Lymphoid Markers Research Articles

Integrative Genomic and Transcriptomic Profiling Reveals Distinct Molecular Subsets in Adult Mixed Phenotype Acute Leukemia

Background: Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia with co-expression of lymphoid and myeloid markers. Genetic alterations are critical for the understanding of pathogenesis, diagnosis, risk stratification and treatment of hematological malignancies. Hitherto, in addition to t(9;22)(q34;q22)/BCR::ABL1 (Ph+) and KMT2A-rearrangements (KMT2A-r), BCL11B and ZNF384 rearrangements have been incorporated into the diagnosis and classification of MPAL. The genetic features in the remaining MPAL merit further investigation. This study aims to apply an integrated genomic and transcriptomic approach to explore the molecular characteristics of adult patients with MPAL. Methods: 176 consecutive cases diagnosed with MPAL at the First Affiliated Hospital of Soochow University between January 2006 and November 2019 were included. Patients’ median age was 48 years old (range:16-87). The diagnoses were made according to the WHO 2016 criteria, 86 (48.9%) cases fulfilled T/My MPAL, NOS, 42 (23.9%) cases fulfilled Ph+ MPAL, 36 (20.5%) cases fulfilled B/My MPAL, NOS, 4 (2.3%) cases fulfilled MPAL with KMT2A-r, and 8 cases (4.5%) fulfilled MPAL, NOS, rare types. We performed targeted sequencing in 104 patients, RNA sequencing in 89 patients and whole genome sequencing in 4 MPAL patients. Gene expression profile data were compared to data from 133 AML, 53 B-ALL and 41 T-ALL cases. 120 of the patients who were treated in our hospital were applied for analysis of treatment response and survival. Results: In total, 74 genes were found mutated with variant allele frequency more than 2% in 94 of 104 (90.4%) MPAL patients, with a total of 239 SNVs and 135 indels. Distinct mutational patterns were observed among different MPAL subtypes. Mutations of CEBPA (41.5% vs. 2.0%, P< 0.0001), DNMT3A (34.0% vs. 0%, P< 0.0001) and NOTCH1 (26.4% vs. 9.8%, P= 0.028) were mostly associated with T/My MPAL compared with in other MPALs. RUNX1 mutation was more common in Ph+ MPAL (51.9% vs. 7.8%, P< 0.0001). NOTCH1 mutation associated with B/T MPAL (60.0% vs. 16.2%, P= 0.041). In comparison to other subtypes of leukemia, 33 of the 89 (37.1%) MPAL cases clustered with B-ALL, 30 (33.7%) clustered with T-ALL, and 26 (29.2%) clustered with AML. By integrating targeted sequencing and RNA sequencing data in 89 of the MPAL patients, we defined 8 molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics (Figure 1A). G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384-rearrangement/ZNF384-rearrangement-like features. With a median follow-up of 49.3 months, the estimated 5-year overall survival (OS) and event-free survival (EFS) in the whole cohort were 37.1 ± 5.1% and 27.9 ± 4.5%, respectively. There was no statistically significant difference in 5-year OS and EFS among patients with T/My, Ph+ and B/My MPAL. Of note, patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) had longer 5-year OS (69.1 ± 7.4% vs. 13.8 ± 5.6%, P< 0.0001, Figure 1B) and EFS (56.2 ± 7.5% vs. 7.4 ± 3.9%, P< 0.0001) than those receiving chemotherapy alone. Moreover, patients receiving allo-HSCT at first complete remission (CR1) had longer 5-year OS than those transplanted at non-CR1 (68.4 ± 11.3% vs. 52.7 ± 14.1%, P= 0.042). There was no significantly difference in the 5-year OS and EFS among the 8 subgroups as a whole (Figure 1C), which might due to the limited number of patients in each subgroup. Conclusions: In this study, we identify 8 molecular subgroups with distinct mutational and gene expression characteristics in adult MPAL. These results indicate that integrative genomic and transcriptomic profiling will provide informative implications on a more precise diagnosis, risk stratification and treatment options for MPAL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

3D organoid modeling of extramedullary hematopoiesis.

