Receptor Rearrangement Research Articles

Bio-P-01 - Prognostic value of the clonal rearrangement of T-cell receptor genes detection in the bone marrow in patients with mycosis fungoides

<h3>Introduction:</h3> Mycosis fungoides constitutes more than 60% of primary cutaneous T-cell lymphomas. The course of disease is progradient: the gradual changes of macular, plaque and tumor stages are noted. The evaluation of the spreading process is critically important for the prognosis determination and therapy choosing. T-cell clonality detection by rearranging genes of the gamma chain of the T-cell receptor in the blood and bone marrow aspirates can be detected at the early stages. It is known to be a prognostically unfavorable factor for diagnostics in peripheral blood, while the value of the rearranging presence of T-cell receptor gene in the bone marrow aspirates has not been fully studied. <h3>Aim:</h3> To evaluate the frequency and prognostic value of the T-cell clonality detection by the gamma chain of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome (MF/SS). <h3>Materials and methods:</h3> The patients with mycosis fungoides/Sézary syndrome were observed at the National Research Centre of Hematology during the period from 2000 to 2015. <h3>Results:</h3> 45 patients diagnosed with mycosis fungoides/Sézary syndrome (MF/SS) corresponded to inclusion criteria. Men/women 1:1. The age of the participants is varied from 20 to 81 years. The diagnosis of mycosis fungoides was made in 36 cases (80%) and the diagnosis of Sézary syndrome was made in 9 cases (20%) respectively. The early stages were diagnosed in 13 patients (29%), the later stages were diagnosed in 32 patients. T-cell clonality by the gamma chain rearrangement of the T-cell receptor was detected in the bone marrow in 15 out of 45 patients (33%). 13 cases were the patients with the late disease stages and clonality at the early Ib stage was detected only in 2 cases. The morphological bone marrow involvement was identified in 5 patients, but in two cases T-cell clonality was not detected. The median survival of patients with morphologically confirmed bone marrow damage was 3 months. The median survival of patients with defined T-cell clonality in the bone marrow was 21 months. The median survival with no molecular damages of bone marrow was 41 months. <h3>Conclusions:</h3> T-cell clonality by gamma chain rearranging of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome is determined in a third of patients mainly at the late MF stages. This is an unfavorable prognostic factor.

Tre-P-24 - Aggressive gamma/delta T-cell lymphoma: successful therapy with encapsulated doxorubicin

An 82-year-old female patient initially presented with a prominent, reddish-livid, sharply circumscribed tumor measuring 10×5.5 cm on the left forearm, as well as multiple small-nodular efflorescences on both arms, the trunk, and the dorsum of the feet. The histologic picture was consistent with peripheral T-cell lymphoma. Immunohistochemical workup showed CD3 expression, high proliferative activity at 70%, and staining with gamma and delta showed the impressive picture of a consistently homogeneous reaction product with delta. No clonal T-cell receptor rearrangement was found in the molecular workup. Initial staging showed no systemic involvement (pT3N0M0). We initially treated with twelve doses of gemcitabine 600 mg fix-dose and local radiotherapy of the tumor node on the left forearm. As the cutaneous manifestations progressed, we switched to off-label therapy with pembrolizumab 200 mg every three weeks in combination with radiotherapy of the tumor nodes on the right forearm, legs and feet on both sides. As the cutaneous tumors progressed again after seven doses of pembrolizumab, therapy was switched to liposomal encapsulated doxorubicin, initially 20 mg/m2 on days 1 and 15, then every four weeks. With this therapy, tumor response has been good after six administrations to date, allowing the dose to be KOF to 10 mg/m2. Incidentally, the patient suffered from arterial hypertension and sigmoid diverticulosis. Primary cutaneous gamma/delta T-cell lymphomas are a heterogeneous group of rare and highly aggressive lymphomas. Median survival is 31 months, as they are largely resistant to radiotherapy and chemotherapy. Allogeneic stem cell transplantation offers the only curative therapy. There is no standard therapy due to the low incidence and lack of prospective studies, so that an individual therapy decision must be made for each patient. Encapsulated doxorubicin may be a good therapeutic alternative to control this highly aggressive lymphoma. As an addititional immunohistochemical workup also showed a homogenous BcL2 expression in our case, the BcL2-inhbitor Venetoclax could offer a future therapeutic option.

