Flow Cytometric Findings Research Articles

Thymoma Involved by Small Lymphocytic Lymphoma (SLL): A Rare and Challenging Diagnosis

Abstract Introduction/Objective Thymoma is a malignant neoplasm arising from the epithelial cells of the thymus. It usually affects patients between the fourth and sixth decades of life. There is a strong association of thymoma with myasthenia gravis and other paraneoplastic syndromes Thymic epithelial tumors are the most common tumors of the anterior mediastinum; however, lymphomas frequently present as mediastinal masses. Concurrent thymoma and lymphoma affecting the thymus is very rare. Methods/Case Report A 79-year-old woman with a history of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) presented with night sweats and weight loss. Chest CT revealed small pulmonary nodules, a 3.3 x cm anterior mediastinal mass and enlarged hilar lymph nodes. Fine needle aspiration (FNA) of the mass revealed a mixture of lymphocytes and epithelial cells. Flow cytometry showed a monoclonal B-cell population, and double CD4/CD8-positive T-cells consistent with thymocytes. Core biopsy showed scattered neoplastic epithelial cells interspersed among densely packed small lymphocytes. Scattered and small clusters of epithelial cells were highlighted by the CK AE1/3 immunostain. CD3 and PAX5 showed the thymocytes and infiltrating B-cells, respectively. Based on cytomorphologic, flow cytometric, histopathologic, and immunohistochemical findings, the diagnosis of thymoma, type B2 involved by SLL was rendered. Results (if a Case Study enter NA) N/A Conclusion A multimodality diagnostic work-up integrating cytomorphology, flow cytometry and immunohistochemistry allowed recognizing this unusual association.

Acellular Cerebrospinal Fluid Preparations with Abnormal Flow Cytometric Findings: A 10-year Institutional Experience

Acellular Cerebrospinal Fluid Preparations with Abnormal Flow Cytometric Findings: A 10-year Institutional Experience

A treatment-refractory aggressive MDS-MLD with multiple highly complex chromosome 5 intrachromosomal rearrangements: a case report

BackgroundA patient with a myelodysplastic neoplasm exhibited a karyotype with multiple complex chromosome 5 rearrangements. This patient appeared to have a catastrophic cytogenetic event that manifested as a treatment-refractory aggressive form of disease, which lead to patient demise within one year. Both the clinical presentation and disease course were unusual based on the medical history and morphologic findings. Such cases of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) with complex abnormalities are not reported in the literature.Case presentationThe patient was a 62-year-old female who presented with pancytopenia and dyspnea. The morphologic appearance of the peripheral blood smear and bone marrow biopsy, along with flow cytometric findings, favored the diagnosis of MDS-MLD unclassifiable. Myelodysplastic syndrome (MDS) with multilineage dysplasia (MDS-MLD), is an MDS characterized by one or more cytopenias and dysplastic changes in two or more of the myeloid lineages (i.e., erythroid, granulocytic, and megakaryocytic). The bone marrow, in particular, showed prominent dysplasia, including the presence of atypical megakaryocytes with small hypolobated morphology reminiscent of those typically seen in MDS with isolated 5q deletion. Cytogenetic analysis, including interphase and metaphase FISH, karyotype and SNP chromosomal microarray were performed, as well as DNA sequencing studies. Cytogenetic analysis showed a very complex karyotype featuring multiple 5q intrachromosomal rearrangements including a pericentric inversion with multiple interspersed deletions and monosomy 7. FISH studies showed a partial deletion of the PDGFRβ gene, and SNP chromosomal microarray and targeted panel-based sequencing identified biallelic loss of function of the TP53 gene. Based on the pathologic findings, the patient was treated for MDS but did not respond to either lenalidomide or azacitidine.ConclusionThe genetic changes described, in particular, the complex intrachromosomal rearrangements of chromosome 5, suggest the occurrence of a sudden catastrophic event that led to an aggressive course in the patient’s disease. Conventional karyotyping, metaphase and interphase FISH, SNP chromosomal microarray and NGS helped to identify the complex genetic changes seen in this case. This highlights the importance of utilizing a multimodality approach to fully characterize complex chromosomal events that may significantly impact disease progression, treatment and survival.

Immunophenotyping Investigation in Acute and Chronic Leukemias as a Gold Standard Test

Immunophenotyping of leukemias and other hematological neoplasms has become one of the most relevant clinical applications of flow cytometry which remains an indispensable tool for the diagnosis, classification, staging as well as hematologic malignancies monitoring. It can provide quantitative data on a number of cell parameters for a large number of cells in the region of cells thousands per second which making it a powerful diagnostic tool. If immunophenotyping is more sensitive significantly, specific and objective than morphological studying, the latter remains the gold standard criterion for creating and establishing the blast percentage in presentation and post treatment together. Moreover, it is important that interpreters of flow cytometric data have the phenotypes exact knowledge of diverse normal cell populations, which can be one of the best tool in recognize deviations from normal as well as are able to discuss the potential clinical significance and knowing look of the flow cytometric findings.