Under certain clinical and experimental conditions hematopoiesis occurs in other site than bone marrow (BM), such as the liver. Here, we develop a 3D organoid that mimics several components of the hematopoietic niche present during liver extramedullary hematopoiesis. To evaluate the capacity of a 3D hematopoietic organoid (3D-HO) to function as a hematopoietic like-niche allowing for blood cell production outside of the BM. The 3D-HO is constituted by liver sinusoidal endothelial cells (LSEC) as the stromal component, BM isolated from 5-FU treated mice (FU-BMCs), collagen microspheres and plasma clot as scaffolds. The ability of the 3D-HO to support the survival and functionality of FU-BMCs was investigated by using confocal microscopy, histology analysis, flow cytometry, and clonogenic assays. After 15 and 30 days, post-ectopic implantation, histological studies of the 3D-HO showed the presence of cells with myeloid and lymphoid lineage morphology. Flow cytometry analysis of these cells showed the presence of cells expressing hematopoietic stem progenitor cells (HSPC) (Sca-1+/c-Kit+), myeloid (Gr-1+) and lymphoid (B220+ and CD19+) markers. Clonogenic assays showed that cells from the 3D-HO formed hematopoietic colonies. Expression of the Sry gene by cells from the 3D-HO, implanted for 30 days in female mice, indicated that male donor cells persist in this model of extramedullary hematopoiesis. The 3D-HO constitutes an extramedullary hematopoietic-like niche which supports the survival and functionality of FU-BMCs. It may constitute an efficient model for investigating, in vitro and in vivo, those factors that control hematopoiesis outside BM.

Adult mixed phenotype acute leukemia (MPAL): B/myeloid MPALisoMPO is distinct from other MPAL subtypes.

Myeloperoxidase (MPO) is considered a specific marker of myeloid/non-monocytic lineage in the diagnosis of mixed phenotype acute leukemia (MPAL). However, the clinical significance of isolated dim MPO expression in otherwise typical B lymphoblastic leukemia (B-ALL; referred to as B/myeloid MPALisoMPO ) in adult patients is unknown. We compared flow cytometric immunophenotype and clinicopathological findings among cases of B/myeloid MPALisoMPO (n=13), other MPAL subtypes (n=10, B/myeloid and T/myeloid MPAL), B-ALL (n=64), and acute myeloid leukemia (AML, n=58), using the 2016 WHO classification. For MPAL cases, MPO was reported as the percent of MPO positive blasts and its intensity (dim or moderate/strong). The pattern of heterogenous antigen expression (inversely coordinated expression between myeloid and lymphoid markers and cell size) was assessed. Cases of B/myeloid MPALisoMPO showed a fairly homogenous single B-lineage blast population with dim MPO expression whereas cases of other MPAL subtypes displayed heterogeneous antigen expression and moderate/strong MPO expression. The percent of MPO positive blasts in these two groups was similar. Expressions of CD15, CD117, and monocytic markers were more common in other MPAL than in B/myeloid MPALisoMPO . B/myeloid MPALisoMPO patients had similar overall and leukemia free survivals as B-ALL patients and better than other MPAL patients. This is the first study to investigate the clinical significance of adult B/myeloid MPALisoMPO using the 2016 WHO classification. Our results suggest that B/myeloid MPALisoMPO clinically behaves more similarly to B-ALL than to other MPAL subtypes, supporting the 2016 WHO classification to segregate this entity from other MPAL subtypes.

Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

Tumor metabolic and secondary lymphoid organ metabolic markers on 18F-fludeoxyglucose positron emission tomography predict prognosis of immune checkpoint inhibitors in advanced lung cancer.