Sigma–1 receptor activation alleviates blood–brain barrier disruption post cerebral ischemia stroke by stimulating the GDNF–GFRα1–RET pathway

Blood–brain barrier (BBB) disruption is one of the most important pathological manifestations of ischemic stroke. Reducing BBB collapse is effective in alleviating brain parenchymal injury and cognitive dysfunction. Our previous study reported that Sigma–1 receptor (Sig–1R) activation in cerebral microvascular endothelial cells (CMECs) ameliorated BBB impairment, but the detailed mechanism remains unclear. In this study, we investigated Sig–1R activation as a BBB integrity promoter via many post ischemic stroke pathways. Sig–1R activation in BBB–associated astrocytes can increase glia–derived neurotrophic factor (GDNF) secretion in bilateral common carotid artery occlusion (BCCAO) mice. Upregulated GDNF activates its receptors in CMECs to promote BBB integrity, and activated Sig–1R in CMECs facilitates this process. In vitro experiments have found that Sig–1R activation in CMECs promotes the interaction between the GDNF α1 receptor and transduction rearrangement gene, increasing PI3K–AKT–junction protein signaling pathway expression. Sig–1R activation could be an effective therapeutic method for preventing BBB damage in ischemic stroke and other neurological conditions.

Testing for EGFR Mutations and ALK Rearrangements in Advanced Non-Small-Cell Lung Cancer: Considerations for Countries in Emerging Markets.

The treatment of patients with advanced non-small-cell lung cancer (NSCLC) in recent years has been increasingly guided by biomarker testing. Testing has centered on driver genetic alterations involving the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements. The presence of these mutations is predictive of response to targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs) and ALK TKIs. However, there are substantial challenges for the implementation of biomarker testing, particularly in emerging countries. Understanding the barriers to testing in NSCLC will be key to improving molecular testing rates worldwide and patient outcomes as a result. In this article, we review EGFR mutations and ALK rearrangements as predictive biomarkers for NSCLC, discuss a selection of appropriate tests and review the literature with respect to the global uptake of EGFR and ALK testing. To help improve testing rates and unify procedures, we review our experiences with biomarker testing in China, South Korea, Russia, Turkey, Brazil, Argentina and Mexico, and propose a set of recommendations that pathologists from emerging countries can apply to assist with the diagnosis of NSCLC.

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders

AbstractCurrent diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.

Targeted genomic profiling revealed a unique clinical phenotype in intrahepatic cholangiocarcinoma with fibroblast growth factor receptor rearrangement

Genomic aberrations (GAs) in fibroblast growth factor receptors (FGFRs) are involved in the pathogenesis of intrahepatic cholangiocarcinoma (ICC), and clinical trials have shown efficacy of FGFR inhibitors in treating ICC patients with FGFR GAs such as FGFR2 rearrangement. To clarify the FGFRs GA profile and corresponding clinicopathological features in Chinese patients with ICC, a total of 257 cases were identified. Fourteen cases (5.45%) were positive for FGFR2 rearrangement. Further analysis on the 110 FGFR2 rearrangement negative cases showed that 13 patients present additional FGFRs GAs, including FGFR3 rearrangement (2.73%), and FGFRs mutations. When compared with patients without FGFRs GAs, those with FGFR2 or FGFR3 rearrangement presented more under the age of 58 years, female sex, HBsAb positivity, CD10 expression, and PD-L1 expression. The clinical characteristics between patients with FGFRs mutation and those without FGFRs GAs were similar, with the exception that cases with FGFRs mutation have more hepatolithiasis. We concluded that FGFR rearrangement is associated with unique clinical phenotypes in ICC.

T-Cell Receptor Gene Rearrangement Clonality, Flow Cytometry Status, and Associated Outcomes in Early-Stage Cutaneous T-Cell Lymphoma

