CD25 Positivity Research Articles

Reevaluating hybrid neurofibroma/schwannoma: Predominance of schwannoma features despite CD34 positivity and initial neurofibroma diagnosis.

Schwannomas consist of both high-cellularity regions (Antoni A area) and hypocellular regions (Antoni B area) in histopathological findings. Neurofibromas characteristically consist of CD34 positive spindle cells with thin, wavy, nuclei and wavy collagen bands. Previous reports have described segments of schwannomas with neurofibroma features as hybrid tumors, although hybrid tumors were diagnosed based on partial CD34 positivity in many previous reports. On the other hand, the Antoni B area of some schwannomas was reported to be positive for CD34. Therefore, the definition of a hybrid tumor has not been clear. The objective of this study was to determine whether only CD34 positive findings in schwannomas could be used to define a hybrid tumor. In the analysis of our patient with schwannomatosis caused by a novel LZTR1 germline mutation, part of the tumor had CD34 positive hypocellular regions. These regions contained no thin, wavy, nuclei, indicating an Antoni B area. Laser microdissection was used to investigate the genetic background and differences in molecular mechanisms between CD34 positive and CD34 negative regions. All mutations identified in CD34 positive regions were also found in CD34 negative regions. Our data could not clear the genetic background of Antoni B which was CD34 positive area. We then reviewed the pathologies of 66 sporadic schwannomas. Histopathological examinations of all schwannomas revealed the absence of thin, wavy, nuclei and wavy collagen bands, and no hybrid tumors were found in any of the cases. Ten of 66 patients were randomly selected for CD34 immunostaining and positivity was found in all cases. Our data suggest that it is difficult to distinguish schwannomas by staining for CD34 alone, as Antoni B areas can also be positive for CD34. Therefore, CD34 staining alone should not be used to diagnose hybrid tumors in patients with schwannomas.

Aberrant CD45 Immunoreactivity in Neuroendocrine Neoplasms: A Diagnostic Pitfall-Report of 10 Specimens and Clinical Recommendations.

Leukocyte common antigen (LCA), or CD45, is classically thought of as a leukocyte-exclusive protein, and as such, CD45 immunohistochemistry (IHC) is often used as a key differentiator between non-Hodgkin lymphomas (NHLs) and morphologically similar neuroendocrine neoplasms (NENs). Herein, we report our experience regarding aberrant CD45 immunoreactivity in a series of NENs. A natural language search was used to retrieve desired archival patient files. All prior NENs which had a positive neuroendocrine diagnosis or IHC results (synaptophysin, chromogranin, CD56, and/or neuron-specific enolase), as well as CD45 staining performed, were reviewed for possible CD45 positivity (n = 686). Among these 686 NENs, 10 were aberrantly positive for CD45 staining. CD45 showed nuclear, cytoplasmic, and/or membranous staining in tumor cells. The significance of such staining is unclear. Albeit for a minority of patients, pathologists should be aware that NENs may aberrantly stain with CD45 and thereby pose a diagnostic pitfall. Therefore, broadening routine IHC panels is recommended to differentiate NENs more clearly from NHLs.

Evaluation of Immunophenotypic Markers in Diagnosis and Prognosis of Atypical Chronic Lymphocytic Leukemia

Abstract: BACKGROUND: Atypical chronic lymphocytic leukemia diagnosis (aCLL) constitutes of monoclonal B lymphocytes of more than 5000 with unique morphological and immunophenotypic features different from classical or typical CLL. By immunophenotyping, aCLL is differentiated from typical CLL in the negative expression of either one or two immunophenotypic markers, mostly CD23 and/or CD5, provided that the patient does not have the diagnostic criteria of any other lymphoid neoplasm. aCLL patients are usually presented clinically in an aggressive manner and have worse outcomes compared to classic CLL. MATERIALS AND METHODS: This cross-sectional study includes 36 patients diagnosed as aCLL compared to 36 patients diagnosed as typical CLL. Diagnosis depends on blood film morphology and immunophenotyping by 8-color flow cytometry, the markers that are used for diagnosis depend on Matutes scoring system of CLL with the addition of CD200 and CD43, in addition to prognostic markers CD38 and CD305. The clinical staging system was applied, and the patients were divided into low-, intermediate-, and high-risk groups. Follow-up program for 6–12 months with evaluation of their hematological and clinical response and assessment of their remission state as was instituted in the hematological center. RESULTS: CD5 and CD23 are negative in 16.6% and 33.3% of aCLL patients, respectively, whereas CD200 and CD43 are positively expressed in 100% and 83.33% of aCLL patients, respectively, co-expression percentage of both markers in 83.33%. aCLL cases were commonly presented with high-risk group and usually have markers that are linked to poor prognosis, including negative expression of CD305 in 83.3% and positive CD38 expression in 75% of cases and only 5.56% of patients achieved remission state. CONCLUSIONS: aCLL is characterized by immunophenotypic differences in comparison to typical CLL. A definitive diagnosis needs additional markers, mainly CD200 and CD43; the prognosis of aCLL is worse than classical CLL.