The purpose of this study was to investigate the predictive value of tumor metabolic parameters in combination with secondary lymphoid metabolic parameters on positron emission tomography (PET)/computed tomography (CT) for immune checkpoint inhibitor (ICI) prognosis in advanced lung cancer. This study retrospectively included 125 patients who underwent 18F-fludeoxyglucose (FDG) PET/CT before ICI therapy, including 41 patients who underwent a second PET/CT scan during ICI treatment. The measured PET/CT parameters included tumor metabolism parameters [maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), total lesion glycolysis (TLG), and total metabolic tumor volume (TMTV)] and secondary lymphoid organ metabolism parameters [spleen-to-liver SUVmax ratio (SLR) and bone marrow-to-liver SUVmax ratio (BLR)]. The correlation of PET/CT metabolic parameters with early ICI treatment response, progression-free survival (PFS), and overall survival (OS) was analyzed. Within a median follow-up of 28.7 months, there were 44 responders and 81 non-responders. The median PFS was 8.6 months (95% confidence interval (CI): 5.872-11.328), and the median OS was 20.4 months (95% CI: 15.526-25.274). Pretreatment tumor metabolic parameters were not associated with early treatment responses. The high bone marrow metabolism (BLR &gt;1.03) was significantly associated with a shorter PFS (p = 0.008). Patients with a high TMTV (&gt;168 mL) and high spleen metabolism (SLR &gt;1.08) had poor OS (p = 0.019 and p = 0.018, respectively). Among the 41 patients who underwent a second PET/CT scan, the ΔSUVmax was significantly lower (p = 0.01) and the SLR was significantly higher (p = 0.0086) in the responders. Populations with low-risk characteristics (low TMTV, low SLR, and ΔSLR &gt; 0) had the longest survival times. High pretreatment TMTV and SLR are associated with poor OS, and increased spleen metabolism after ICI therapy predicts treatment benefit. This indicates that the combination of tumor and spleen metabolic parameters is a valuable prognostic strategy.

Immunophenotypic Characterisation Along with Aberrant Expression of CD markers in Morphologically Diagnosed Cases of Acute Myeloid Leukemia

Immunophenotyping of leukemia cells is useful for detecting leukemia cell line, determining maturation stage and identifying aberrant antigens which act for individual treatment monitoring and detection of residual disease. A total of 104 newly diagnosed cases of acute myeloid leukemia were identified at hematology department in National Institute of Cancer Research and Hospital from January 2020 to December 2021.We detect Immunophenotypic pattern in newly diagnosed cases of acute myeloid leukemia. We also determine the frequency and pattern of aberrant expression of CD markers in acute myeloid leukemia patients. Mean age of patients was 35 years (SD±16 years) with male to female ratio was 1.53:1. Most frequent morphologic subtype was AMLM2 constituting 33.6% of all AML cases. Lineage specific markers HLADR, CD13, CD33, MPO, CD117 and CD34 were expressed in 80%, 89%, 95%, 77%, 74% and 62% cases of all AML cases respectively. Among 104 AML patient, aberrant CD expression was observed in 36% cases. The most frequently observed aberrant markers were CD7 and CD19 lymphoid markers, that were expressed in 15.38% and 14.42% cases respectively. Less frequent aberrant cCD3, CD10, CD5 and cCD79a antigens were expressed in 2.88%, 1.92%, 0.96% and 0.96% cases respectively. Immunophenotyping is essential in diagnosis and sub-classification of AML and expression of aberrant CD antigens is common in acute myeloid leukemia. These findings suggest the necessity for a more extensive study to evaluate the prognostic significance of aberrant CD marker expression in AML and to improve the accuracy of diagnosis and classification of AML. J MEDICINE 2022; 23: 106-111

A rare case of mixed phenotype acute leukemia: acute myeloid leukemia to early T-cell precursor acute lymphoblastic leukemia transformation

Mixed phenotypic acute leukemia (MPAL) comprises of both lymphoid and myeloid markers or blasts in a single population. Diagnostic criteria hinges on classifications provided by identifying these lineages based on cytogenetic markers taken throughout the disease course. We describe an interesting presentation of a patient who had first presented with Acute Myeloid leukemia (AML) but 8 weeks later transformed into Early precursor T cell ALL (ETP-ALL). Cytogenetics were taken throughout the course of the cancer and confirmed the presence of a CD34 precursor cell marker. This transformation and cytogenic markers indicated a pluripotent progenitor cell origin confirming the diagnosis of MAPL. This case highlights a pluripotent progenitor origin with initial presentation as AML (myeloid clone) and later as ALL after initial partial response to AML therapy due to clonal evolution.

