Monoclonal Population Research Articles

S2095 Gastric Extramedullary Plasmacytoma Presenting as a Solitary Gastric Mass

INTRODUCTION: Extramedullary plasmacytomas (EMP) are a subcategory of plasma cell neoplasm that involves organs outside the bone marrow. We report a case of an aggressive extramedullary myeloma invading the stomach. CASE DESCRIPTION/METHODS: An 84 year old lady with PMHx of DM, hypothyroidism, HTN and GERD presented to the ED with abdominal pain, nausea and vomiting for 2 days. Patient was found to be anemic with Hgb of 10.7 g/dL, hematocrit 32.4%. Patient’s electrolytes and renal function tests were within normal limits. Imaging (US) of the abdomen showed gallbladder calculus with positive murphy’s sign and no pericholecystic fluid. A CT scan of the abdomen and pelvis showed a large hiatal hernia with suspicion of cholelithiasis. A HIDA scan showed no evidence of cystic duct obstruction or distal biliary obstruction. After cholecystitis was ruled out GI was consulted and a EGD was planned for intractable abdominal pain with intermittent vomiting. During the EGD findings [Figure 1] were noted for a 4 cm non-bleeding, non-ulcerated mass on a very large stalk that appeared to be blocking the pylorus. The pylorus was normal appearing once past the mass. The polyp was partially resected. A small polyp was resected but the larger polyp was not able to be removed due to getting stuck at the cricopharyngeus so it remained in the stomach. Biopsy findings were noted predominantly of well-differentiated plasma cells with some of the cells extending to the mucosa. The immunohistochemical stains were positive for CD138, CD 79 [Figure 2] and showed a low Ki-67 proliferation index of < 5%. The neoplastic cells were lambda light chain restricted [Figure 3]. Rare plasma cells are positive for kappa. They were negative for PAX5, CD20, CD56, c-KIT, cyclin D1 and pankeratin. CD3 highlights background T-cells. Based on the findings a plasmacytoma or secondary involvement by multiple myeloma was favored. DISCUSSION: Plasmacytoma can be primary or secondary with secondary form more common. The most commonly involved sites are the liver, spleen, and lymph nodes. The prevalence of GI system involvement was found to be 0.9% in a recent systematic review done with 2,584 Multiple myeloma (MM) patients. This is associated with a poor survival of less than seven months from diagnosis. [1] Favorable outcomes in cases of EMP have been linked to tumor size < 4 cm, age < 50 years, patients with head and neck EMP, and serum M protein negativity. [2]. We have described a rare case of gastric plasmacytoma without symptoms of MM.Figure 1.: EGD finding of a mass obstructing the pylorus, injected with epinephrine and clamped before excision.Figure 2.: Cells stained with CD79A.Figure 3.: Monoclonal population lambda +.

Modeling How Heterogeneity in Cell Cycle Length Affects Cancer Cell Growth Dynamics in Response to Treatment.

Tumors are complex, dynamic, and adaptive biological systems characterized by high heterogeneity at genetic, epigenetic, phenotypic, as well as tissue microenvironmental level. In this work, utilizing cellular automata methods, we focus on intrinsic heterogeneity with respect to cell cycle duration and explore whether and to what extent this heterogeneity affects cancer cell growth dynamics when cytotoxic treatment is applied. We assume that treatment acts on cancer cells specifically during mitosis and compare it with a (cell cycle-non-specific) cytotoxic treatment that acts randomly regardless of the cell cycle phase. We simulate the spatiotemporal evolution of tumor cells with different initial spatial configurations and different cell length probability distributions. We observed that in heterogeneous populations, strong selection forces act on cancer cells favoring the faster cells, when the death rates are lower than the proliferation rates. However, at higher mitotic death rates, selection of the slower proliferative cells is favored, leading to slower post-treatment regrowth rates, as compared to untreated growth. Of note, random cell death progressively eliminates the slower proliferative cells, consistently, favoring highly proliferative phenotypes. Interestingly, compared to the monoclonal populations that exhibit complete response at high random death rates, emergent resistance arises naturally in heterogeneous populations during treatment. As divergent selection forces may act on a heterogeneous cancer cell population, we argue that treatment plan selection can considerably alter the post-treatment tumor dynamics, cell survival, and emergence of resistance, proving its significant biological and therapeutic impact.