A comparison and review of the flow cytometric findings in classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, T cell/histiocyte rich large B cell lymphoma, and primary mediastinal large B cell lymphoma.

The "Hodgkin-like" lymphomas including classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, T cell/histiocyte rich large B cell lymphoma, and primary mediastinal large B cell lymphoma have been shown to be pathobiologically related. With the exception of primary mediastinal large B cell lymphoma, these lymphomas have similar morphologic growth patterns with occasional neoplastic cells within a prominent reactive cell background. Historically, distinguishing these entities was difficult by flow cytometry; however, over the past 15 years, our laboratory has developed antibody-fluorochrome combinations capable of accurately distinguishing these entities by their immunoprofile. Additionally, an algorithmic approach based on characterization of the background reactive B-cell and T-cell populations can aid in narrowing the differential diagnosis. This review summarizes both the morphologic and immunophenotypic features and the current flow cytometric insights of the neoplastic and reactive populations found in this unique subset of lymphomas.

Immune Profiling of Multiple Myeloma Patients Treated with Daratumumab Monotherapy: Acquired and Primary Daratumumab-Resistance Is Associated with NK Cell Exhaustion

Immune Profiling of Multiple Myeloma Patients Treated with Daratumumab Monotherapy: Acquired and Primary Daratumumab-Resistance Is Associated with NK Cell Exhaustion

Flow Cytometric Findings in Clonal Cytopenia of Undetermined Significance.

To examine flow cytometric (FCM) findings in clonal cytopenia of undetermined significance (CCUS) in relation to variant allele fraction (VAF) and mutation risk. Nine FCM parameters, including 5 FCM metrics (Meyerson-Alayed scoring scheme [MASS] parameters) we previously used to identify myelodysplastic syndromes (MDS), were compared among 96 CCUS samples, 100 low-grade MDS samples and 100 samples from patients without somatic alterations (controls). FCM findings did not differ between CCUS samples with less than 20% VAF and controls. CCUS samples with more than 20% VAF (CCUS >20% VAF) demonstrated more than 1 abnormal FCM parameter at a frequency between MDS and controls. Abnormalities in CCUS with high-risk alterations (CCUS(hi)) were similar to MDS, with no statistical difference in the percentage of cases with more than 1 FCM abnormality or a positive MASS score. The positive predictive value (PPV) for clinically significant myeloid processes; MDS, CCUS(hi), and CCUS >20% VAF compared with other CCUS samples and controls was 94.8%, with 96.5% specificity and 61% sensitivity using a modified MASS score. A subset of MDS (43%) was distinguished from CCUS(hi) and CCUS >20% VAF using 3 parameters, with a 93.5% PPV and 83.3% specificity. FCM abnormalities can distinguish high-risk CCUS based on VAF or alteration type from low-risk CCUS and MDS in many cases. The findings are of potential utility in the evaluation of patients with cytopenias.

Correlation of Myelodysplasia-Related Flow Cytometric Findings with Mutations in Patients Undergoing Diagnostics for Suspected Myelodysplastic Syndrome