Solving the mystery of Reed Sternberg like cells in a composite Peripheral T Cell Lymphoma with Epstein Barr Virus and Epstein Barr Virus driven Lymphoproliferative Disorder

Abstract Introduction/Objective Nodal PTCL with EBV positivity can occasionally contain Reed Sternberg-like cells, and EBV- LPD can mimic Hodgkin lymphoma. Coexistence of these two unusual lymphomas in the same lymph node is very rare. We present a case of a 56-year-old male with Composite EBV+ PTCL NOS and EBV LPD, with positive B-cell gene rearrangement and negative T-cell gene rearrangement presenting a significant diagnostic challenge. Methods/Case Report A 56-year-old male with a history of abdominal tuberculosis presented with lethargy, fatigue, and poor appetite. Physical examination revealed generalized lymphadenopathy. CT scan confirmed lymphadenopathy in bilateral axilla, supraclavicular lymph nodes along with lytic lesions on illiac crest. Ill defined splenic lesions raised concerns for metastasis. Cervical lymph node and bone marrow biopsies were performed. Flow cytometry on the lymph node was not done due to tuberculosis history. Flow cytometry on the bone marrow showed an inverted CD4:CD8 ratio. H&E sections of the lymph node revealed a polymorphic lymphoid infiltrate with prominent Reed Sternberg-like cells. Immunohistochemical staining showed CD30-positive and CD45-negative large cells. T-cell and B-cell gene rearrangements were performed. Initially, B-cell gene rearrangements were positive, while T-gamma was negative. Further testing by IHC and morphological evaluation revealed two distinct cell populations, one with CD3, CD5, and CD7 positivity with partial weak positivity for PD1, CD43, and CD8 and the other consisting of B-cells expressing CD30, EBV, MUM1, and partial expression of BCL6 rare CD79a, and partial weak Pax5. Outside molecular testing showed positive T-cell gene rearrangements. The diagnosis of Composite EBV+ Peripheral T Cell Lymphoma, not otherwise specified, with cytotoxic profile along with EBV driven LPD was made. Bone marrow studies confirmed involvement by the same lymphoma. Results (if a Case Study enter NA) NA Conclusion Identifying EBV-positive Reed Sternberg-like cells in both EBV LPD and PTCL-NOS in a composite lymphoma can be challenging. In the absence of flow cytometry, a composite lymphoma can potentially be misdiagnosed, leading to under-treatment. Repeat testing can be helpful in case of any doubt. Although EBV-positive PTCL with cytotoxic profile and EBV-positive LPD has been reported, the combination of these two lymphomas together is exceedingly rare.

CD33-Deleted Hematopoietic Cells (Trem-Cel) Are Protected from CD33xCD3 Bispecific Antibody Treatment and Produce Significantly Reduced Levels of Inflammatory Cytokines in Preclinical Studies

INTRODUCTION: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia. Currently, allogeneic hematopoietic stem cell transplant (HCT) is the standard of care for high-risk patients yet ~40% patients will relapse, emphasizing the need for new therapeutic approaches. Effective targeted therapy in AML has been hampered by a lack of cancer specific targets. One described AML target is CD33, a glycoprotein expressed predominantly on normal myeloid cells and the majority of AML cells. However, CD33-directed therapies have been challenged by cytopenias due to on-target, off-tumor toxicity and, in the case of bispecific antibodies, cytokine release syndrome (CRS). Tremtelectogene empogeditemcel (trem-cel) is a gene-edited hematopoietic stem and progenitor cell (HSPC) transplant product currently in clinical trial (NCT04849910) whereby CD33, shown to be dispensable for normal hematopoiesis and function, is deleted to allow for post-HCT treatment with CD33-directed immunotherapies with reduced myelotoxicity as the reconstituted hematopoietic compartment lacks CD33. JNJ-67571244, a bispecific antibody that binds to both CD3 and CD33, is capable of inducing T cell recruitment for CD33-directedtumor cytotoxicity. This study examines whether CD33 neg hematopoietic cells are protected from JNJ-67571244 and if CD33 neg human HSPC (hHSPC) xenotransplanted mice have reduced levels of inflammatory cytokines associated with CRS when treated with JNJ-67571244. METHODS: In vitro proof-of-concept (POC) studies were carried out using CD3 + T cells co-cultured with JNJ-67571244 and AML target cell lines. Further studies were conducted with CD34 + hHSPCs that were either CRISPR/Cas9 gene edited to delete CD33 or treated with a non-targeting gRNA control (gCtrl) followed by differentiation towards the monocytic lineage. These in vitro differentiated monocytes (MIVD) were assayed for editing frequency, CD33protein expression and myeloid phenotype by flow cytometry prior to co-culture with donor matched T cells and JNJ-67571244 ranging from 0.5 pM to 500 nM. Cytotoxicity was analyzed using flow cytometry at 48 and 72 hr and cytokine concentrations in the supernatants were measured using Luminex. For in vivo POC studies, CD33 or gCtrl-edited CD34 + hHSPCs were xenotransplanted into NSG-SGM3 mice followed by treatment with either control bispecific (CD3xNull) or JNJ-67571244 at 0.5 mg/kg. Plasma cytokine concentration were measured via Luminex and hematopoietic tissues were analyzed by flow cytometry to measure protection from JNJ-67571244 cytotoxic killing. RESULTS: Annexin V and Live/Dead staining showed that JNJ-67571244 was able to induce AML target cell killing by human T cells. Importantly CD33 neg MIVD cells co-cultured with T cells and JNJ-67571244 were significantly more viable at all doses compared to gCtrl cells. The EC 50 of gCtrl MIVD was 0.0208 nM whereas the CD33 neg MIVD EC 50 was at least 20,000-fold higher (>430.8 nM). T cell activation, measured by CD25 and CD69 positivity, and release of cytokines that are associated with CRS such as IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in the CD33 neg MIVD cells treated with JNJ-67571244 compared to the gCtrl group at all doses. Analysis of blood and bone marrow from xenotransplant studies revealed that JNJ-67571244 was effective at killing CD33 + cells as well as CD14 + monocytes in the gCtrl group treated with JNJ-67571244. Conversely, CD14 + monocytes and CD15 + neutrophils derived from CD33-edited hHSPCs, were not significantly reduced in cell number when treated with JNJ-67571244 suggesting that they were protected from JNJ-67571244 specific killing. IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in mice transplanted with CD33-edited hHSPCs compared to gCtrl group in response to JNJ-67571244. CONCLUSIONS: Taken together, these studies demonstrate that CD33 deleted hematopoietic compartment is protected from the CD33 directed immuno-therapy JNJ-67571244 both in in vitro cytotoxicity assays and preclinical xenotransplantation studies, with decreased concentrations of inflammatory cytokines associated with CRS. These findings enable the development of a next-generation AML treatment strategy by pairing trem-cel transplant with a subsequent CD33-directed bispecific compound to potentially improve safety and efficacy while minimizing myelotoxicity.