Abstract 239: Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment

Abstract Background: Entinostat, a selective histone deacetylase 1/3 inhibitor has been shown to promote a robust anti-tumor response and increased neoantigen expression and antigen-specific T cell activation in preclinical tumor models. Entinostat plays a role in neutralizing myeloid derived suppressor cells (MDSC) to improve anti-tumor immune response. Entinostat is currently being tested in later-stage clinical trials in various types of cancer. Using a novel 3D ex-vivo platform with fresh patient tumor samples we assessed the therapeutic efficacy of Entinostat with intact stromal components and tumor immune microenvironment. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples from renal cell carcinoma (RCC) patients using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Entinostat for 48h to detect treatment-mediated changes in tumor immune cell composition including lymphoid and myeloid markers and their activation status. Results: Tumoroids treated with Entinostat increased NK, NKT, CD4 and CD8+T cells compared to untreated control. Ex vivo treatment with Entinostat led to an increase in CD69 and Granzyme B expression in tumor resident CD4+ T cells. Immune checkpoint receptors, 41BB and PD-1 expression was increased in CD8+T cells followed by Entinostat treatment. Decreased monocytic MDSC was seen in Entinostat treated tumoroids compared to untreated control and this was associated with decreased GM-CSF, IL-4, IL-6 cytokine levels and increased IFN-γ and IL-2, quantified by multiplex cytokine assay. Interestingly, the tumor cell killing study revealed a higher percentage of death in early stage MDSC (eMDSC) indicating a direct effect of Entinostat on MDSC survival. Conclusions: These results demonstrate that Entinostat treatment leads to a broad range of immune cell activation and MDSC inhibition in RCC. These data provide a mechanistic rationale utilizing our 3D tumoroid platform for discovery of potential biomarkers of drug sensitivity and to identify novel immunotherapeutic approaches. Clinical applications of 3D-EXplore may help to identify RCC patients who may likely benefit from Entinostat treatment. Citation Format: Krithika Nandakumar Kodumudi, Brittany Bunch, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 239.

Mycobacterium tuberculosis infection drives a type I IFN signature in lung lymphocytes.

SUMMARYMycobacterium tuberculosis (Mtb) infects 25% of the world’s population and causes tuberculosis (TB), which is a leading cause of death globally. A clear understanding of the dynamics of immune response at the cellular level is crucial to design better strategies to control TB. We use the single-cell RNA sequencing approach on lung lymphocytes derived from healthy and Mtb-infected mice. Our results show the enrichment of the type I IFN signature among the lymphoid cell clusters, as well as heat shock responses in natural killer (NK) cells from Mtb-infected mice lungs. We identify Ly6A as a lymphoid cell activation marker and validate its upregulation in activated lymphoid cells following infection. The cross-analysis of the type I IFN signature in human TB-infected peripheral blood samples further validates our results. These findings contribute toward understanding and characterizing the transcriptional parameters at a single-cell depth in a highly relevant and reproducible mouse model of TB.

Cell-free RNA in liquid biopsy and biomarkers profiling of hematologic and solid tumors.

3047 Background: Expressed RNA can capture mutations, changes in expression levels due to methylation, and provide information on cell of origin, growth, and proliferation status. We developed an approach to isolate fragmented RNA from peripheral blood plasma and explored its potential to be used in liquid biopsy. Methods: Peripheral blood cfRNA was extracted from patients with neoplasms in B-cell (#105), T-cell (#16), Myeloid (#73), and from solid tumors (#44), Normal individuals (#51), and reactive post-transplant (#137). RNA was sequenced using a 1459-gene panel. Expression profile was generated using Cufflinks. Results: cfRNA levels of various solid tumor biomarkers (CA-125, CA-15-3, CEA 8, Keratin19, Keratin6A...) were significantly higher (P &lt; 0.0001) in samples from solid tumors as compared with normal control. Similarly, cfRNA lymphoid markers (CD19, CD22, CD79A, and CD79B...) and cfRNA myeloid markers (CD33, CD14, CD117, CD56...) were all higher in B-cell lymphoid neoplasms and myeloid neoplasms, respectively (P &lt; 0.0001), as compared with control. In evaluating the host immune system, cfRNA CD4:CD8B and CD3D:CD19 ratios in normal controls were as expected (median: 5.92 and 6.87, respectively) and were significantly lower in solid tumors (median 3.40 and 2.23, respectively, P &lt; 0.0002). Solid tumor cfRNA showed CTLA4:CD8B ratio significantly higher in tumors than in normal (median 0.74 vs 0.19, P = 0.0001), while there was no difference in cfRNA PD-L1:CD8B ratio (median 1.45 vs 1.77, P = 0.96). Similar distinct patterns are noted for various cytokine and chemokines. cfRNA was highly predictive of diagnosis (AUC &gt; 0.98) of solid tumors, B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, and myeloid neoplasms as compared with normal control. When a specific neoplastic disease was considered against all cases including control and other neoplasms, the AUC varied between 0.77 and 0.949. Conclusions: This data shows that liquid biopsy using targeted sequencing of cfRNA in patients with various types of cancer provides comprehensive and reliable information on the neoplastic disease as well as the host. [Table: see text]