The prognostic significance of clonal T-cell receptor (TCR) rearrangement or low-level blood involvement as assessed by flow cytometry for patients with early-stage cutaneous T-cell lymphoma (CTCL) is not clear. To assess the association of low-level blood involvement by TCR clonality and flow cytometry with outcomes for patients with early-stage CTCL. A retrospective cohort analysis was conducted from September 1, 2019, to February 29, 2020, of 322 patients with early-stage (I-IIA) CTCL seen at the Winship Cancer Institute of Emory University and Grady Memorial Hospital. T-cell receptor gene rearrangement and flow cytometry records from the peripheral blood were documented at initial assessment. T-cell receptor clonality and peripheral blood flow cytometry. Univariate and multivariable models and Kaplan-Meier assessments were analyzed for overall survival (OS) and time to next treatment. The primary outcome was OS from diagnosis and time to next treatment, and the hypotheses were formulated prior to data collection. A total of 322 patients (166 female patients [51.6%]; median age at diagnosis, 53.8 years [range, 8.6-87.4 years]) with early-stage CTCL diagnosed from 1990 to 2018 were identified; of these, 258 had data available for both flow cytometry and TCR. Positive results for both TCR clonality and flow cytometry were associated with inferior OS in early-stage CTCL compared with both having negative results (hazard ratio [HR], 2.86; 95% CI, 1.02-8.06; P = .046). Positive results for only TCR clonality or only flow cytometry were not associated with OS (TCR clonality: HR, 1.31; 95% CI, 0.70-2.47; P = .40; flow cytometry: HR, 1.21; 95% CI, 0.58-2.52; P = .61) or time to next treatment (TCR clonality: HR, 1.05; 95% CI, 0.77-1.43; P = .76; flow cytometry: HR, 0.74; 95% CI, 0.47-1.16; P = .12). However, positive flow cytometry results were associated with reduced OS in the stage IIA subgroup (n = 94; HR, 1.17; 95% CI, 1.18-8.74; P = .02). Covariates associated with reduced survival included advanced age at diagnosis, male sex, and higher disease stage. This cohort study of patients with early-stage CTCL suggests that low-level blood involvement as indicated by positive results for both TCR gene rearrangement and flow cytometry was associated with inferior OS, whereas positive results for either flow cytometry or TCR clonality was not. More precise measurements of blood involvement in CTCL and larger multi-institutional cohorts are needed to validate the prognostic significance of low-level blood involvement in early-stage CTCL.

NOTCH Activation at the Definitive Mesoderm Stage Facilitates Efficient Generation of T Cells with High Proliferation Potential from Human Pluripotent Stem Cells

Adoptive T cell therapies show promise in the treatment of several types of blood cancers. Developing off-the-shelf T cell products will further advance immunotherapies to the clinic and broaden their application. Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. However, production and expansion of T cells from hPSCs remains inefficient. In order to improve T cell production from hPSCs it is essential to identify cell subsets that are highly enriched in T cell progenitors, and those stages of development at which NOTCH activation induces the most potent T cells. Previously, we have developed both OP9-based and chemically defined systems for hematopoietic differentiation from iPSCs (Vodyanik et al., 2006 and Uenishi et. al, 2014). In these differentiation systems, hPSCs undergo stepwise progression towards APLNR+PDGFRa+ mesoderm with hemangioblast colony forming cells (HB-CFCs) that reflect primitive hematopoiesis (day 3 of differentiation), KDRbrightPDGFRalow/- hematovascular mesodermal progenitors (HVMP) with definitive hematopoietic potential, VE-cadherin (VEC)+CD43-CD73- HE with definitive hematopoietic potential (day 4-5 of differentiation) and CD43+ hematopoietic progenitors, including CD235+CD41+ erythromegakaryocytic progenitors (E-MkP) and CD235a/41a-CD45+/- multipotent hematopoietic progenitors (MHP) that have lin-CD34+CD90+CD38-CD45RA- hematopoietic stem progenitor cells (HSPC) phenotype (days 6-8 of differentiation). To assess the stage at which NOTCH activation induces the most potent T cells, we isolated the aforementioned blood forming populations and cultured them in T cell conditions on OP9-DLL4. We found that Day 3 APLNR+PDGFRa+ primitive posterior mesodermal cells did not produce T cells, while all downstream subsets except CD235a+CD41a+CD45- cells do produce T cells when cultured on OP9-DLL4. As determined by limiting dilution assay, the highest frequency of T cell precursors was detected from day 4 HVMP (1 in 14 HVMP). The frequency of T cell precursors in day 5 HE and day 8 HPs was 1 in 16 HEs and 1 in 20 MHPs, respectively. In addition, we found that T cells generated from HVMPs have the capacity to proliferate for 8 weeks, in comparison to HEs and MHPs subsets, which could only be expanded for 4-5 weeks. T cell differentiation from hPSCs proceeded through a CD5+CD7+ progenitor stage that eventually transitions into CD8+CD4+ double positive cells (~90%), CD3+TCRa/b+ and CD3+TCRg/d+ cells. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. In vitro generated T-cells were functionally active and proliferated upon stimulation with PMA and IL-2. Upon activation, the cells express CD25+CD69+ (~73%) markers, cytokines (IFN-γ ~87%, TNFa~22%, IL2 ~34.5%) and cytolytic proteins (Perforin~37%). We also demonstrated that CD5+CD7+ T cell progenitors can be genetically modified to express CD19 CARs and eventually differentiate into CAR T cells with significant cytotoxic effect on Raji cells. Overall, our studies may aid in establishing protocols for the efficient off-the shelf production and expansion of PSC-derived CAR T cells for treating hematologic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Assessing the Utility of Immunoglobulin and T Cell Receptor Gene Rearrangement Studies for Cerebrospinal Fluid Specimens