Cluster of Differentiation 38 Protein Significantly Adds to the Prognostic Value of Cytogenetic Risk in Patients with Acute Myeloid Leukemia.

There is some controversy regarding the clinical significance of cluster of differentiation 38 (CD38) protein in acute myeloid leukemia (AML). We aimed to study the prognostic value of CD38 expression in relation to the standard cytogenetic risk stratification in AML patients. In this prospective study, adult patients with AML were diagnosed based on the finding of ≥ 20% blasts in the bone marrow and the flow cytometry. The control group was selected from normal bone marrow samples examined at the same period in the lab. The CD38 expression levels were assessed by immunophenotyping the bone marrow samples of the patients prior to therapy, as well as those from the controls. The subjects were N = 52 newly diagnosed adult AML patients, while n = 25 sex-and-age matched normal volunteers served as the control. The CD38 expression among AML patients ranged from 3.6% to 79.9%, significantly higher levels than in the control population (p =0.001). Positive CD38 expression was higher in AML patients with favorable cytogenetics when compared to those with intermediate and poor cytogenetics (75.0%, 57.1%, and 68.2%, respectively) (p =0.007). Median CD38 expression in AML patients was higher in the favorable cytogenetic group and lower in intermediate and poor cytogenetic groups, though lacking in significance (p =0.578). Patients with poor cytogenetic risk were associated with significantly shorter median overall survival when compared to favorable and intermediate cytogenetic risk (p =0.010). The expression of CD38 significantly adds to the prognostic value of cytogenetic risk stratification at diagnosis of AML patients.

New Prognostic Indicators in CLL: Relation to Disease Burden

Abstract Background Identifying prognostic markers is important for the clinical and pathological course of chronic lymphocytic leukemia (CLL). Cluster of differentiation CD49d expression and percentage of smudge cells in CLL patients have recently been reported as newer prognostic factor. Aim of the Study To estimate CD49d expression as an independent prognostic marker in CLL patients and to investigate its relation to the known prognostic factors Methods Thirty adults with CLL were enrolled. Smudge cells percentage was calculated by microscopic evaluation of blood smears. Testing of cluster of differentiation CD49d expression was done by flowcytometry and detection of ataxia telangiectasia mutated (ATM) gene, protein 53(P53) gene deletions and trisomy 12 were performed using fluorescent in situ hybridization (FISH). Results CD49d expression was associated with lymphadenopathy, splenomegaly, advanced staging, high total leucocytic count, immunophenotyping scoring, CD38 positivity, short LDT, low smear cell percentage, and presence of p53 deletion. Low percentage of Smear cell (<30%) showed a significantly higher incidence of lymphadenopathy and organomegaly. Additionally, it was associated with a high total leucocytic count, low platelet count, high atypical lymphocytic count and typical IPT scoring, shorter LDT, positive CD38 expression and CD49d expression and ATM and P53 deletions. Conclusion CD49d expression and low smudge cell percent were consistently associated with poor prognostic indicators, suggesting that they could be considered as adverse predictors and they should be merged together to improve the power of the prognostic panels of CLL in the clinical setting.

Pregnancy Outcomes, Immunophenotyping and Immunohistochemical Findings in a Cohort of Pregnant Patients with COVID-19—A Prospective Study

(1) Background: SARS-CoV-2 infection during pregnancy could determine important maternal and fetal complications. We aimed to prospectively assess placental immunohistochemical changes, immunophenotyping alterations, and pregnancy outcomes in a cohort of patients with COVID-19; (2) Methods: 52 pregnant patients admitted to a tertiary maternity center between October 2020 and November 2021 were segregated into two equal groups, depending on the presence of SARS-CoV-2 infection. Blood samples, fragments of umbilical cord, amniotic membranes, and placental along with clinical data were collected. Descriptive statistics and a conditional logistic regression model were used for data analysis; (3) Results: Adverse pregnancy outcomes such as preterm labor and neonatal intensive care unit admission did not significantly differ between groups. The immunophenotyping analysis indicated that patients with moderate-severe forms of COVID-19 had a significantly reduced population of T lymphocytes, CD4+ T cells, CD8+ T cells (only numeric), CD4+/CD8+ index, B lymphocytes, and natural killer (NK) cells. Our immunohistochemistry analysis of tissue samples failed to demonstrate positivity for CD19, CD3, CD4, CD8, and CD56 markers; (4) Conclusions: Immunophenotyping analysis could be useful for risk stratification of pregnant patients, while further studies are needed to determine the extent of immunological decidual response in patients with various forms of COVID-19.