Alveolar rhabdomyosarcoma with massive bone marrow infiltration and disseminated intravascular coagulation mimicking acute leukemia.

A 27-year-old man was admitted with a 2-month history of general fatigue, appetite loss, and back pain, leading to a worsening of his general condition. On admission, laboratory tests showed an increased white blood cell count (15.9 × 109/L), with leucoerythroblastosis (myeloblasts, 1.0%; promyelocytes, 0%; myelocytes, 2.5%; metamyelocytes, 1.5%; band neutrophils, 3.5%; segmented neutrophils, 67.0%; lymphocytes, 18%; erythroblasts, 48%), severe anemia (hemoglobin, 45.0 g/L), and thrombocytopenia (platelet count, 21.0 × 109/L). Biochemical tests revealed elevated lactate dehydrogenase (2582 U/L) level, and coagulation tests showed a prolonged prothrombin time (20.3 s; reference interval: 10.0–13.0), normal fibrinogen (2.24 g/L), elevated fibrinogen-fibrin degradation product (331.9 mg/L), plasmin inhibitor-plasmin complex (9.05 mg/L), thrombin-antithrombin complex (41.2 μg/L), and decreased antithrombin (75.3%) levels. A computed tomography scan revealed systemic lymphadenopathy, splenomegaly, bilateral pleural effusions, and massive ascites. A bone marrow aspirate revealed massive infiltration by large abnormal cells with a basophilic cytoplasm and cytoplasmic vacuoles (Figure 1A). Flow cytometry analysis showed that the abnormal cells expressed only CD56, but no myeloid or lymphoid cell markers. Because he suffered severe disseminated intravascular coagulation (DIC) and worsening hypoxemia due to increased pleural effusions, initial treatment for acute leukemia comprised prednisolone and cytarabine. On Day 2 postadmission, biopsy of the right axillary lymph node was performed. Immunohistological findings from a bone marrow clot and biopsy tissue from the right axillary lymph node revealed diffuse proliferation of abnormal round cells, which expressed desmin (Figure 1B), myogenin, vimentin, and CD56 (Figure 1C), but not cytokeratin and leukocyte antigen. Therefore, a diagnosis of alveolar rhabdomyosarcoma was made. Subsequently, treatment with vincristine, doxorubicin, and cyclophosphamide was initiated, which ameliorated the lymphadenopathy, ascites, and DIC. Although a leukemic phase or bone marrow infiltration is an uncommon manifestation of alveolar rhabdomyosarcoma, flow cytometry and immunohistological analyses of bone marrow specimens can facilitate correct diagnosis. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. AS, HK, HU, and SK contributed to patient's care. SK performed pathological diagnosis. All authors wrote and reviewed the manuscript. Written informed consent for publication was obtained from the patient. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent for publication was obtained from the patient. All data can be assessed by contacting the corresponding author.