Introduction: Diagnostic procedures in patients with suspected myelodysplastic syndromes (MDS) have been broadened recently by adding flow cytometry and mutation analysis to cytomorphology and cytogenetics. In parallel the concept of pre-MDS conditions has been widely accepted in analogy to MGUS preceding multiple myeloma. Thus, idiopathic cytopenia of undetermined significance (ICUS) has been defined by cytopenia with minimal dysplasia in the absence of mutations and clonal cytopenia of undetermined significance (CCUS) is separated from ICUS by the presence of at least one MDS-related mutation. While this concept implies the outgrowth of a mutated cell clone and the acquisition of additional mutations as well as the gradual manifestation of the dysplastic phenotype as proven by cytomorphology and flow cytometry no comprehensive data is available yet on a multidisciplinary diagnostic approach taking into consideration ICUS and CCUS. Aim: To describe the correlation of the presence of mutations with the occurrence of flow cytometric aberrations in the context of cytomorphologic and cytogenetic findings in patients with reactive conditions, ICUS, CCUS, MDS and related malignancies. Patients and methods: In a retrospective analysis patients were identified in whom cytomorphology, cytogenetics, flow cytometry and mutation analysis had been performed in parallel for suspected MDS. Flow cytometric procedures followed ELN criteria. Mutation analysis was performed by next generation sequencing targeting the genes ASXL1, CALR, CBL, CSNK1A1, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2 . A total of 1607 patients were identified (median age 72 years, range 18-93; 1007 males, 600 females). Results: Cytomorphologic evaluation revealed reactive conditions in 522 (33%) patients, ICUS was diagnosed in 248 (15%), MDS in 631 (39%), MDS/MPN in 63 (4%), CMML in 108 (7%) and 35 (2%) were not evaluable. An aberrant karyotype was found in 350 (22%) patients. The numbers of flow cytometric aberrations were as follows: 0, 90 patients (6%); 1 to 2, 496 (31%); 3 to 4, 456 (28%); 5 to 7, 421 (26%); ≥8, 144 (9%). The myeloid progenitor cell compartment was not affected in 1292 (80%) and showed 1 aberration in 210 (13%), 2 in 86 (5%) and 3 to 4 in 19 (1%). The respective number for the granulocytic compartment were 0, 425 (26%); 1, 438 (27%); 2, 364 (23%); 3, 226 (14%) and 4 to 6, 154 (10%) and for the monocytic compartment 0, 451 (28%); 1, 513 (32%); 2, 307 (19%); 3, 161 (10%) and 4 to 7, 175 (11%). In 847 (53%) the erythroid compartment was not affected and in 760 (47%) 1 aberration was observed. The numbers of mutations were as follows: 0, 739 patients (46%); 1, 347 (22%); 2, 295 (18%); 3, 157 (10%); 4 to 6, 69 (4%). The genes most frequently mutated were TET2 (342, 21%), ASXL1 (226, 14%), SRSF2 (215, 13%), DNMT3A (129, 8%), SF3B1 (133, 8%), RUNX1 (108, 7%), TP53 (89, 6%), and U2AF1 (83, 5%). The number of flow cytometric aberrations was strongly correlated to the number of mutations (r=0.494, p Mean (±SD) numbers of flow cytometric aberrations and mutations, respectively, were highest in CMML (6.2±2.3, 2.6±1.2), MDS/MPN (5.0±2.7, 1.9±1.3) and MDS (4.5±2.4, 1.3±1.2). In contrast, patients with reactive conditions had the lowest numbers (2.4±2.0, 0.3±0.7). Interestingly, numbers for ICUS cases were in between (3.7±2.3, 1.0±1.1). Importantly, re-classifying ICUS cases as CCUS based on the presence of at least one mutation (n=141) not only by definition increased the number of mutations (1.8±1.0) but also increased the number of flow cytometric aberrations (4.4±2.4) to levels observed in MDS while the remaining ICUS cases had levels observed in those with reactive conditions (2.9±1.7). Mean (±SD) numbers of flow cytometric aberrations and mutations, respectively, in patients with and without cytogenetic aberrations were 4.7±2.5 vs. 3.7±2.4 and 1.4±1.2 vs. 1.0±1.2 (p Conclusions: This data confirms the concept of ICUS and CCUS preceding MDS and suggests to broaden this concept by integrating flow cytometric data showing strong correlation to observed mutations. Confirmatory analyses and follow up analyses correlating the findings of the different applied methods with the patients´ outcome should substantiate these results and build the basis for a clinically orientated classification of patients with cytopenia and MDS-related conditions. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Pathogenic Mutations and Atypical Flow Cytometric Findings Characterize the Majority of Unclassifiable Myelodysplastic/Myeloproliferative Neoplasms.

Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy.

Two Atypical Cases of Classical Hairy Cell Leukaemia and Hairy Cell Leukaemia Variant: A Case Study

Introduction: Classical Hairy Cell Leukaemia (cHCL) and Hairy Cell Leukaemia variant (HCL-v) are both rare and slow-growing mature B cell neoplasms. According to flowcytometry data, they fall into the group classified as CD5- CD10- B cell lymphoproliferative disorders. Methods: Two cases with features atypical to two neoplasms at the time of diagnosis were studied. Results: Case 1 was a 15 year old male with right cervical lymph nodes (1x1 cm) in the posterior triangle, a few ecchymotic patches on the arm and a massive splenomegaly. C-reactive protein (CRP) level was 53 mg/dL. Erythrocyte Sedimentation Rate (ESR) was 98 mm/1 st hour. Full Blood Count (FBC) revealed typical features of pancytopenia with monocytopenia. The liver and renal profiles were normal. Morphology of bone marrow was suggestive of cHCL. Flowcytometry and BRAF V600E mutation was positive confirming the diagnosis of cHCL. Case 2 was a 55 year old male presenting with moderate splenomegaly and absolute lymphocytosis. The FBC revealed leukocytosis which is commonly seen with monocytopenia. Blood pictures revealed many hairy cells with moderately basophilic cytoplasm and visible nucleoli suggesting HCL-v. Flowcytometry findings and negative BRAF V600E mutation confirmed HCL-v. Conclusions: Clinical findings, blood images, morphology of bone marrow, flowcytometric findings and positive BRAF V600E mutation confirmed the diagnosis of cHCL in case 1 (15 year old boy) making it as a very rare case. The morphological findings on blood, the presence of characteristic CD markers on flowcytometry and negativity of BRAF V600E confirmed the case 2 as HCL-v, despite having CD10 positivity and monocytopenia.Keywords: Flowcytometric immunophenotyping, Hairy cell leukaemia, Hairy cell leukaemia variant