INTRODUCTION : Immunoglobulin (IGH and IGK) and T cell receptor (TCRG) rearrangements (-R) by polymerase chain reaction (PCR) have emerged as important ancillary tools for assessing clonality of B-cell and T-cell processes, respectively, in specimens such as lymph node, bone marrow and peripheral blood. However, the utility of these gene rearrangement studies in cerebrospinal fluid (CSF) samples has not yet been systematically examined. Cytomorphology and flow cytometry (FC) are currently the standard tools for the assessment of the CSF for leptomeningeal involvement by hematological malignancies. There are inherent limitations to both of these diagnostic methods; thus, it is important to establish the role of IGH/IGK- R and TCRG- R studies in the examination of CSF.METHODS : Pathology reports of 87 consecutive CSF specimens from 61 patients submitted for IGH, IGK and/or TCRG -R studies in the past three years (2015-2017) were retrospectively reviewed. PCR for IGH (using FR3 and JH primers), IGK and TCRG rearrangements was performed using the BIOMED2 assays. The PCR products for all three reactions were analyzed via polyacrylamide gel or capillary electrophoresis. The interpretation was rendered by an attending molecular pathologist. These results were assessed in the context of the corresponding flow cytometry report.RESULTS : Majority of the patients (82% of patients) had a prior diagnosis of a hematologic malignancy (Table 1), including diffuse large B-cell lymphoma (31% of specimens) and acute leukemias (17%), while the remaining patients presented with CNS symptoms with/without a brain mass. Nearly equal numbers of cases were requested by the clinicians concurrently with flow cytometry (46 cases; 53%) or by the hematopathologist evaluating the CSF FC results (41 cases; 47%). In the latter group, PCR was requested due to an atypical finding based on CSF flow cytometry, which most commonly was an abnormal population with an immunophenotype was different from the diagnostic specimen or a suspicious population that was too small to be diagnostic.Of the 61 IGH- R and 35 IGK -R tests requested, the majority of the specimens (74% and 63%, respectively) demonstrated a pseudoclonal pattern or poor amplification because of the paucity of B cells. Of the cases with sufficient cellular material, a monoclonal population was detected by IGH- R and IGK -R in more than half of the cases (56% and 54%, respectively). In contrast, in 75% (24/32) of the cases where TCRG-R was requested, there was an adequate number of T-cells for analysis; of these 24 informative cases, 9 (38%) were monoclonal and 15 (62%) were polyclonal.Overall, 37 of the 87 cases (43%) yielded an informative result for one or more of IGH, IGK, and/or TCRG -R. 38% (14/37) of these cases showed a result that clarified or supported an atypical/suspicious finding by FC, whether submitted by the pathologist (70%) or treating clinician (30%). Eight of the 37 cases (22%) were submitted by the pathologist to confirm the FC findings. In 7 of these 8 cases, the PCR findings corroborated the FC results; there was one discordant case in which a polyclonal T-cell population was seen in a positive FC case of T-lymphoblastic lymphoma/leukemia. Remainder of the cases (14/37, 38%) were submitted by the treating clinicians; if they were submitted together with a concurrent FC analysis (12 of 14 cases), the PCR results confirmed unambiguous FC results (whether negative or positive). In 6 of the 14 cases in which a IGH -R and/or IGK -R identified a monoclonal population, the atypical or neoplastic population constituted <5% of the analyzed cells by FC.CONCLUSIONS : The paucicellular nature of the CSF specimens provides a challenge not only for cytomorphology and FC but also for PCR for IGH, IGK, and TCRG rearrangements. Nonetheless, an informative clonality result was observed in 43% of the cases submitted for PCR analysis. The results of 60% of these cases (26% of the total submitted cases) were helpful for clarifying and/or supporting an FC finding. Thus, PCR for IGH, IGK and TCRG -R of CSF specimens is a useful ancillary diagnostic aid and can help in the interpretation and confirmation of flow cytometry results, particularly when the neoplastic lymphoid cells are of low abundance or exhibit an immunophenotype that is distinct from the diagnostic tumor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow-Derived Mesenchymal Stromal Cells Promote Thymopoiesis and Function Recovery in Acute Graft-Versus-Host Disease Murine Models

