. B-ALL diagnosis, molecular profiles, and clinical outcomes were abstracted by chart review. Descriptive statistics were used to summarize baseline characteristics and outcomes. Flow cytometry analysis was performed using 4- and 10-color leukemia screening panel. Median and geometric mean of CD45 antigen intensity was analyzed using FCS Express V6 software. Statistical analysis was performed using SPSS statistical package. Results: A total of 148 BALL patients were included in the analysis. 19 (12.8%) were adults (&amp;gt;18 years old) and 13 (8.7%) were Philadelphia chromosome positive. Average Median CD45 expression and CD45 Geometric Mean were 22.9 and 21.9 respectively for the entire cohort. Relevant clinical outcomes collected included complete remission (CR) at days 14 and 29, presence of minimal residual disease (MRD) at days 7 and 29, relapse of disease and death. Mann-Whitney Test was used to establish association between CD45 levels and clinical outcomes. A significant association was found between achieving CR at 29 days and lower levels of CD45 expression (as defined by median and geometric mean, p=0.03 for both). The association was was maintained when excluding Philadelphia positive cases (p=0.05 for both) and excluding patients over 30 years old (p=0.05 for both). There were no significant correlations between CD45 expression and other clinical variables. Conclusions: Lower CD45 expression as determined by flow cytometry is associated with higher rates of CR at 29 days. This finding is significant across different age groups and Philadelphia negative cohorts. This finding needs to be explored in further studies. CD45 protein expression levels could be an important biomarker for adjusting induction chemotherapy to achieve complete remission. Disclosures No relevant conflicts of interest to declare.

Transformation of de novo high-grade B cell lymphoma with MYC and BCL2 rearrangements to double-hit B lymphoblastic leukemia/lymphoma: a case report and review of literature

High-grade B cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (“double-hit” lymphoma (DHL)/“triple-hit” lymphoma (THL)) are mature B cell neoplasms with highly aggressive clinical behavior and poor response to therapy. Lymphoblastic transformation of mature B cell neoplasms is an uncommon event that is best recognized for follicular lymphoma (FL). To our knowledge, only one case of “B lymphoblastic” transformation of de novo DHL has been reported in the literature. Here, we describe another case of such transformation. The patient was a 57-year-old man who presented with hypercalcemia, high lactate dehydrogenase (LDH), and multiple lytic bone lesions and was diagnosed with high-grade B cell lymphoma with MYC and BCL2 rearrangements. The neoplastic cells were positive for CD45, CD19, CD20, CD79a, PAX5, CD10, BCL6, and BCL2 and negative for CD34, CD99, and terminal deoxynucleotidyl transferase (TdT). The patient received chemotherapy followed by autologous stem cell transplant and achieved complete remission. Nine months after the initial presentation, he developed general weakness and found to have cytopenias and circulating blasts. Bone marrow examination revealed extensive involvement by a high-grade B cell neoplasm co-expressing PAX5, CD10, and BCL2, in addition to precursor markers TdT and CD99. There was loss of CD20, CD79a, and BCL6 expression, and CD34 remained negative. A diagnosis of B lymphoblastic leukemia/lymphoma (B-LBL) was established. Similar MYC and BCL2 rearrangements were identified. IGH gene rearrangement studies confirmed clonal relatedness. The patient passed away 3 days after bone marrow examination. This case represents an extremely rare case of DHL transformation to B-LBL.

Improved Recognition of Hematogones From Precursor B-Lymphoblastic Leukemia by a Single Tube Flow Cytometric Analysis.

To improve diagnostic accuracy in differentiating hematogones from leukemic blasts in cases of precursor B-lymphoblastic leukemia/lymphoma (B-ALL), particularly those that are posttreatment or after bone marrow transplant, and to provide an algorithmic approach to this diagnostic challenge. A seven-color antibody panel including CD10, CD19, CD45, CD38, CD34, CD58, and CD81 was generated to assess the feasibility of a single tube panel and provide an algorithmic approach to distinguish hematogones from B-ALL. Fifty-three cases were analyzed, and results were correlated with histology and ancillary studies. There was a significant difference in mean fluorescent intensity (MFI) for CD81 and CD58 when comparing hematogones and B-ALL populations (P < .001). B-ALL cases had a mean (SD) MFI of 24.6 (27.5; range, 2-125) for CD81 and 135.6 (72.6; range, 48-328) for CD58. Hematogones cases had a mean (SD) MFI of 70.2 (19.2; range, 42-123) for CD81 and 38.8 (9.4; range, 23-58) for CD58. The flow cytometry panel with the above markers and utilization of the proposed algorithmic approach provide differentiation of hematogones from B-ALL. This includes rare cases of hematogones and B-ALL overlap where additional ancillary studies are necessary.