An Unusual Case Of Disseminated Histiocytic Sarcoma Preceded By Myeloid Sarcoma With Concomitant Acute Myeloid Leukemia

Abstract Introduction/Objective Histiocytic sarcoma (HS) is rare (<1% of hematolymphoid neoplasms), and can present extranodally as disseminated disease. Immunophenotypically, the cells express CD163, CD68, lysozyme and CD45. HS often occurs as a secondary event following B-cell lymphomas, acute lymphoblastic leukemia or acute myeloid leukemia (AML) typically with monocytic differentiation retaining the same molecular/cytogenetic abnormalities as the primary tumor. Results Our patient, a 47 year old male was diagnosed with myeloid sarcoma (MS) following FNA of a new neck mass. A bone marrow biopsy revealed AML without monocytic differentiation. Flow cytometric findings of both marrow and neck mass were similar (positive for CD34, CD117, CD33, CD11b, CD13, CD15, CD64, CD7; negative for CD4, CD14, CD56). Karyotypic and FLT3 ITD mutation analysis were normal. CNS involvement was diagnosed 2 months later, while a marrow biopsy (status post therapy) confirmed resolution of AML. A hypermetabolic left perinephric mass noted by PET CT, when biopsied, showed large epithelioid polygonal cells with amphophilic cytoplasm and atypical vesicular nuclei (positive for CD68, PU.1; negative for LCA, CD163, CD34, CD4, pankeratin). A diagnosis of atypical epithelioid neoplasm suggestive of HS was rendered, although negativity for LCA and CD163 was unusual. No treatment was given for HS. A month later, the patient presented with a cheek mass diagnosed again as being suggestive of HS. His AML also relapsed. Next-generation sequencing (37 genes including BRAF) from both marrow and tissue samples detected the presence of a nonsense mutation in exon 7 of WT1 (p.Ser169). Conclusion Our case appears to be the first reported one of disseminated HS preceded by MS and concomitant AML, lacking monocytic differentiation. The findings overall support the hypothesis of origin as being from a common progenitor cell differentiating along both myeloid and histiocytic/other cell lineages at different time points.

Flow Cytometry: An Important Diagnostic Tool in Critically Ill Preterm Neonates with Suspected Sepsis.

Sepsis is a major cause of neonatal mortality. The gold standard for diagnosis is blood culture which suffers from low sensitivity and huge turn-around time. Flow cytometry has been extensively applied to malignant disorders and is an upcoming tool for diagnosis of nonmalignant disorders due to its rapidity and accuracy in detecting cells, cell products, and their functional states. The aim of this study was to investigate the utility of flow cytometric expression of neutrophil CD64, monocyte human leukocyte antigen (HLA-DR) and CD16 in diagnosis in suspected preterm neonates. In total, 100 preterm neonates with clinical signs of sepsis were enrolled in the study. Blood culture, C-reactive protein (CRP) and flow cytometry for nCD64, mHLA-DR, and mCD16 were performed. The neonates were divided into two groups: culture positive and culture negative and CRP and flow cytometric findings compared. ROC analysis was performed to determine the best cut-off for nCD64, mHLA-DR, and mCD16 values along with estimation of sensitivity, specificity, and predictive values. Probability of <0.05 was taken as significant. Out of the 100 enrolled neonates, 34 (34%) were culture positive. CRP was not found to be significantly different in the two groups. Expression of nCD64 (p = 0.03) was significantly upregulated in the blood culture positive cases with a cut-off mean fluorescence intensity (MFI) value = 4.72 and sensitivity of 92% and specificity of 52%. Expression of mCD16 (p = 0.02) was also upregulated in the blood culture positive cases with a cut-off MFI value = 4.9, with sensitivity of 41%, specificity of 83%. The study concluded that nCD64 and mCD16 can be potential biomarkers for early diagnosis of neonatal sepsis with a high sensitivity and specificity. · Neutrophil CD64 significantly upregulated in septic neonates.. · Monocyte CD16 significantly upregulated in septic neonates.. · C-reactive protein values were not significantly different in septic versus nonseptic neonates..