Pattern of Immunophenotypic Aberrant Expression in De-Novo Acute Leukaemia

Background: Aberrant expression (AE) of Acute Leukaemia (AL) is essential to confirm the diagnosis of AL patients whether it is biphenotypic/mix phenotypic AL or it is AL with AE. Objectives: This study is conducted to observe the diversity of aberrant immunophenotypic expressions among the patients of acute leukaemia with varying frequencies, to find out the correlation between aberrantly expressed immunophenotypic markers with different variety of French American British (FAB) sub classification of acute leukaemia and any correlation of clinical presentation of AL patients with aberrantly expressed immunophenotypic markers. Methodology: This cross-sectional observational study was carried out in the department of Haematology, BSMMU, Bangladesh on 50 patients from 14 to 65 years of age of both sex of newly diagnosed de- novo untreated AL patients from January 2017 to June 2018. Informed written consent &amp; clinical history is taken and physical examinations were done in a predesigned data collection sheet. Then Bone marrow (BM) with peripheral blood sample for morphology and immunophenotyping were done in the laboratory of the Haematology department of BSMMU. After collection of data, these data were analysed for the final result. Result: In this study, 24 (48%) patients of de-novo Acute Leukaemia have Immunophenotypic aberrant expressions. Among them 12 (24%) patients were AML with AE, 9 (18%) patients were B ALL with AE, 3 (6%) patients were T ALL with AE and 2 (4%) patients were MPAL. In case of AML the most frequent Lymphoid aberrant marker is CD7 (12%), In B ALL the most frequent aberrant marker is CD5 (10%) and in T ALL the most frequent aberrant marker is CD10 (4%). Conclusion: In de-novo acute leukaemia, there is significant number of patients have aberrant expression which should be differentiated from biphenotypic/mix phenotypic AL for therapeutic and prognostic implications.

Quantitative assessment of the immune microenvironment in African American Triple Negative Breast Cancer: a case\u2013control study

PurposeTriple negative breast cancer (TNBC) is more common in African American (AA) than Non-AA (NAA) population. We hypothesize that tumor microenvironment (TME) contributes to this disparity. Here, we use multiplex quantitative immunofluorescence to characterize the expression of immunologic biomarkers in the TME in both populations.Patients and methodsTNBC tumor resection specimen tissues from a 100-patient case: control cohort including 49 AA and 51 NAA were collected. TME markers including CD45, CD14, CD68, CD206, CD4, CD8, CD20, CD3, Ki67, GzB, Thy1, FAP, aSMA, CD34, Col4, VWF and PD-L1 we quantitatively assessed in every field of view. Mean expression levels were compared between cases and controls.ResultsAlthough no significant differences were detected in individual lymphoid and myeloid markers, we found that infiltration with CD45+ immune cells (p = 0.0102) was higher in TNBC in AA population. AA TNBC tumors also had significantly higher level of lymphocytic infiltration defined as CD45+ CD14− cells (p = 0.0081). CD3+ T-cells in AA tumors expressed significantly higher levels of Ki67 (0.0066) compared to NAAs, indicating that a higher percentage of AA tumors contained activated T-cells. All other biomarkers showed no significant differences between the AA and NAA group.ConclusionsWhile the TME in TNBC is rich in immune cells in both racial groups, there is a numerical increase in lymphoid infiltration in AA compared to NAA TNBC. Significantly, higher activated T cells seen in AA patients raises the possibility that there may be a subset of AA patients with improved response to immunotherapy.

Immunohistochemical and pathological changes in BALB/c mice immunized with whole sonicated Listeria monocytogenes antigens and the effect of probiotics

The current study was undertaken to investigate the role of macrophages as a cellular immune function against immunization with whole sonicated Listeria monocytogenes antigens (WSLMAgs) and the effect of probiotics. A preparation of WSLMAgs containing whole L. monocytogenes cell, after two subcutaneous immunization of BALB/c mice with 0.5ml WSLMAgs (0.5mg/ml) at an interval of two weeks. The bacterial identification was conducted by a conventional culture method using Listeria selective media PALCAM and confirmed by Polymerase Chain Reaction (PCR), As well as, the immunohistochemical and pathological change of it was studied in vivo by inoculating mice with pre-challenge WSLMAgs and post-challenge with virulent L. monocytogenes (1×108 CFU/mL). The results revealed the cellular immune function against pre- and post-immunization in spleen organ via lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles and marker F4/80+ show the immune-positive cells in aggregation adjacent to lymphoid follicle or focal aggregation of macrophages between follicles. In conclusion, the effectiveness of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for applicants.