Morphological and immunofenotypic features of the monoclonal population of B-lymphocytes in chronic lymphocytic leukemia

Purpose of the study. To evaluate the features of morphological and immunophenotypic characteristics of the lymphoid population with different restriction of light chains of immunoglobulins in patients with chronic lymphocytic leukemia (CLL).Materials and methods. The study included 30 CLL patients aged 47–79 years (20 men and 10 women). All patients underwent a General clinical blood test (SysmexXE 2100, Japan), morphological examination of the bone marrow (BioVision; Micros, Austria), immunophenotyping of bone marrow and peripheral blood by flow cytofluorometry (Navios10/3, Beckman Coulter, USA). B-cell clonality established by detection of restriction of light chains of surface immunoglobulins kappa or lambda. Morphological analysis of lymphocytes that differ in the expression of light chains of surface immunoglobulins: kappa (k) — group I (22 people — 73,3%), lambda (λ) — group II (8 people — 26,7%).Results. Determination of cell types by values of direct (FSC) and lateral (SSC) light scattering during immunophenotyping of peripheral blood and bone marrow samples showed that in patients of group I (CD19k+/CD5+/CD23+) on the light scattering diagram, the lymphoid population had low parameters: on the FSC scale — from 200 to 400, on the SSC — from 10 to 160 units, which indicates morphological uniformity of cells. In group II (CD19λ+/CD5+/CD23+), on the contrary, on the light scattering sketogram, the lymphoid zone was heterogeneous and stretched: on the FSC scale — from 200 to 1000, on the SSC — from 10 to 400 units, which indicates morphological polymorphism of cells. There were also differences in the expression of the common leukocyte antigen CD45. In group I, the expression is higher: the population of B-lymphocytes in terms of fluorescence intensity is on the dot graph on the CD45 scale in the second half of the third decade and in the fourth decade — to the right, than in group II, in which B-lymphocytes lie in the third decade. The data indicate that the CD19k+/CD5+/CD23+ population is represented by Mature cells, while the Cd19k+/CD5+/CD23+ population is represented by less Mature and / or intermediate forms. Significant morphological differences in lymphocyte populations were also observed in microscopic studies of blood and bone marrow preparations.Conclusion. The established immunophenotypic and morphological differences in lymphoid populations expressing either kappa — or lambda-light chains of immunoglobulins may be important for identifying risk groups among patients with biologically heterogeneous variants of chronic lymphocytic leukemia.

Cancer-associated mutations in normal human endometrium: Surprise or expected?

The human endometrium is an essential component in human reproduction that has the unique characteristic of undergoing cyclic regeneration during each menstrual cycle. Vigorous regeneration after shedding may be sustained by stem/progenitor cells, for which molecular markers have not been fully identified. Although clonality analysis using X chromosome inactivation patterns has shown that normal human endometrial glands are composed of a monoclonal cell population, whether clonal expansion is derived from stem/progenitor cells remains unclear. Remarkable advances in next‐generation sequencing technology over the past decade have enabled somatic mutations to be detected in not only cancers, but also normal solid tissues. Unexpectedly frequent cancer‐associated mutations have been detected in a variety of normal tissues, and recent studies have clarified the mutational landscape of normal human endometrium. In epithelial glandular cells, representative cancer‐associated mutations are frequently observed in an age‐dependent manner, presumably leading to growth advantage. However, the extremely high mutation loads attributed to DNA mismatch repair deficiency and POLE mutations, as well as structural and copy number alterations, are specific to endometrial cancer, not to normal epithelial cells. The malignant conversion of normal epithelial cells requires these additional genetic hits, which are presumably accumulated during aging, and may therefore be a rare life event. These discoveries could be expected to shed light on the physiology and pathogenesis of the human endometrium and urge caution against the application of genetic screening for the early detection of endometrial cancer.

Pleural fluid MYD88 L265P mutation supporting diagnosis and decision to treat extramedullary Waldenstrom\u2019s macroglobulinemia: a\xa0case report