Cord Blood Low-Density Granulocytes Correspond to an Immature Granulocytic Subset with Low Expression of S100A12.

Although substantial progress has been achieved concerning neonatal sepsis, its lethality remains considerably high, and further insights into peculiarities and malfunctions of neonatal immunity are needed. This study aims to contribute to a better understanding of the role of human neonatal granulocyte subpopulations and calgranulin C (S100A12). For this purpose, we gathered 136 human cord blood (CB) samples. CD66b+ CB low-density granulocytes (LDG) and CB normal-density granulocytes were isolated and functionally and phenotypically compared with healthy adult control granulocytes. We could identify CB-LDG as CD66bbright CD64high CD16low CD35low CD10low S100A12med-low and, based on these markers, recovered in whole CB stainings. Consistent with flow cytometric findings, microscopic imaging supported an immature phenotype of CB-LDG with decreased S100A12 expression. In CB serum of healthy neonates, S100A12 was found to be higher in female newborns when compared with males. Additionally, S100A12 levels correlated positively with gestational age independently from sex. We could solidify functional deficits of CB-LDG concerning phagocytosis and generation of neutrophil extracellular traps. Our study reveals that previously described suppressive effects of CB-LDG on CD4+ T cell proliferation are exclusively due to phagocytosis of stimulation beads used in cocultures and absent when using soluble or coated Abs. In conclusion, we characterize CB-LDG as immature neutrophils with functional deficits and decreased expression and storage of S100A12. Concerning their cross-talk with the adaptive immunity, we found no direct inhibitory effect of LDG. Neonatal LDG may thus represent a distinct population that differs from LDG populations found in adults.

Platelet-rich plasma promotes recruitment of macrophages in the process of tendon healing.

IntroductionResearchers have investigated the use of platelet-rich plasma (PRP) therapy. However, the mechanisms through which PRP affects tissue repair remain unclear. We hypothesize that PRP promotes tissue repair through not only via direct manner on the local cells but also via indirect manner that encourage the recruitment of reparative cells such as macrophages (MPs), and it depends on the quality of PRP including the concentration of leukocytes. The aim of this study is to elucidate the actions of the MPs in the mechanisms of PRP on tissue repair processes.MethodsLeukocyte-rich (LR) PRP and leukocyte-poor (LP) PRP were prepared from 12-week-old C57BL6 mice. Full-thickness defects were created in central third of patellar tendons of 12-week-old C57BL/6 mice for histologic analysis (n = 36) and 12-week-old B6.129P-Cx3cr1tm1Litt/J mice for flow cytometry analysis (n = 108). B6.129P-Cx3cr1tm1Litt/J mouse is GFP-positive only in the MP-linage cells thus MPs recruited to the repair tissue can be distinguished whether it had originated from administrated PRP or recruited from host mouse. Mice were treated either with LR-PRP, LP-PRP, or without PRP (control group). Histological analyses were performed to evaluate the tendon healing using Bonar score as semi-quantitative histological scoring system. Flow cytometric analyses were performed to count the number of GFP-positive cells around repaired patellar tendon. In addition, the ratio of pro-inflammatory MPs (M1)/anti-inflammatory MPs (M2) were analyzed in those GFP-positive cells. The statistical analysis was performed using GraphPad Prism ver6. P values < 0.05 were considered statistically significant.ResultsIn LR-PRP and LP-PRP groups, all variables in Bonar score such as cell morphology, cellularity, vascularity, and collagen arrangement were significantly improved in comparison with control group, indicating that both PRPs promote tendon hearing. LP-PRP promoted the tendon healing significantly faster than that of LR-PRP on postoperative day 28 (P < 0.001). LR-PRP enhanced angiogenesis (vascularity: P < 0.001), while LP-PRP improved the collagen arrangement on postoperative day 28 (collagen arrangement: P < 0.01). In other variables such as cell morphology and cellularity score, there were no significant differences between LR-PRP and LP-PRP groups in any time points. Flow cytometric findings showed that recruitment of GFP-positive MPs in the LR and LP-PRP groups were significantly increased from postoperative day 4 compared with control group without PRP treatment (P < 0.001). The majority of GFP-positive MPs were M1 at the initiation of tendon healing phase, and M2 were gradually increased from postoperative day 4. The number of M1 was significantly high both in the LP- and LR-PRP groups (day 4 and 7, p < 0.001), but the number of M2 was high only in the LP-PRP group (day 7 and 14, P < 0.05) when it compared with control group. The M1/M2 ratio on postoperative day 7 was significantly lower in the LP-PRP group than those in the control group (P < 0.05).ConclusionsThis study demonstrated that PRP enhanced the tendon healing and promoted the recruitment of MPs to the injured tissue. The subtypes of MPs were different depends on the types of PRPs, suggesting that leukocytes in PRP influence the effect of PRP therapy.