Malignant Gastrointestinal Neuroectodermal Tumor: a case report and a review of the literature

Abstract Introduction/Objective Malignant gastrointestinal neuroectodermal tumor (GNET) is a rare soft tissue tumor arising in the wall of the gastrointestinal tract. The GNET was first described as an osteoclast rich tumor of the gastrointestinal tract with features resembling clear cell sarcoma, with only few cases reported in the literature. Methods/Case Report We report a case of a 71-year-old man with a past medical history of hypertension, hyperlipidemia, and benign prostatic hyperplasia presented with complaints of dyspnea, dizziness, fatigue, black stools, and a recent syncopal episode. Laboratory testing revealed anemia (HB 5.4 g/dL). Esophagogastroduodenoscopy demonstrated a submucosal gastric mass. An abdominal CT scan confirmed a 7.8 cm mass along the gastric cardia and fundus. Results (if a Case Study enter NA) Biopsy rendered a gastrointestinal neuroectodermal tumor (GNET). Microscopically, the tumor cells were spindle with eosinophilic cytoplasm and arranged in fascicules. They were positive for CD56, CD99, and Fli-1, synaptophysin, and negative for chromogranin, TTF-1, SMA, desmin, CD34, EMA, pan-cytokeratin, and lymphoid markers. Two months later, further imaging confirmed metastasis to the liver and spleen. GNETs typically arise within the muscularis propria of the gastrointestinal tract and often extend into the submucosa and subserosa. Conclusion The most important differential of GNET is the clear cell sarcoma of the gastrointestinal tract (CCS-GI). Both share similar morphological as well as molecular features and show S100 positivity; however, the lack of melanocytic differentiation in GNET distinguishes it from CCS-GI. Both typically show rearrangements of the EWSR1 gene, with t(12;22) (q13;q12) EWSR1-ATF1 or t(2;22)(q34;q12) EWSR1-CREB1 fusions. Pathologists should be aware of GNET diagnostic entity due to its aggressive behavior and high rate of recurrence and mortality even after complete resection.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells

AbstractMixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

S3059 Clear Cell Sarcoma of the Jejunum: Dissecting an Obscure Malignancy

Introduction: Clear Cell Sarcoma (CCS) of the jejunum is an extremely rare and mortiferous presentation of a poorly understood malignancy. CCS is a cancer of the soft tissue and aponeuroses of tendons, affecting young individuals with predilection towards the ankle. It is often confused for malignant melanoma, as it is also derived from neural crest origin and shares a similar histological appearance. Currently, the only definitive treatment is surgical resection. Even so, CCS is known to recur. Due to the sheer modicum of cases, our medical knowledge is limited. Case Description/Methods: We present a case of a 49-year-old gentleman from Cameroon with a past medical history of hypertension and diabetes mellitus type II, who presented to the emergency department with complaints of abdominal pain, nausea, and vomiting for a one-month duration. CT scan of the abdomen and pelvis revealed left-sided small bowel obstruction. The patient underwent diagnostic laparoscopy, which converted to open surgery, with small bowel resection and umbilical hernia repair. A 2.6 cm circumferential obstructing jejunal mass was identified and resected. The margins of resection were negative for malignancy. There was no lymph vascular invasion, and 12 lymph nodes were negative for malignancy as well. Immunohistochemistry stains resulted positive for S100, SOX 10, and EMA. FISH was positive for EWSR1. Recent studies have revealed a chromosomal translocation, t(12;22) (q13;q12), also known as the EWSR1 and ATF1 gene, respectively, which has been widely accepted as pathognomonic for CCS. At 2 years of follow-up, our patient had a drastic improvement in his symptoms, with active surveillance and close follow-up, as CCS has a high recurrence rate. Discussion: This case report aims to describe the clinical presentation and evolution of this unique soft-tissue malignancy, which can be found in the GI tract in extraordinary cases. We hope this rare case of Clear Cell Sarcoma can enhance our knowledge about this obscure cancer that we still have so much to learn.Figure 1.: This 2.6 cm tumor is present in the submucosa, muscularis propria and subserosa and shows nests and cords of patterns by network of delicate vascular septa. Neoplastic cells are plump with epithelioid, clear cell and focal spindle cell appearance. Mitotic figures are about 5/10HPF. Immunostains show that tumor cells are positive for S100, SOX-10, and EMA but negative for HMB-45, Melan A, carcinoma markers (AE1/AE3, CAM5.2, CK7 and CK20), gastrointestinal stromal tumor markers (CKIT, CD34, and DOG1), lymphoid markers (CD3 and CD20), and desmin and ASMA. EWSR1 is positive by FISH studies. A) (Magnification x40, lower power, hematoxylin-eosin) and B) (Magnification x400, high power, hematoxylin-eosin) show epithelioid and clear cell appearance. C) (Magnification x400, antibody for S100) and D) (Magnification x400, antibody for SOX-10) reveal positivity.