Background: The thymus is the primary lymphoid organ of T-cells development and plays a crucial role for immune tolerance following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The toxicity of chemoradiotherapy conditioning regimen and acute graft-versus-host disease (aGVHD) damage the thymic structure and function, which result in the production of autoreactive T-cells and the reduction of natural regulatory T cells (nTregs) to aggravate the development of aGVHD. Nowadays, mesenchymal stromal cells (MSCs) as multipotent stem cells, have been widely recognized as a promising therapeutic strategy for aGVHD. However, the well-known mechanisms of MSCs ameliorating aGVHD mainly focus on their immunoregulatory effect on peripheral immune compartments. The contribution of MSCs to thymus, the central immunological organs, is rarely reported.Methods: In this study, we carried out studies in a murine aGVHD models of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). We randomly divided aGVHD mice into two groups, including MSCs group and control group without MSCs treatment. In MSCs group, aGVHD mice were intravenously administrated GFP-labelled bone marrow (BM) derived MSCs at a dose of 5×105 cells/mice at 7 day after allo-HSCT. The mice were killed at 1d, 2d, 4d and 20d after MSCs injection in MSCs group and the corresponding time in control group, respectively. Thymic tissues were collected and manufactured into frozen sections or thymic single-cell suspension. And then we analyzed the distribution of GFP-MSCs in thymus and thymic size, the expression of thymic T cells subsets (CD4+CD8-, CD4-CD8+, CD4+CD8+ T, CD4+CD25+Foxp3+Tregs), thymic epithelial cells (TECs) substes (CD45-CD326+Ly51+ cortical TECs and CD45-CD326+UEA-1+ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus between the two groups to evaluate the repair effect of MSCs for thymus.Results: We found that MSCs treated mice have longer survival and alleviate the clinical signs of aGVHD compared to the control group (P<0.05). Next, we observed that GFP-MSCs can home to thymic tissue and mainly locate in the corticomedullary boundary at 2d, 4da after MSCs infusion. MSCs treated thymus exhibited a more organized corticomedullary architecture, compared with the conrol group. Significant increases in thymus size and weight, as well as the number of total thymocytes were seen in MSCs group. To further investigate whether MSCs promote thymopoiesis and ameliorate thymic functions after allo-HSCT, we found that in MSCs group the proportion of thymic T cells subets composition (CD4+CD8-, CD4-CD8+, CD4+CD8+), especially Foxp3+ Tregs, and the expression of TECs, especially cTECs, were significantly higher than control group at 20d after the MSCs infusion (P<0.001). Furthermore, MSCs administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 20d after the MSCs infusion compared with the control group (P=0.007).Conclusions: Collectively, our data proposed a new mechanism for MSCs ameliorating aGVHD by central immune organ, including promoting thymopoiesis and enhancing thymic output. DisclosuresNo relevant conflicts of interest to declare.

Label-Free Analyses of Minimal Residual Disease in ALL Using Deep Learning and Imaging Flow Cytometry

MD/MC and PR/JI contributed equallyMinimal residual disease is an important response biomarker for risk stratification in acute lymphoblastic leukaemia (ALL) and is assessed by two methodologies, molecular analyses of patient-specific, clonal antigen receptor rearrangements or flow cytometric analyses of aberrant leukaemia-associated immunophenotypes. Some clinical regimens use both methodologies and sequential samples of bone marrow and/or peripheral blood are assessed during, at the end of induction therapy and at time points beyond. Both MRD methods are highly specialised, laborious and for resource poor countries, are not affordable. In addition, a proportion of patients are not evaluable by either method.Technological advances in cytometry have led to imaging flow cytometers (IFC) which deliver all standard flow cytometric parameters, as well as a high quality image of each cell. We have previously shown that machine learning of IFC data can classify cell cycle phases within a cell, using only data from bright field (BF) and dark field (DF)1 and we hypothesised that this might be possible in the MRD setting. Thus, paired diagnostic and follow up bone marrow aspirates taken during remission induction from children with ALL (n=23, 6 at day 8 or day 15 or day 28 following the start of treatment) were stained with CD19 APC, CD10PE, CD34Texas Red, CD45APCH7 and DAPI and ran on an imaging flow cytometer (Amnis, Imagestream X Mark II) at x40 magnification. A standard sequential gating technique identified ALL cells and levels (range 90-0.01%) correlated with that assessed by traditional flow cytometry. Next, 200 morphological features were extracted from BF and DF images of cells including pixel intensities, size, shapes, textures and subcellular components and both supervised machine and deep learning algorithms were developed to correctly classify ALL cells in clinical samples. The classifiers were trained initially using all fluorescent and BF and DF parameters but then with a sequential leave-one-parameter-out. In parallel, iteration of the training-testing sets on 23 datasets, with a leave-one-sample-out approach was performed, to ensure prediction accuracy across samples. Correct cell classifiers peaked at 98.21% when all parameters were considered but even without antibody-conjugated signals, relying only in DAPI, DF and BF signals, correct cell classification averaged 85.36%. Finally, DF/BF in combination and BF alone still gave rise to correct cell call rates of 81.62% and 71.26% respectively.Thus, we report a new MRD detection method that is label free, using only the morphological features extracted from BF and DF cell images. The method is cheap, quick, and highly applicable and with a technology that could be simplified to be laser-free and allow point of care testing. Transferability of this unique methodological approach to other types of leukaemia is also feasible and may be further developed to give functional information e.g. cell cycle status.