Preclinical Development of Non-Edited CD7 CAR-Modified Invariant NKT Cell Therapy for T-Cell Malignancies and Acute Myeloid Leukemia

The major hurdle in developing chimeric antigen receptor T (CAR-T) cell therapy for T-cell malignancies is CAR-T cell fratricide. This refers to the self-killing of the CAR-T cells due to shared expression of targetable antigens (e.g., CD7) between CAR-T and malignant target T cells. This CAR-T fratricide creates difficulties in producing sufficient numbers of CAR-T cells for infusion and can limit survival of the cells in vivo. Nuclease (e.g., CRISPR)-mediated genomic disruption of CD7 or T cell receptor alpha chain expression provides an alternative approach to enabling expansion of CD7 CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based genome editing remain unclear. Thus, other approaches to development of allogeneic, non-edited CD7 CAR-T cell therapy for relapsed and refractory T-cell malignancies is warranted. We unexpectedly found that human invariant natural killer T (iNKT) cells in normal blood donors displayed on average approximately 56% CD7 positivity (range 38 - 74%, n = 9 donors). Therefore, we hypothesize that CD7- subsets in iNKT cells expressing CD7 CAR can partially avoid fratricide without nuclease-mediated genome editing to delete CD7 and be expanded for adoptive cell therapy of CD7+ malignancies. By leveraging our proprietary 6000-10000-fold iNKT cell expansion platform, lentiviral transduced CD7 CAR iNKT (CD7 CAR-iNKT) cells can be expanded to clinical scale (n=2) (Fig. 1A). Based on CD19 CAR- and CD7 CAR-iNKT expansion rate (n=5), 100-500 doses (assuming 1-3 infusions per patient) of CAR-iNKT cells can be manufactured from a single donor-derived leukocyte concentrate. CD7 CAR-iNKT cells (n=5, 64-98% CAR+) specifically killed luciferase-expressing CD7+ T-cell acute lymphoblastic leukemia (T-ALL) (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1) at E/T ratios ranging from 20:1 to 2.2:1 compared to mock iNKT cells and CD19 CAR-iNKT cells (p < 0.0001). Control CD19 CAR-iNKT cells (n=3) specifically killed CD19+ B-cell tumor cells but not CD19- T-ALL and AML cells (p < 0.0001). Mock iNKT cells showed minimal cytotoxicity to all target cells except for CD1d+ Jurkat. CD7 CAR-iNKT cells (n=5) also produced significant amounts of IFN-γ in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1, Kasumi-6) compared to mock (n=5, p < 0.001) and CD19 CAR-iNKT cells (p < 0.05 except for Jurkat, p < 0.01). CD19 CAR-iNKT cells (n=3) produced significant amounts of IFN-γ to CD19+ B-cell leukemia and lymphoma cell lines (Nalm6, Daudi, Raji) compared to mock and CD7 CAR-iNKT cells (p < 0.0001). As expected, CD7 CAR-iNKT cells significantly eliminated almost all CD7+ cells (mean ± SD 0.4% ± 0.1, n=4) compared to mock (61.6% ± 24.4, n=4) and CD19 or ROR1 CAR-iNKT cells (69.9% ± 24.4, n=3) (p < 0.0001). Furthermore, expanded CD7 CAR-iNKT cells were >95% CD3+TCRVα24-Jα18+ and specifically responded to a-galactosylceramide or 7DW8-5 lipid antigens presented by CD1d molecules. The anti-leukemic effect of CD7 CAR-modified iNKT cells was assessed in HSB2 T-ALL and KG1 AML xenograft models (n=5 per group). Three i.v. infusions of thawed CD7 CAR-iNKT cells (5x106 cells/mouse) at days 0, 3, and 6 into leukemia (5x105/mouse, i.v. at day -2)-bearing mice significantly suppressed T-ALL growth by bioluminescent imaging analysis at days 3 (p < 0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024) and 17 (p < 0.0001) compared to PBS and mock controls (Fig. 1B). A significant survival advantage was observed after CD7 CAR-iNKT cell therapy compared to PBS and mock controls (p = 0.0009, Fig. 1C). Two i.v. infusions of thawed CD7 CAR-iNKT cells into pre-established AML mice significantly suppressed AML growth at days 3 (p = 0.0096), 6 (p < 0.0001), 10 (p = 0.0026), 17 (p < 0.0001), 24 (p < 0.0001), 31 (p < 0.0001), 38 (p < 0.0001) and 45 (p = 0.0003) compared to mock control (Fig. 1D). CD7 CAR-iNKT cell therapy also significantly prolonged the animal survival (p = 0.0069, Fig. 1E). In conclusion, these results demonstrate that CD7 CAR-iNKT cells without genome editing can be manufactured to therapeutic doses and mount anti-CD7+ tumor responses in vitro and in vivo. Our CD7 CAR-iNKT cells can be used as allogeneic, off-the-shelf products to treat all subtypes of T-ALL and 30% of CD7+ AML patients. In addition, our CD7 CAR-iNKT cells are likely more effective in 70% CD7+CD1d+ T-ALL patients as they provide dual target specificity and reduce the likelihood of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trial-in-Progress: AMC 112: Axicabtagene Ciloleucel in Relapsed or Refractory HIV-Associated Aggressive B-Cell Non-Hodgkin Lymphoma (NCT05077527)