BackgroundOur case of a patient with untreated lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia with extramedullary pleural effusion is the first documented case of pleural fluid MYD88 L265P mutation status in a community hospital setting. Our patient was intolerant to 420 mg ibrutinib, but still achieved a lasting complete remission, as defined by National Comprehensive Cancer Network guidelines, with a dose reduction to 240 mg of ibrutinib.Case presentationA 72-year-old Caucasian (white) man diagnosed with monoclonal immunoglobin M kappa lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia monitored without treatment for 2 years, presented with dyspnea and a left pleural effusion. At presentation, computed tomography scans of his chest, abdomen, and pelvis showed layering left pleural effusion and para-aortic lymphadenopathy. Pleural fluid cytology demonstrated B-cell lymphoma of the lymphoplasmacytic subtype, with monoclonal kappa B-cell population on flow and a positive MYD88 L265P mutation. The pleural effusion recurred post-thoracentesis and he achieved a lasting complete remission as defined by National Comprehensive Cancer Network guideline with 240 mg ibrutinib.ConclusionsOur discussion details a comprehensive literature review of extramedullary pulmonary involvement in Waldenstrom’s macroglobulinemia. Establishing a malignant etiology for pleural effusion in Waldenstrom’s macroglobulinemia can be challenging, as standard techniques may be insensitive. Allele-specific polymerase chain reaction for detecting MYD88 L265P mutations is more sensitive for confirming lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia in pleural fluid. Extramedullary pulmonary involvement usually presents post-diagnosis of Waldenstrom’s macroglobulinemia and responds well to Waldenstrom’s macroglobulinemia-directed treatment regimens. Allele-specific polymerase chain reaction is a sensitive assay for detecting MYD88 L265P mutations in pleural fluid to support the diagnosis of malignant pleural effusion in the setting of Waldenstrom’s macroglobulinemia and helps guide the treatment decision to use ibrutinib. Although intolerant of ibrutinib 420 mg, our patient achieved complete and sustained remission of pleural effusion with a dose of 240 mg with progression free survival of over 30 months.

Successful Treatment for Solitary Bone Plasmacytoma of the Maxilla: A Rare Case Report

Introduction: Plasma cell tumors are monoclonal neoplastic proliferation of plasma cells which are divided into three groups: solitary bone plasmacytoma (SBP) and extramedullary plasmacytoma (EMP) which are localized forms, and multiple myeloma (MM) which is the disseminated form. Skull, long bones, and vertebrae are common sites for SBP and its presence in maxillofacial area is rare. There is a possibility that SBP converts to MM and some authors believe that it can be the early stage of MM. Case Presentation: Here, we present a 59-year old male patient with a mass in maxillary bone enlarging after tooth extraction. Cone beam computed tomography (CBCT) confirmed the existence of a poorly defined lesion in the left maxilla. The microscopic evaluation and immunohistochemical studies confirmed the diffuse and monoclonal population of cells, indicating the diagnosis of plasma cell tumor. There was also no evidence of bone marrow involvement in the aspiration. Conclusions: Early and precise diagnosis of SBP is a crucial element to rule out the possibility of MM. It must be kept in mind that there is possibility of recurrence or progression to MM after treatment which can completely change the course of disease, and this emphasizes the importance of regular follow up.

Maladies des chaînes lourdes

In most plasma cell disorders, monoclonal proteins have a structure similar to normal molecules but in abnormal amounts. Heavy chain disease (HCD) is a rare pathology defined by the presence of a monoclonal immunoglobulin (Ig) of abnormal structure, consisting only of an incomplete fragment of the heavy chain (truncated) and devoid of light chains, remarkable for the frequency of associated immunological or haematological diseases.Abnormal lymphocytes or plasma cells secrete the different types of heavy chains (alpha [α], gamma [γ], mu [μ] or delta [δ]) without light chains. The majority of heavy chain proteins are fragments of their normal counterparts with internal deletions of variable length. These deletions seem to result from structural mutations.There are 4 types of MCL according to the Ig heavy chain class produced by clonal cells: α, γ, μ or δ. The clinical presentation is closer to that of lymphoma than to multiple myeloma.The diagnosis of HCD and related diseases is based on the histological study of affected tissues and the analysis of serum or urine. The histological analysis with immunostaining shows a positive monoclonal population for a heavy chain, but negative for the two kappa (κ) and lambda (λ) light chains.The frequency of detection of the monoclonal peak is variable and depends on the type of HCD. The prognosis of these diseases is very variable from one patient to another and the treatment is not standardized.

Genetically Engineered Human Kidney Cells for Real-Time Cytotoxicity Testing In Vitro.

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.

Conjunctival Pediatric-Type Follicular Lymphoma.