Immature Terminal Deoxynucleotidyl Transferase Positive B Cells are Detected in a Subset of Adult and Pediatric Liver Biopsies.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear enzyme restricted to precursor lymphoid cells and their malignant counterparts; immunohistochemical TdT labeling is helpful in recognition of lymphoblasts, which can resemble mature lymphocytes. The diagnosis of B-lymphoblastic leukemia/lymphoma (B-ALL) is occasionally first encountered on liver core biopsy, but TdT immunostain specificity for B-ALL is not clearly established in this setting, which can be problematic when only a few TdT-positive cells are identified. In this study, we evaluated the incidence and distribution of immature B lymphocytes coexpressing TdT and PAX-5, in pediatric and adult liver biopsies, to determine whether a normal complement of hepatic immature B cells can be detected, which must be recognized in a workup to exclude B-ALL. We selected 41 pediatric and adult liver biopsies with a significant portal and/or sinusoidal hematolymphoid infiltrate and performed immunohistochemical stains for TdT and PAX-5 to identify and categorize distribution of immature B cells. TdT-positive cells were detected in 40% of pediatric liver biopsies with a significant hematolymphoid infiltrate (4/10), which included all biopsies from neonates (and infants under 9 wk of age). In adults, immature B-cell infiltrates were less common (6%, 2/31). Dual immunostaining was performed on 2 cases of neonatal hepatitis, which documented B-cell lineage in at least a subset of TdT-positive cells and there was no colabeling with CD3. Immature B cells can be detected in liver biopsies in a variety of clinical settings, most commonly in children, and presence of a few TdT-positive cells cannot be considered entirely specific for involvement by B-ALL. Further workup for B-ALL can be warranted if there is more extensive multifocal portal and/or sinusoidal involvement by blasts with TdT labeling.

49. A single institution experience of CRLF2 rearranged pediatric acute lymphoblastic leukemia/lymphoma

B-lymphoblastic leukemia/lymphoma (B-ALL) BCR-ABL1-like (Ph-like) is a provisional entity in the 2016 revision of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. The incidence of Ph-like ALL is 15-30% in children and young adults with B-ALL. Genetic abnormalities associated with Ph-like ALL are heterogenous. Approximately 50% of Ph-like ALL patients may have cytokine receptor like factor 2 (CRLF2) gene overexpression, JAK2 mutations with activation of JAK-STAT pathway and a poor clinical outcome. Due to recent advances in the genomic testing of B-ALL, patients can be stratified into different risk groups based on early treatment response to drugs and minimal residual disease (MRD).

B lymphoblastic leukemia/lymphoma: new insights into genetics, molecular aberrations, subclassification and targeted therapy.

B lymphoblastic leukemia/lymphoma (B-ALL) is a clonal hematopoietic stem cell neoplasm derived from B-cell progenitors, which mostly occurs in children and adolescents and is regarded as one of top leading causes of death related to malignancies in this population. Despite the majority of patients with B-ALL have fairly good response to conventional chemotherapeutic interventions followed by hematopoietic stem cell transplant for the last decades, a subpopulation of patients show chemo-resistance and a high relapse rate. Adult B-ALL exhibits similar clinical course but worse prognosis in comparison to younger individuals. Ample evidences have shown that the clinical behavior, response rate and clinical outcome of B-ALL rely largely on its genetic and molecular profiles, such as the presence of BCR-ABL1 fusion gene which is an independent negative prognostic predictor. New B-ALL subtypes have been recognized with recurrent genetic abnormalities, including B-ALL with intrachromosomal amplification of chromosome 21 (iAMP21), B-ALL with translocations involving tyrosine kinases or cytokine receptors (“BCR-ABL1-like ALL”). Genome-wide genetic profiling studies on B-ALL have extended our understanding of genomic landscape of B-ALL, and genetic mutations involved in various key pathways have been illustrated. These include CRLF2 and PAX5 alterations, TP53, CREBBP and ERG mutations, characteristic genetic aberrations in BCR-ABL1-like B-ALL and others. The review further provides new insights into clinical implication of the genetic aberrations in regard to targeted therapy development.

B-lymphoblastic leukemia/lymphoma arising in treated plasma cell myeloma: A rare second malignancy

Plasma cell myeloma (PCM) is a neoplasm of plasma cells, end-stage post germinal center B cells, which shows a variable clinical spectrum from asymptomatic to aggressive; the nature of the disease is generally progressive. Rarely, leukemia arises treated plasma cell myeloma. Acute myeloid leukemia/Myelodysplastic syndrome (AML/MDS) is most common, but rare cases of B lymphoblastic leukemia/lymphoma (B-ALL) have been described. The diagnosis of transformation or secondary B-ALL is supported by ancillary studies such as flow cytometry, immunohistochemistry, and molecular studies. Once the diagnosis of a B-ALL is made, consideration should is given as to whether the malignancy is a clonally related transformation of the original PCM or treatment related second leukemia. We present a case of B-ALL after PCM which does not appear to show a clonal relationship with the original PCM suggesting a treatment related B-ALL.

54 - Development of a Custom, Sensitive and Specific PCR Strategy for the Detection of ERG Deletions in Pediatric B- Lymphoblastic Leukemia/Lymphoma (B-ALL)

54 - Development of a Custom, Sensitive and Specific PCR Strategy for the Detection of ERG Deletions in Pediatric B- Lymphoblastic Leukemia/Lymphoma (B-ALL)