Background: Persons living with HIV (PLWH) have an increased risk of B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL), even with well-controlled HIV. The lifetime cancer risk in PLWH is 25% to 40%1-3, and cancer is the leading cause of death in PLWH in economically advanced countries. Non-Hodgkin lymphoma (NHL) is the most common cancer in PLWH in the United States. Despite aggressive frontline treatments, about 20-30% of patients with HIV-associated B-NHL will not be cured with current therapies4, comparable to the non-PLWH B-NHL population. Axicabtagene ciloleucel (axi-cel) is an FDA-approved CD19-directed CAR-T cell product for the treatment of patients with relapsed or refractory (R/R) DLBCL based on the ZUMA-1 (>2 prior lines of therapy5) and ZUMA-7 (primary refractory or relapse within 12 months of initial therapy6) trials. As patients with HIV were excluded from registrational studies, only sparse case reports or small case series7 exist to guide treatment in this context. With regards to safety concerns, a study of CD4 cell count trends in a subset of HIV-negative patients receiving axi-cel demonstrated low CD4 cell counts (median baseline 220/μL) that decreased over time and remained persistently low after lymphodepletion and axi-cel therapy (median CD4 cell count 155/μL at 1 year post treatment).8 This critical gap in evidence can potentially lead to lives lost in patients with HIV, typically aged 25 to 65 years. We hypothesize that axi-cel will be safe and feasible in treating R/R HIV-associated DLBCL. Study Design and Methods: This is a multicenter phase I study to define the safety and feasibility of axi-cel in R/R HIV-associated DLBCL (NCT05077527). Concurrent correlative studies will investigate immune reconstitution and HIV virologic response. All participants will receive the standard dose of axi-cel at AMC sites following conditioning chemotherapy; bridging therapy may be given at the discretion of the treating physician (Figure 1). Key eligibility criteria include HIV positivity with viral load <50 copies/mL; diagnosis of R/R DLBCL, high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma, or follicular lymphoma grade 3B; either post 2 prior lines of therapy or refractory/relapsed within 12 months of frontline treatment; evaluable disease; adequate organ/marrow function; and CD19 positivity confirmed if previously received CD19-directed therapy. The study will enroll 20 participants, including at least 6 participants with a CD4 cell count <100/μL. A safety assessment of participants with CD4 cell count <100/μL will be done after 6 participants in that cohort have been enrolled in this study (Figure 2). If >2/6 participants experience dose-limiting toxicity, the study will halt recruitment of participants with CD4 cell count <100/μL and continue to recruit participants with CD4 cell count of ≥100/μL. The primary endpoint of the study is safety and feasibility. Safety is defined by blood count recovery of neutrophils to grade ≤2, and platelets to grade ≤2 within 6 weeks of axi-cel infusion, incidence/severity of infection, and incidence/severity CRS/ICANS9 among patients who receive axi-cel. The incidence of toxicity will be summarized using frequency and percentage and presented overall and for each CD4 cell count cohort (<100 versus ≥100/μL). The feasibility assessment will be evaluated as the proportion of participants who receive axi-cel among all enrolled participants who undergo leukapheresis. Based on published data10 from patients without HIV (success rate ~85%) and anticipating potential difficulties in clinical progression, leukapheresis, and manufacture, we assigned >50% as the acceptable feasibility cutoff for our study population. Key secondary endpoints include 1) complete response (CR) rate per Lugano criteria,11 2) event-free survival (time from enrollment to earliest of disease progression; commencement of new anti-lymphoma therapy; or death), and 3) response duration (time from date when response criteria are met to relapse or progression). Key exploratory endpoints include 1) differences in cytokine profiles between patients who achieve CR and those achieving <CR per Lugano criteria (tested for statistical significance using Wilcoxon rank sum test) and 2) associations between cytokine profile and CRS and ICANS severity (≥ grade 3 vs < grade 3, tested for statistical significance using Wilcoxon rank sum test). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissecting Ph-like ALL: The Role of Genomic Lesion and Minimal Residual Disease in Refining Outcome