Imaging features of unilateral renal T‐cell lymphoma occurring simultaneously in the urinary bladder wall and contralateral ureter with secondary polycythaemia in a dog

AbstractA six‐year‐old neutered male Labrador Retriever was presented with haematuria and inappetence of one‐week duration. Physical examination revealed mild abdominal pain and the blood cell count showed moderate polycythaemia, with a slight increase of the erythropoietin levels. Urinalysis demonstrated proteinuria, haematuria and isosthenuria and urinary sediment revealed mild amount of lymphoid cells. After performing a complete imaging study (radiographs, ultrasound and CT scan), a unilateral renal lymphoma with secondary extranodal lymphoma of the urinary tract (bladder wall and left ureter) was suspected. Fine‐needle aspirates of both kidneys, ureter and bladder masses were obtained, and a tentative diagnosis of lymphoma was made. Positive PCR antigen receptor rearrangement indicated a T‐cell origin. The owners opted for euthanasia and declined necropsy. CT was essential for the evaluation of this case – it not only depicted renal lesions, but also identified accurately extension to adjacent anatomic structures helping to determine the systemic spread of the disease.

Novel methods to assess cell-free circulating tumor DNA in acute lymphoblastic leukemia.

10034 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relatively high relapse rate, which is associated with poor prognosis. Currently, minimal residual disease (MRD) at the end of induction and consolidation therapy is the best predictor of patient relapse, however obtaining bone marrow aspirate is invasive and not always accurate. Another major concern in ALL is the presence of central nervous system (CNS) disease, which is often present long before clinical diagnosis can be made by flow cytometry. To circumvent these clinical challenges, we developed a new assay quantifying cell-free, circulating tumor (cfDNA) as a biomarker of disease progression, which can be correlated with MRD status as a predictor of relapse. cfDNA is frequently used to monitor progression of solid tumors, but pediatric leukemias lack common mutations that can be used to distinguish leukemic cfDNA from normal cfDNA. Methods: We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We developed a novel workflow for identifying VDJ rearrangements in leukemia cells and tracking their presence in cfDNA. We collected bone marrow, blood, and CSF samples from newly diagnosed patients, and cfDNA was isolated from blood and CSF samples throughout treatment. Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements of leukemic clones in genomic DNA. The MinION assay relies on patient-specific sequencing. We are also in the process of developing a universal assay that utilizes recurrent methylation changes in ALL to identify leukemic cfDNA in patient samples. Results: The MinION workflow was used to follow leukemic cfDNA throughout the course of treatment, and accurately identified MRD and CNS disease in patients. This workflow performed equivalent or better at detecting leukemic clones compared to MiSeq and droplet digital polymerase chain reaction (ddPCR), and is faster and less expensive than traditional Illumina sequencing. Methylation analysis of 865 ALL and 79 healthy samples yielded 55 regions and 19 specific methylation sites that were uniquely present in ALL samples. We are validating these sites by ddPCR to establish a panel of biomarkers to track ALL over time via cfDNA. Conclusions: The end goal of our study is provide a more sensitive and less invasive method for tracking MRD and CNS disease than current approaches. Results will ultimately be correlated with patient response to therapy, the presence of relapse or CNS disease, and overall outcomes determined by standard clinical diagnostic procedures.

Molecular pathology in the cancer clinic - where are we now and where are we headed?