Common phenotypic dynamics of tumor-infiltrating lymphocytes across different histologies upon checkpoint inhibition: impact on clinical outcome

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized the cancer therapeutic landscape and our perception of interactions between the immune system and tumor cells. Despite remarkable progress, disease relapse and primary resistance are not uncommon. Understanding the biological processes that tumor-infiltrating lymphocytes (TILs) undergo during ICI, how this affects the tumor microenvironment (TME) and, ultimately, clinical outcome is, therefore, necessary to further improve treatment efficacy. AimIn the current study, we sought to characterize TILs from patients with metastatic solid tumors undergoing ICI correlating flowcytometric findings with clinical outcome. MethodsIn total, 20 patients with 10 different metastatic solid tumors treated with ICIs targeting programmed-cell death-1 (PD-1)/PD-L1 axis were included in this study. The phenotype of T cells deriving from biopsies obtained prior to treatment initiation and on-treatment was investigaded. Analyses were focused on T cells’ degree of differentiation and activity and how they correlate with transcriptomic changes in the TME. ResultsData indicate that patients benefitting from ICIs accumulate CD8+central memory T cells. TILs developed an effector-like phenotype over time, which was also associated with a cytolytic gene signature. In terms of modulation of T-cell responses, we observed that high expression of checkpoint molecules pre-treatment (i.e., PD-1, lymphocyte activation gene-3 [LAG-3], B and T-lymphocyte attenuator [BTLA] and T-cell immunoglobulin and mucin domain containing-3 [TIM-3]) was associated with similar gene signature and correlated to treatment benefit. Increasing expression of LAG-3 and BTLA in the CD8 compartment and their co-expression with PD-1 during treatment were, however, a common feature for patients who failed to respond to ICIs. ConclusionsBesides identifying immune profiles suggestive of response to ICI, our results provide a more nuanced picture regarding expression of checkpoint molecules that goes beyond T-cell anergy.

Eosinophils are the main cellular targets for oral gene delivery using Lactic acid bacteria

Lactic acid bacteria have been studied as a vehicle for the delivery of plasmid DNA to the gastrointestinal tract. However, low levels of gene expression in vivo limit their practical use. Furthermore, it is still unclear how the orally administrated bacteria transfer their harbored plasmid DNA to host intestinal cells. To more easily track the delivery of plasmid DNA for eukaryotic expression in the intestine, we constructed an L. lactis-E. coli shuttle plasmid (pLEC) that allowed significantly elevated expression of the target protein of interest in eukaryotic cells. We first demonstrated its usefulness for delivery from L. lactis to Caco-2 cells in vitro. We then investigated the cellular target for the L. lactis DNA delivery system in vivo. Mice were orally administrated with LL/pLEC:EGFP, an L. lactis strain carrying pLEC for EGFP expression, and immunofluorescent analyses of frozen sections prepared from their small intestines identified a number of EGFP-expressing cells in the lamina propria and some in the sub-epithelial dome of the Peyer’s patches. Flow cytometric analysis revealed that these EGFP-expressing cells were both CD11c- and F4/80-positive but CX3CR1-negative, suggesting that they are eosinophils. Immunostaining of the sections with an antibody against Siglec-F, a marker protein of eosinophils, confirmed the flow cytometric findings. Thus, the target cells of DNA delivery from L. lactis in the intestines are mainly eosinophils in the lamina propria and Peyer’s patches. This finding may open a new approach to the development of DNA vaccines for oral administration.

Abstract P5-04-24: Differential expression of novel immune checkpoint receptors expressed on tumor-infiltrating lymphocytes (TIL) in patients with early breast cancer