Cardinali D and Della Starza I contributed equally Foà R and Chiaretti S contributed equally Background. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a subgroup of B-lineage ALL characterized by a gene expression profile (GEP) that resembles that of Ph-positive ALL but lacks the BCR/ABL1 transcript. Ph-like ALL cases are characterized by CRLF2 overexpression, JAK-STAT pathway mutations, IKZF1 deletions and rearrangements involving cytokine receptors and tyrosine kinases. To identify Ph-like ALL cases, our group developed a predictive tool named "BCR/ABL1-like predictor", which is based on the quantification by quantitative PCR of 10 transcripts, specifically overexpressed by Ph-like ALL cases1. ALL is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring is pivotal for guiding therapeutic choices. Real-time-quantitative PCR (RQ-PCR) of IG/TR gene rearrangements is the most widely used molecular method for MRD assessment. The GIMEMA LAL2317 is a clinical trial designed for newly diagnosed adult B-lineage Ph-negative ALL that includes two cycles of blinatumomab in the consolidation phase. MRD is evaluated at established time points (TPs), the most important being TP2, i.e., after the first consolidation cycle with high-dose chemotherapy, and TP3, after the first cycle of blinatumomab. Aim of this study. To refine risk relapse categories of Ph-like ALL cases based on their genomic features at presentation in combination with the MRD status. Methods. We performed this sub-analysis on a cohort of Ph-like ALL cases enrolled in the GIMEMA LAL2317 protocol identified according to the BCR/ABL1-like predictor assay and that underwent a centralized comprehensive molecular screening at diagnosis by IG/TR gene rearrangements and targeted DNA/RNA sequencing. MRD status was evaluated at specific TPs, i.e., TP2 and TP3, by RQ-PCR and digital droplet PCR (ddPCR). Results. A Ph-like profile was documented in 31/109 evaluable patients (28.4%); 11 of them (35.5%) experienced a relapse. By targeted RNA sequencing, 9/11 presented a gene fusion at presentation: in 4/9 a CRLF2-P2RY8 gene fusion was identified, whereas the remaining cases had PAX5-ZCCHC7, ETV6-BCL21L4, IKZF1-DDC, JAK2-r and ABL-class fusion in 1 case each (Figure 1A). The MRD status was evaluated at TP2 and TP3 by RQ-PCR in 23/31 patients: 15/23 were negative at both TPs, while 8/23 were positive at TP2 and negative at TP3; in the remaining 8 cases, MRD was not evaluated because of refractoriness (n=3), early death (n=2) or loss to follow-up (n=3). DdPCR analysis was performed in 9 patients and compared to RQ-PCR: 6/9 were concordant by both methods, while 3/9 resulted discordant with RQ-PCR being negative at TP3 while being positive by ddPCR. On the basis of the Ph-like signature and MRD results, we defined 3 subgroups: i) Ph-like ALL cases (n=12, 39%) that did not relapse, being MRD-negative at both TP2 and TP3; 3 cases were positive for fusion genes; ii) relapsed Ph-like ALL cases (n=8, 26%) who were MRD-positive at TP2 but became MRD negative at TP3, with 7 harboring a fusion gene; iii) relapsed Ph-like ALL cases (n=3, 10%), always MRD-negative at both TPs, with 2/3 positive for fusion genes (Figure 1B). Finally, by flow cytometry analysis, available in 7 patients at relapse, 6 maintained CD19 positivity and only 1 proved CD19-negative. Conclusions. Ph-like ALL patients tend to relapse early even after blinatumomab treatment and despite becoming MRD-negative. Relapse is not related to a mechanism of CD19 escape. Based on the gene fusions at presentation and on the MRD status, we could identify 3 subgroups of Ph-like ALL. These findings suggest that Ph-like ALL cases should be followed with different markers in addition to IG/TR, particularly in cases with well-defined fusion genes, who are at a very high risk of relapse. In this sense, a refined and rapid genetic characterization at presentation of Ph-like ALL cases is warranted for a more personalized and targeted patient management. 1. Chiaretti S et al., BJH 2018 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Frequency Of CD34 Expression In Acute Lymphoblastic Leukaemia And Its Correlation With Clinicopathological Characteristics: A Single Centre Experience From Pakistan.

This study was carried out to determine the frequency of CD34 positivity in acute lymphoblastic leukaemia (B-ALL) in our population and to report its association with the clinicopathological profile at the time of diagnosis. The cross-sectional study was conducted at National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, Pakistan, from March 2020 till December 2020.Newly diagnosed patients were selected, from both genders and all age groups. Relevant history and findings of physical examination were recorded. Immunohistochemistry was done on trephine biopsy and molecular studies were carried on bone marrow aspirates or peripheral blood samples. Out of 105 patients enrolled, 67 (63.8%) were males, with a male to female ratio (M: F) 1.8:1. Of the total patients, 62 (59.04%) were above 15 years of age. CD34 was expressed in 73 (69.5%) cases. Lymphadenopathy, splenomegaly, and hepatomegaly were separately noted in context to CD 34 expression in 22 (66.6%), 24 (64.8%), and 14 (58.3%) patients, respectively. CNS disease was seen in a total of 3(2.75%) subjects, in which 2 (66.6%) of the patients had CD34 expression. Total 81 patients in our study fall into the high-risk group out of which CD 34 expression was seen in 58(71.6%) subjects. Cytogenetic analysis, BCR-ABL p190, and MLL gene rearrangement were investigated in all participants. Cytogenetic analysis revealed an abnormality in 20 (19%) cases out of which 13 (17.8%) cases were from CD34 positive group. Our study reported CD34 expression in more than two-thirds of cases. High-risk disease was significantly associated with CD34 expression.

Prognostic significance of CD25 expression in dogs with a noninvasive diagnosis of B-cell lymphoma treated with CHOP chemotherapy.

Prior studies have identified high CD25 expression in canine diffuse large B-cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B-cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B-cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196-348 days) and median PFS was 196 days (95% CI, 172-220 days). Higher percentage of B-cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p=.02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed.

Study of infiltration patterns of lymphomas in bone marrow trephine biopsy and immunohistochemical analysis in a tertiary care institute

Lymphomas are malignant neoplasms of lymphoid cells present usually as solid mass in the lymphoid organs of the body. They have a tendency to metastasize to different parts of the body and bone marrow is one of the most frequent sites. They infiltrate particular parts of the bone marrow and hence form various different patterns of infiltration. Each lymphoma has its characteristic one or more patterns of infiltration. Recognition of such a pattern can often help in diagnosis of such lymphoma. This was a prospective descriptive study of one year duration enrolling known cases of lymphoma diagnosed in a tertiary care center and subsequently bone marrow trephine biopsy was done and studied for pattern of infiltration. Also immunohistochemistry was applied to differentiate B cell and T cell lymphomas. Paratrabecular pattern was the most common pattern seen solely or in mixed in the majority of the lymphoma cases. Also the frequencies of B cell lymphoma with CD-19 positivity were higher than T cell lymphoma with CD-3 positivity. Recognition of pattern of infiltration especially when no lymphomatous cells are identified on blood and or bone marrow aspiration smears are very helpful in diagnosis of lymphoma.