Systemic Inflammation Associates With a Myeloid Inflamed Tumor Microenvironment in Primary Resected Colon Cancer—May Cold Tumors Simply Be Too Hot?

Systemic inflammation measured by the acute-phase protein CRP associates with poor outcome across cancer types. In contrast, local tumor-associated inflammation, primarily evaluated by T-lymphocytes, correlates with favorable prognosis. Yet, little is known whether these two responses are related or opposing processes and why elevated CRP in relation to cancer is detrimental for clinical outcome. As proof of concept, we developed a platform combining multiplexed IHC and digital imaging, enabling a virtual readout of both lymphoid and myeloid immune markers and their spatial patterns in the primary tumors of resected stage II and III colon cancer (CC) patients with and without accompanying systemic inflammation. Twenty-one patients with elevated CRP (>30 mg/l) and 15 patients with low CRP (<10 mg/l) were included in the analyses. Whole slides from the primary tumors were stained for markers of adaptive (CD8+, CD4+, foxp3 regulatory T cells, CD20+ B cells) and innate (CD68+ macrophages, CD66b+ neutrophils) immunity and the immune checkpoint molecule PD-L1. Associations between individual immune markers, preoperative CRP values, mismatch repair status (MMR), and risk of recurrence or death were assessed. Unsupervised hierarchical clustering was used to explore whether distinct immune phenotypes were present. Tumors from systemically inflamed patients (CRP >30 mg/l) displayed significantly more myeloid features in terms of higher densities of CD66b+neutrophils (p = 0.001) and CD68+macrophages (p = 0.04) and less lymphoid features (lower CD8 T cell, p = 0.03, and foxp3 regulatory T cell densities, p = 0.03) regardless of MMR status. Additionally, systemically inflamed patients harbored lower mean distances between neutrophils and tumor cells within the TME. Intriguingly, microsatellite instable (MSI) tumor status correlated with systemic inflammation. However, using a combinatorial approach, we found that regardless of an adaptive composite score (compounded CD4+ and CD8+ T cells), a high innate score (CD66b+ neutrophils and CD68+ macrophages) associated significantly with elevated CRP. In conclusion, tumor-associated systemic inflammation correlated with a myeloid-dominated TME in a small cohort of resectable CC patients. Our data highlight the importance of a comprehensive immune classification of tumors including players of innate immunity and support a role for CRP as an informative biomarker of the immune response taking place at the tumor site.

Traversing the Spectrum of Non-Langerhans Cell Histiocytosis: A Case of Rosai-Dorfman Disease with Features of Necrobiotic Xanthogranuloma

Introduction. Non-Langerhans cell histiocytoses (non-LCH) are a group of rare diseases with varied clinical manifestations and overlapping features seen among the subtypes. Here, we present a case of Rosai-Dorfman disease with features of necrobiotic xanthogranuloma.&#x0D; Case. A 45-year-old female presented with a 10-year history of an enlarging neck mass with normal overlying skin accompanied by dysphagia and multiple asymptomatic pink to yellowish-brown papules, nodules, and plaques on the face, trunk and extremities. Biopsies of a skin nodule and plaque revealed granulomatous dermal infiltrates (lymphocytes, foamy histiocytes, and Touton giant cells), emperipolesis and areas of necrosis. CD1A and Fite-Faraco staining showed negative results while CD68 and S100 positively stained the tissues of interest. Histopathology of the neck mass paralleled these findings in addition to being negative for lymphoid markers. Patient had monoclonal gammopathy and thyromegaly with enlarged cervical lymph nodes on further tests and imaging. Intralesional and systemic steroids were given which led to flattening of skin lesions and improvement in dysphagia, respectively.&#x0D; Conclusion. Diagnosis and classification of a particular type of non-LCH may be difficult due to similarities across its subtypes. Hence, it is our belief that these diseases may occur on a spectrum. Treatment involves a multidisciplinary approach for the best possible care.