Molecular pathology is a developing sub-microscopic discipline of pathology that studies the effects of molecular variations and mutations on disease processes. The ultimate goal of molecular pathology in cancer is to predict risk, facilitate diagnosis and improve prognostication based on a complete understanding of the biological impact of specific molecular variations, mutations and dysregulations. This knowledge will provide the basis for customised cancer treatment, so-called precision medicine. Rapid developments in genomics have placed this field at the forefront of clinical molecular pathology and there are already a number of well-established genetic tests available for clinical use including PCR of antigen receptor rearrangement and KIT mutational analysis. Moving beyond tests assessing a single gene, there are significant research efforts utilising genomics to predict cancer risk, forecast aggressive behaviour and identify druggable mutations and therapeutic biomarkers. Researchers are also investigating the use of circulating cells and nucleic acid for clinically useful low morbidity genomic assessments. If we are to realise the full potential of molecular pathology and precision medicine there are a number of challenges to overcome. These include developing our understanding of the underlying biology (in particular intra-tumoural heterogeneity), methodological standardisation of assays, provision of adequate infrastructure and production of novel therapeutics backed by high-quality clinical data supporting the precision medicine approach. The era of molecular pathology holds the potential to revolutionise veterinary cancer care, but its impact on clinical practice will depend upon the extent to which the inherent challenges can be overcome.

Prognostic value and characterization of NTRK1 variation by fluorescence in situ hybridization in esophageal squamous cell carcinoma.

Rearrangement of the neurotrophic tyrosine kinase receptor (NTRK) 1 gene is a target of tropomyosin receptor kinase A (TRKA) inhibitors, and its targeted drug (larotrectinib) has been approved by the US Food and Drug Administration. We investigated the existence and prognostic importance of NTRK1 variation in esophageal squamous cell carcinoma (ESCC). Fluorescence in situ hybridization of a NTRK1 rearrangement was conducted on 523 ESCC samples through tissue microarrays. Kaplan-Meier curves with log-rank tests were used to evaluate survival. We identified 8 (1.5%), 35(6.7%) and 109 (20.8%) cases with a NTRK1 rearrangement using 15%, 10% and 5% as cut-off values, respectively. We observed copy number (CN) variation of NTRK1 in some cases: 79 (15.1%) cases had a gain in NTRK1 CN ≥ 3, and 24 (4.6%) cases had NTRK1 CN ≥ 4. A NTRK1 rearrangement at the above-mentioned thresholds was not related to disease-free survival (DFS, P = 0.45, 0.47, 0.87) and overall survival (OS, P = 0.80, 0.74, 0.57), respectively. Gain in NTRK1 CN was associated with a poor prognosis irrespective of whether NTRK1 CN ≥ 4 (DFS, P = 0.015; OS, P = 0.035) or NTRK1 CN ≥ 3 (DFS, P = 0.039; OS, P = 0.025). A NTRK1 rearrangement occurred rarely in ESCC. The increased CN of NTRK1 might be a prognostic indicator for DFS and OS in patients with ESCC.

Minimal Disease Monitoring in Pediatric Non-Hodgkin’s Lymphoma: Current Clinical Application and Future Challenges

Simple SummaryMinimal residual disease (MRD) describes the detection of a few remaining malignant cells in blood or bone marrow with molecular methods or flow cytometry. The detection of MRD in patients with leukemia during therapy indicates the risk of relapse. Its use for treatment stratification is state of the art in all modern leukemia treatment protocols. In pediatric non-Hodgkin lymphoma patients, minimal amounts of tumor cells, so-called minimal disseminated disease (MDD), can often be detected in blood or bone marrow at diagnosis. In children with ALK-positive anaplastic large cell lymphoma, MDD and MRD detected in the blood or bone marrow is associated with high relapse risk. For patients with Burkitt lymphoma or -leukemia or lymphoblastic lymphoma, the meaning of MDD and MRD is less clear. This review summarizes the current knowledge, techniques, application, and challenges in minimal disease detection in pediatric non-Hodgkin lymphoma.Minimal residual disease (MRD) detection is established routine practice for treatment stratification in leukemia and used for treatment optimization in adult lymphomas. Minimal disease studies in childhood non-Hodgkin lymphomas are challenged by stratified treatment in different subtypes, high cure rates, low patient numbers, limited initial tumor material, and early progression. Current clinical applications differ between the subtypes. A prognostic value of minimal disseminated disease (MDD) could not yet be clearly established for lymphoblastic lymphoma using flow cytometry and PCR-based methods for T-cell receptor (TCR) or immunoglobulin (IG) rearrangements. MYC–IGH fusion sequences or IG rearrangements enable minimal disease detection in Burkitt lymphoma and -leukemia. An additional prognostic value of MDD in Burkitt lymphoma and early MRD in Burkitt leukemia is implicated by single studies with risk-adapted therapy. MDD and MRD determined by PCR for ALK-fusion transcripts are independent prognostic parameters for patients with ALK-positive anaplastic large cell lymphoma (ALCL). They are introduced in routine clinical practice and used for patient stratification in clinical studies. Early MRD might serve as an endpoint for clinical trials and for guiding individual therapy. Validation of MDD and MRD as prognostic parameters is required for all subtypes but ALCL. Next-generation sequencing-based methods may provide new options and applications for minimal disease evaluation in childhood lymphomas.