PB2210: FAVORABLE OUTCOME AFTER SECOND CD19 CAR T INFUSION OF AN IDENTICAL PRODUCT: HOST ENVIRONMENT AND DISEASE CHARACTERISTICS DETERMINE THE OUTCOME OF TREATMENT

Background: CD19-targeted CAR T cell therapy has revolutionized treatment options for patients with relapsed/refractory large B-cell lymphoma (r/r LBCL). However, a significant proportion of patients are refractory or relapse after treatment. So far, no consented treatment recommendations for patients failing CAR T cell therapy exist. Patients who relapse after initial response to therapy may be candidates for a second infusion, but data on the efficacy of this approach is still limited (Gauthier et al., Blood 2021). Aims: We sought to analyze differences in CAR T associated parameters that contribute to a favorable outcome after 2nd CAR T infusion of the identical CAR T product in a patient with r/r LBCL. Methods: Here, we present the clinical course of a 39-year-old patient with r/r LBCL, transformed from splenic marginal zone lymphoma (sMZL), who relapsed 11 months post their first CAR T cell infusion (CART1; October 2019) and received a second CAR T cell infusion afterwards (CART2; November 2020). Results: LBCL relapse was confirmed by histology, and IHC demonstrated positivity for CD19 and PD-L1. After lymphodepletion, the patient received a second dose (CART2; November 2020) of Tisagenlecleucel (Tisa-cel). Prior (day -27) and post (day 20) CART2 the patient received pembrolizumab (200 mg). Toxicity during CART1 was substantial, with ASTCT grade III CRS, grade I ICANS, and prolonged cytopenia (ANC <500/µl at day 90). After CART2 infusion, neither CRS nor ICANS were observed, and no longer-lasting cytopenias occurred. The patient achieved a CR following CART2, which has been ongoing for >15 months. Although CART1 and CART2 were identical products, derived from the same apheresis and production run, baseline proinflammatory markers (Ferritin) and tumor volume (LDH, MTV) differed between the two CART transfusions. The proinflammatory markers were significantly higher prior to CART1 vs CART2 (Ferritin 1582 vs 529 ng/ml) and the same trend was observed for tumor volume (LDH 262 vs 133 U/l; STLV 23,3 vs 2,6 cm3). Summary/Conclusion: Second CAR infusions represent a feasible salvage strategy in patients with late relapse after first CAR T infusion. The case illustrates that pretreatment factors, including patient inflammatory state and tumor volume may determine CAR T outcome in this setting. Peri-CAR-T checkpoint blockade may ameliorate tumor-intrinsic resistance mechanisms.

CD20 and CD19 expression loss in relapsed or refractory b-cell non-Hodgkin lymphoma: A retrospective cohort.

e19537 Background: CD20-targeted therapies such as rituximab are widely used in the management of B-cell non-Hodgkin lymphoma (NHL). Re-treatment with anti-CD20 agents is common, however previous research has demonstrated loss of CD20 expression at time of relapse or refractory disease in a subset of patients. There is also amplified interest in alternative targets, specifically CD19, in the relapsed or refractory setting. Incidence of CD19 loss has been explored in post-CAR T cell therapy patients but not described elsewhere. We hypothesized that CD marker loss may be higher than previous reports due to expanded use of CD20- and CD19-targeted therapy. Methods: The present study is a retrospective cohort analysis of 229 patients with B-cell NHL from Harbor-UCLA Medical Center in Los Angeles, CA from 2006 to 2022. Charts were retrospectively reviewed for cases of B-cell NHL, and data was collected about demographics, diagnosis, treatment, and relapses. CD20 and CD19 expression analysis was performed by immunohistochemistry and / or flow cytometry at diagnosis and at each relapse whenever possible. The primary endpoint of the study was the rate of CD marker expression loss at time of relapse. Results: The cohort of 229 patients was composed of 54.1% diffuse large B-cell lymphoma (DLBCL), 14.4% follicular lymphoma (FL), 8.3% high-grade B-cell lymphoma (HGBCL), 7.0% marginal zone lymphoma (MZL), 4.8% mantle cell lymphoma (MCL), and 11.2% other. Overall, 28.8% (66/229) patients had relapsed or refractory disease. The rate of CD20 expression loss was 7.9% (5/63) across all relapses with two patients converting at first relapse (1 FL relapsing as HGBCL, 1 MZL), two patients at 2nd relapse (1 FL relapsing as DLBCL, 1 MZL relapsing as DLBCL), and one patient at 5th relapse (1 MZL). All five patients with CD20 phenotypic conversion received rituximab, and three patients received two or more rounds of anti-CD20 therapy. The median OS after CD20 expression loss was 4 months (95% CI 3.3 – 4.7 months). All patients (14/14) who had CD19 testing performed at relapse maintained CD19 positivity. Conclusions: This retrospective study demonstrates a rate of CD20 expression loss at relapse of 7.9%, which is less than previously reported rates ranging between 11-60%. CD20 expression loss seems to be associated with transformation to high-grade lymphoma, poor overall survival, and marginal zone histology. The conservation of CD19 expression across relapses supports the use of novel anti-CD19 targeted therapy in the relapsed or refractory setting. Whenever possible, repeat biopsy should be done at time of relapse prior to treatment with CD-targeted therapy. This study is of notable clinical relevance with expanded use of anti-CD20 agents and novel CD19-targeted therapies in the management of B-cell NHL.