Pediatric-type follicular lymphoma is an uncommon B-cell lymphoma that primarily involves the lymph nodes of the head and neck of children and young adults. Ocular involvement is rare, with only 5 well-documented cases in the literature, all occurring in the conjunctiva. The authors describe a 20-year-old African American man with enlarging bilateral medial conjunctival nodules. Excisional biopsy of the larger left conjunctival lesion, clinically presumed to be pyogenic granuloma, revealed pediatric-type follicular lymphoma composed of expansile follicles with germinal centers containing mitotically active blastoid cells, which coexpressed CD20, CD10, and BCL6 and lacked expression of BCL2. Polymerase chain reaction for immunoglobulin heavy chain rearrangement identified a monoclonal B-cell population. Fluorescence in situ hybridization studies failed to demonstrate BCL2, BCL6, and MALT1 gene rearrangements. Next-generation sequencing revealed a pathogenic MAP2K1 mutation, known to be enriched in pediatric-type follicular lymphoma. This report summarizes the clinical and pathologic diagnostic findings, biologic behavior, and management of conjunctival pediatric-type follicular lymphoma.

Localised pulmonary crystal-storing histiocytosis with underlying extranodal marginal zone lymphoma: A case report

Crystal-storing histiocytosis (CSH) is a rare entity, composed of a proliferation of non-neoplastic histiocytes with an accumulation of intracytoplasmic crystalline material. It is often associated with an underlying lymphoproliferative disorder, most often those that express monoclonal immunoglobulins, such as multiple myeloma and lymphoplasmacytic lymphoma. Here we report a case of localised pulmonary CSH in an asymptomatic 54-year-old man, found to have an incidental solid lesion in the right lung on computed tomography (CT). The lesion was composed of sheets of CD68 positive histiocytes that contained refractile eosinophilic crystalline inclusions, scattered lymphoid follicles of CD20 positive B lymphocytes, and occasional plasma cells. PCR for immunoglobulin heavy chain gene rearrangement confirmed the presence of a monoclonal population of B cells. These findings were supportive of a diagnosis of CSH with an underlying B cell/ plasma cell lymphoproliferative disorder, which was favoured to be an extranodal marginal zone lymphoma after clinical and radiological workup. Recognition of this rare entity is important as it may obscure an underlying lymphoproliferative disorder. A diagnosis of CSH should prompt careful exclusion of neoplasia, including clinical workup for systemic disease. In this case report we also discuss clinical features and diagnostic pitfalls.

Large Granular Lymphocytosis and Its Impact on Long Term Clinical Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation: 14-Year Follow-up Data

Introduction Several studies have attempted to describe the characteristics associated with Large granular lymphocytosis (LGL) lymphocytosis following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its clinical significance. The aim of current study to define the cumulative incidence of LGL lymphocytosis, to identify associations with transplant-related clinical parameters and to assess the impact on transplant related outcomes. Patients and Methods During a 14-year follow up period (2005-2017) in this unicentric cohort study, we identified 19 patients (2.8%) with a significant LGL lymphocytosis, among 667 consecutive adult patients who underwent allo-HSCT. Results A total of 19 (Female/ male: 10 [52.6 %]/ 9 [47.4 %]) patients included into the study met the morphological criteria for LGL lymphocytosis. The median onset of LGL lymphocytosis was 11.5 (2.1- 55.7) months and median lymphocyte count at the time of diagnosis of LGL lymphocytosis was 5400/ mL (5170- 8700/ mL). None of the patient showed palpable splenomegaly, and none of them had typical signs or symptoms of an autoimmune disease. GvHD, viral infections, disease relapse and loss of donor chimerism were excluded during lymphocytosis. Flow cytometry studies confirmed a T cell phenotype of LGLs in the majority of patients (n=12). Two patients presented with LGLs consistent with NK cells and seven showed properties of a mixed NK/T-cell lineage. A monoclonal LGL population of T-cell origin was identified in eight (42.1%) of these patients. None of the 19 patients experiencing LGL lymphocytosis had shown unexplained fever, night sweats, weight loss or lymphadenopathy suggestive of lymphoproliferative neoplasms. In subgroup analysis, we compared the OS of monoclonal and oligoclonal LGL lymphocytosis and 1-year-OS was longer but non-significantly in monoclonal LGL lymphocytosis group; 75 ± 1.6% vs. 44.4 ± 2.2%, respectively. Median PFS was 28.8 months in oligoclonal LGL lymphocytosis group and 8.3 months in monoclonal LGL lymphocytosis group but the number of patients in this group does not provide enough statistical power to confirm whether the differences in PFS were statistically significant. At the time of this report, 2 of 3 patients died of TRM (10.5%). Discussion In conclusion, we observed LGL lymphocytosis in 2.8 % of a large cohort of post allo-HSCT survivors. Regardless of clonality, all patients were clinically asymptomatic with respect to LGL lymphocytosis. Our data indicate that, even if monoclonal, post-transplantation LGL expansion may be considered as an expression of chronic stimulation triggered by GVHD or relapse of the disease rather than the result of a malignant transformation. Whether the presence of LGL expansion is the expression of a balanced chronic immune response with a possible effect after allogeneic HSCT should be evaluated in a larger cohort of patients.