Abstract Introduction: High expression of immune checkpoint receptors in tumor microenvironment reduces antitumor immunity and cause immune evasion of tumor cells. In recent years, immunotherapy trials using PD-1 or PDL-1 inhibitors in advanced triple negative (TN) breast cancer evolved very rapidly. The differential expression of novel immune checkpoint receptors such as TIM-3, LAG-3 and TIGIT in addition to PD-1, and CTLA-4 on tumor infiltrating lymphocytes (TIL) in patients with early breast cancer was investigated. Material and methods: TIL were isolated by using a Tumor Dissociation Kit from fresh tumoral tissue. Flow-cytometric analyses were performed by using CD8, CD16, CD56, PD-1, CTLA-4, TIM-3, LAG-3 and TIGIT specific monoclonal antibodies on isolated TIL. Correlations were estimated between biological and clinical characteristics of tumors and demographic features of patients and flow cytometric findings. Results: Median age was 47 (range 28-68). There were 7 patients (35%) with HER2+ or triple negative tumors, whereas 13 patients (65%) had HER2 (-) luminal cancers. Our findings showed that patients younger than 45 years were more likely to express high levels of CTLA-4 (p=0.013) and TIGIT (p=0.007) on CD56+ natural killer (NK) cells and TIM-3 (p=0.043) on CD16+ lymphocytes (Table 1), whereas the other high expressions including LAG-3 (p=0,08) and TIM-3 (p=0.06) on CD56+ NK cells did not reach the statistical significance. Furthermore, patients with high Ki-67 proliferation index &amp;gt;35% were found to express higher CTLA-4 (p=0.011) on CD16+ lymphocytes. Patients with Stage II disease expressed higher levels of PD-1 (p=0.018) and LAG-3 (p=0.04) on CD8+ cytotoxic T lymphocytes than patients with Stage I disease. Similarly, patients with lymph node metastasis had higher TIGIT (p=0.04) and PD-1 (p=0.05) levels on CD16+ and CD56+ lymphocytes, respectively. No other significant associations could be found between immune check receptors and other parameters. Conclusion: Our results suggest TIL in patients with more advanced stages and younger than 45 years old are more likely to express higher levels of immune checkpoint receptors such as LAG-3, TIM-3, CTLA-4, TIGIT and PD-1. Interestingly, no difference could be found in immune checkpoint receptor expressions in TIL between patients with luminal and TN or HER2+, that would justify immunotherapeutical approaches in selected luminal breast cancers in future trials. Table 1. Significant correlations between immune check point receptor expression and demographic and tumor features CD8PD1CD8LAG3CD16CTLA4CD16TIGITCD16TIM3CD56CTLA4CD56TIGITCD56PD1meanpmeanpmeanpmeanpmeanpmeanpmeanpmeanpAge&amp;lt;45 (n:7)11.930.427120.40513.210.13212.290.3214.140.043150.01315.360.00710.290.905&amp;gt;45 (n:13)9.739.699.049.548.548.087.8810.62Ki-67 (cut off %35)&amp;lt;%35 (n:12)10.710.84711.710.26313.250.01110.920.69810.540.96911.920.18911.880.20312.040.153≥%35 (n:8)10.198.696.389.8810.448,388.448.19N stageN0 (n:13)10.120.69210.770.7819.810.4758.580.04710.270.8129,580.3419.960.5798.620.05N1 (n:7)11.211011.7914.0710.9312,2111.514StageStage 1 (n:5)5.10.0185.90.0447.40.17611.40.69311.20.7690.51312.50.3826.40.073Stage 2 (n:15)12.312.0311.5310.210.27119.8311.87 All statistical analyses were evaluated using the Mann Whitney U test. Citation Format: Baran Mollavelioglu, Esin Aktas Cetin, Neslihan Cabioglu, Aykhan Abbasov, Semen Onder, Selman Emiroglu, Mustafa Tukenmez, Mahmut Muslumanoglu, Abdullah Igci, Gunnur Deniz, Vahit Ozmen. Differential expression of novel immune checkpoint receptors expressed on tumor-infiltrating lymphocytes (TIL) in patients with early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-24.

Clinical Laboratory Validation and Implementation of Quantitative, Real-Time PCR-based Detection of NPM1 Type A Mutation

NPM1 mutations have prognostic significance in acute myeloid leukemia (AML) and monitoring mutant NPM1 levels during and after therapy has been described to predict relapse and survival. Despite the published significance of this molecular biomarker, routine monitoring for mutant NPM1 levels has not been widely adopted in academic clinical laboratories. Therefore, our objective was to validate a quantitative, reverse transcription-PCR assay for the detection of NPM1 Type A mutant transcripts for use in the clinical laboratory. A quantitative, real-time, reverse-transcription PCR-based method for the detection of NPM1 Type A mutant transcripts was validated for use in routine clinical practice. Results from this assay were compared to results from orthogonal methods, including next generation sequencing and digital droplet PCR. This real-time, reverse-transcription PCR-based method is sensitive (limit of detection: 0.0150% NCN and reproducible (≤ 0.5 log10-fold variation). We summarize the rigorous validation results and share observations that will help other clinical laboratories that may wish to implement this testing. We show the superior sensitivity of this assay compared to other assays (e.g., 45 gene Myeloid Next Generation Sequencing panel) and present a representative case which highlights the assay's utility in the pathologic assessment of cases with borderline morphologic or flow cytometric findings. As molecular testing for residual disease in AML continues to expand, this sensitive and reproducible method will be an appropriate testing option for the detection of NPM1 Type A mutant transcripts in clinical practice.