Canine Multicentric Large B Cell Lymphoma with Increased Mott Cells Diagnosed by Flow Cytometry

A 12-year-old dog was referred due to multiple superficial lymphadenopathy. On cytology, each lymph node showed different cell populations where some of them consisted of intermediate to large lymphocytes with frequent Mott cells. Presence of Mott cells along with immature lymphocytes made the cytological diagnosis challenging, and therefore, supplementary diagnostic tests including PCR for Antigen Receptor Rearrangement (PARR) assay and flow cytometry were performed. This case report illustrates the value of flow cytometry in the diagnosis of lymphadenopathy with ambiguous cytologic findings.

Thymectomy in Juvenile Myasthenia Gravis Is Safe Regarding Long Term Immunological Effects

Thymectomy is an established treatment in adult MG and also recommended for the treatment of post-pubertal onset juvenile MG. Whether the youngest children should be thymectomized is still debated. Signs of premature aging of the immune system have been shown in studies on early perioperative thymectomy in children with congenital heart defect. In this retrospective cohort study the objective was to investigate the long-term effects of treatment related thymectomy on T cell subsets and T cell receptor rearrangement excision circles (TRECs) in peripheral blood of juvenile myasthenia gravis (MG) patients, as well as clinical occurrence of autoimmune disorders, malignancies and infectious diseases. Forty-seven patients with onset of myasthenia gravis before the age of 19 years were included; 32 (68.1%) had been thymectomized and 15 (31.8%) had not. They were studied at varying times after thymectomy (7–26 years). We found a significant lower number of naïve helper T cells (CD4+CD45RA+) with an increased proportion of memory helper T cells (CD4+CD45RO+), and a significant lower number of naïve cytotoxic T cells (CD8+CD27+CD28+) in the thymectomized patients. In addition they showed a significant reduction in the number of TRECs and proportion of recent thymic emigrants (RTE) compared to non-thymectomized patients. In none of them an increased frequency of malignancies or infections was found. Our findings indicate a premature aging of the immune system after thymectomy in juvenile MG, but associated clinical consequences could not be verified.

Telomere length and signal joint T-cell receptor rearrangement excision circles as biomarkers for chronological age estimation

Background Chronological age estimation is a challenging marker in the field of forensic medicine. The current study aimed to investigate the accuracy of signal joint T-cell receptor rearrangement excision circles (sjTRECs) quantification and telomere length measurement as methods for estimating chronological age. Methods Telomere length was estimated in the DNA derived from the buccal cells through estimating the telomeric restriction fragment (TRF) length using TeloTTAGGG Telomere Length Assay while the sjTRECs quantification was carried out on DNA isolated from the blood samples using qPCR. Results The TRF length was shortened with increased age (r = −0.722, p < 0.001). The sjTRECs were also decreased with increased age (r = −0.831, p < 0.001). Stronger coefficient and lower standard error of the estimate was obtained when multiple regression analysis for age prediction based on the values of both methods was applied (r = −0.876, p < 0.001).

Feline and canine Merkel cell carcinoma: A case series and discussion on cellular origin.

Merkel cell carcinoma (MCC) is in humans and cats a malignant cutaneous neuroendocrine carcinoma, whereas in dogs it possibly has a more benign behaviour. It may be cytologically confused with round cell tumours such as lymphoma because of its striking cytomorphologic similarity. Although MCC is considered to arise from Merkel cells, recent findings indicated that primitive (epi‐)dermal stem cells, early B‐cells or dermal fibroblasts were the origin of human MCC. The aim of our study was to evaluate a possible lymphoid origin in feline and canine MCCs. Specific analysis of CD3, PAX‐5, KIT and PARR assay were performed in 3 feline and 3 canine MCCs. All MCCs (6/6) were negative for CD3 and PAX‐5. KIT was expressed in all MCCs (6/6). Assessment of clonality by PARR assay exhibited a polyclonal B‐ and T‐cell receptor rearrangement in all five cases tested. In conclusion, a lymphoid origin of feline and canine MCCs could not be demonstrated. This is in contrast with human MCCs, that often express early B‐cell lineage markers.