A Closer Evaluation for Refractory Atrial Fibrillation with Heart Failure, a Case Report

<h3>Introduction</h3> Atrial fibrillation (AF) is highly prevalent after the age of 55 years. However, it can be associated with infiltrative cardiomyopathy. <h3>Case Report</h3> A 63 years old had new onset of palpitation and shortness of breath. He presented to ED with rapid AF at rate of 140 bpm treated with metoprolol, diltiazem and rivaroxiban. The patient started to develop lower limb swelling, dizziness, shortness of breath with walking short distance. His blood pressure was 110/60 mmHg off antihypertensive medication. He had NYHA class III symptoms and significant peripheral edema. A TTE showed a moderate concentric left ventricle hypertrophy IVS 16 mm with mild global systolic dysfunction LVEF 50% restrictive diastolic function and pericardial effusion. The patient had significant myocardial hypertrophy, HFpEF and AF with intolerance to medical therapy we proceeded with PYP bone scan which was equivocal for TTR amyloidosis. The bone to heart ration was 1.27. Cardiac MRI showed progressive decline in LVEF to 27% with severe RV systolic dysfunction EF 28% . There was a diffused subendocardial delayed enhanced lesion. Given these findings hematology workup sent which showed serum Free Lambda of 147mg/L; Free Kappa 6.71mg/L and Kappa/Lambda ratio of 0.05 (0.26 - 1.65). Bone marrow biopsy showed plasma cell percentage up to 40%. Flowcytometry showed lambda restricted plasma cell population together with positive CD38, CD138, CD56 and negative CD19. Fat Pad biopsy positive for Congo red stain consistent of amyloid deposits. He received chemotherapy CyBorDex 4 cycles followed by reduction in plasma cells to 2% in the bone marrow. He had cardiac function recovery. <h3>Summary</h3> AL cardiac amyloidosis can present with cardiac manifestation. High suspicion of underlying infiltrative disease should be investigated in refractor cases AF and HFpEF.

CD13 is a useful tool in the differential diagnosis of meningiomas with potential biological and prognostic implications

Meningiomas are common tumors of the central nervous system. Although their histological diagnosis is usually straightforward, their differential diagnosis versus other tumors may be challenging at times. The objective of this study is to assess the diagnostic value of CD13 immunoexpression in the differential diagnosis between meningiomas and their morphological mimics. Immunohistochemical analysis for CD13, epithelial membrane antigen, SOX10, and STAT6 was carried out in a large cohort of primary meningeal tumors comprising 225 meningiomas, 15 schwannomas, and 20 solitary fibrous tumor/hemangiopericytomas. Within the meningioma group, the expression of CD13 and epithelial membrane antigen was distinguished in three categories using a semiquantitative score. Most of meningiomas expressed CD13 (94%) and epithelial membrane antigen (96%) while none of the schwannomas nor of the solitary fibrous tumor/hemangiopericytomas was positive for either the two markers. Diffuse positivity for CD13 and epithelial membrane antigen was more common in low-grade meningiomas than in anaplastic ones, which were also more often negative for such markers, especially for CD13 (32%). CD13 is a helpful immunohistochemical marker for the differential diagnosis of meningiomas and their mimics, achieving in combination with epithelial membrane antigen maximal sensitivity (100%) and showing statistically relevant difference of expression in comparison with both schwannomas (p < 0.0001) and solitary fibrous tumor/hemangiopericytomas (p < 0.0001). Furthermore, loss of CD13 expression could be related to outcome as it is associated with worrisome histological findings, mainly in the setting of anaplastic meningiomas.

ZNF384 rearrangement in acute lymphocytic leukemia with renal involvement as the first manifestation is associated with a poor prognosis: a case report

BackgroundNovel fusion genes such as ZNF384, have been identified in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in recent years. Patients harboring ZNF384 rearrangements have a distinctive immunophenotype with weak CD10 and aberrant CD13 and/or CD33 expression. Thus, ZNF384-rearranged ALL is a unique subtype of BCP-ALL. However, research on the prognostic significance of ZNF384 rearrangements has been limited to date, especially in adolescents.Case presentationWe described a 17-year-old adolescent who was diagnosed with ALL and had renal involvement as the first manifestation, which was very rare in the existing studies. FISH analysis indicated a rearrangement of ZNF384 according to its probe. The patient had a typical characteristic immunophenotype of ZNF384 rearrangement, with CD10 negativity and CD13 and CD33 positivity. She had an unfavorable prognosis because she responded poorly to chemotherapy and developed a relapse shortly after reaching CR.ConclusionThe importance of ZNF384 rearrangements in terms of prognosis remains unclear. We reported an adolescent who was diagnosed with ZNF384-rearranged ALL with renal involvement. She underwent different therapies, but her prognosis remained poor. Since ZNF384 rearrangements may act as a prognostic predictor in children or adolescents, early detection based on its characteristic immunophenotype is of great necessity.