Long Term Responses to Venetoclax and Ibrutinib in Mantle Cell Lymphoma Are Associated with Immunological Recovery and Prognostic Changes in Inflammatory Biomarkers

Combination Ibrutinib (IB) plus Venetoclax (Ven) for the treatment of Mantle Cell Lymphoma (MCL) has demonstrated efficacy in the relapsed/refractory setting. The AIM trial treated 24 patients with IB monotherapy for 4 weeks (560mg/day) with Ven added (stepwise to 400mg/day) thereafter resulting in an overall response rate of 71% at 16 weeks (Tam et al, NEJM 2018). 5 patients discontinued treatment within 6 months due to resistant disease and 3 patients relapsed. 16 patients remained on treatment for more than 1 year. In this study we examined the longitudinal changes in peripheral blood (PB) immunology in patients treated in the AIM trial in order to identify immunological and inflammatory biomarkers associated with response and to examine if stable control of MCL without further chemotherapy exposure was associated with immunological recovery. Multiparameter flow cytometry of cryopreserved PBMCs collected at enrolment (baseline), and weeks 4, 16, 56 and 110 of treatment was used to assess the proportions of immune subsets (CD4 and CD8 memory subsets, γδ T cells and CD16+ NK cells) in AIM patients and compared to 13 age matched healthy donor (HD) samples. At baseline samples from MCL patients exhibited alterations in multiple immune subsets - most notably skewing in CD8+ memory subsets (Figure 1). Despite reduction of circulating B cell numbers consistent with clinical responses, there were no significant changes to PB immune subset distributions between baseline and 16 weeks. However at the later time points of 56 and 110 weeks there was evidence in responding patients of PB immunology normalisation to HD levels in T cell subsets including a shift in CD8 T cells from Naïve and Central Memory (CM) to Effector Memory (EM) and Terminally differentiated EM (TEMRA) phenotype and a shift in CD4 T cells from Naïve and CM to EM phenotype (Figure 2). Increased proportions of γδ T cells (Baseline 2.8 ± 3.0% week 56 4.4 ± 3.4% P&lt;0.05) and CD16+ NK cells (Baseline 40.1 ± 28.0% week 56 79.1 ± 21.0% P&lt;0.01) were also observed. T cell function assessed by intracellular IFNγ, TNFα and IL-2 in CD4 and CD8 T cells in response to T cell stimulation beads showed no change in IFNγ or TNFα production by CD4 or CD8 cells following therapy. In patients with elevated IL2+ CD4 and CD8 populations at early time points, these normalised by week 56 to HD levels. To further examine changes in immune function pathways, targeted multiplex gene expression profiling using 770 gene NanoString nCounter® PanCancer Immune Profiling Panel was performed on PBMCs at baseline and weeks 4, 16 and 56 and analyzed using nSolver advanced analysis module. Consistent with the flow cytometric findings there were minimal changes in gene expression at week 16 compared to baseline. The greatest changes were observed at week 56 with alterations in multiple pathways including increased signalling through complement, adhesion, TNF superfamily and transporter pathways in responding patients. The top upregulated genes at week 56 were CTSS (Cathepsin S), FCER1G (Fc fragment of IgE), S100A8 (S100 calcium binding protein A8) and CD14 while the top downregulated gene was CD22 (reflecting the change in B cell burden). Increased expression of C1QA and C1QB at week 16 was significantly associated with disease non-response or relapse on IB + Ven and warrants further investigation in an independent cohort to assess its applicability as a predicative biomarker for patient response. Our findings show that long term treatment with combination IB and Ven in MCL is associated with immune recovery and changes in the expression of inflammatory biomarkers. This study demonstrates that while short term assessments can be used to examine clinical disease responses, long term assessments are required to determine the immunological consequences of small molecule inhibitors. Restoration of immune system function in these patients may allow responses to subsequent immunotherapies and suggests that, in contrast to conventional chemotherapy-based regimens, this combination targeted therapy may result in beneficial impacts on immunological recovery. Disclosures Handunnetti: Gilead: Honoraria. Anderson:Walter and Eliza Hall Institute: Employment, Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; BeiGene: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees. Seymour:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Tam:Janssen: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; AbbVie: Honoraria, Research Funding; BeiGene: Honoraria; Novartis: Honoraria. Ritchie:Sanofi: Honoraria; Novartis: Honoraria; Imago: Research Funding; Beigene: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding. Koldej:NanoString Technologies: Other: Travel grant.

Correlation of Flow Cytometric Aberrations with Cytogenetic, Molecular Genetic, and Morphology in Patients with Unexplained Cytopenias

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had &gt;1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.