Generation and characterization of a mitotane-resistant adrenocortical cell line.

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Morphological and immunohistochemical approaches to the diagnosis of Hashimoto thyroiditis and mucosa associated lymphoid tissue lymphomas: An audit.

Most cases of autoimmune hypothyroidism are due to Hashimoto thyroiditis (HT). It is sometimes difficult to distinguish HT from mucosa associated lymphoid tissue (MALT) lymphoma using cytology or histology alone. This has necessitated the use of immunohistochemistry and other molecular techniques. A descriptive study was undertaken to determine the prevalence of MALT lymphoma and other associations of HT using histopathological and immunohistochemical methods. For testing of kappa and lambda antibodies, paraffin sections were prepared for immunohistochemical staining using a peroxidase-antiperoxidase immune complex method. Immunostaining in HT, demonstrated a polyclonal lymphoid population, as evidenced by dual positivity for kappa and lambda staining cells, whereas MALT lymphoma revealed a monoclonal lymphoid population, with strong positivity for kappa immunostaining and lack of lambda light chain expression. The proportion of MALT lymphoma in surgically treated cases of HT in the present study was 3.5% (95% CI: 0%–8.3%). The common malignancy associated with HT was papillary carcinoma of the thyroid (8.8%). Among the benign conditions, nodular colloid goiter (7%) was more commonly associated. Although modern molecular techniques are available for the confirmation of lymphomas, they are more expensive, time consuming and are available only in a few centers. Strict morphological criteria can differentiate HT from MALT lymphoma, but in suspicious cases, paraffin section immunohistochemistry using light chain restriction can offer comparable and reliable results. Since papillary carcinoma and MALT lymphoma have been associated with HT, these patients require careful surveillance.

Quantitation and Comparison of Phenotypic Heterogeneity Among Single Cells of Monoclonal Microbial Populations.

Phenotypic heterogeneity within microbial populations arises even when the cells are exposed to putatively constant and homogeneous conditions. The outcome of this phenomenon can affect the whole function of the population, resulting in, for example, new “adapted” metabolic strategies and impacting its fitness at given environmental conditions. Accounting for phenotypic heterogeneity becomes thus necessary, due to its relevance in medical and applied microbiology as well as in environmental processes. Still, a comprehensive evaluation of this phenomenon requires a common and unique method of quantitation, which allows for the comparison between different studies carried out with different approaches. Consequently, in this study, two widely applicable indices for quantitation of heterogeneity were developed. The heterogeneity coefficient (HC) is valid when the population follows unimodal activity, while the differentiation tendency index (DTI) accounts for heterogeneity implying outbreak of subpopulations and multimodal activity. We demonstrated the applicability of HC and DTI for heterogeneity quantitation on stable isotope probing with nanoscale secondary ion mass spectrometry (SIP–nanoSIMS), flow cytometry, and optical microscopy datasets. The HC was found to provide a more accurate and precise measure of heterogeneity, being at the same time consistent with the coefficient of variation (CV) applied so far. The DTI is able to describe the differentiation in single-cell activity within monoclonal populations resolving subpopulations with low cell abundance, individual cells with similar phenotypic features (e.g., isotopic content close to natural abundance, as detected with nanoSIMS). The developed quantitation approach allows for a better understanding on the impact and the implications of phenotypic heterogeneity in environmental, medical and applied microbiology, microbial ecology, cell biology, and biotechnology.

Stochastic cell renewal process and lengthening of cell cycle

Evolution of the stem cell population responsible for homeostatic cell renewal processes is analyzed. We assume that this regime is the product of a delicate balance between symmetric divisions that, after each cell cycle, originates a new stem cell or its disappearance (through cell differentiation). This dynamics leads to a monoclonal population, that is for an initial homogeneous set of stem cells, fixation of each clone is equiprobable. In this work we show that if there is an altered stem cell with a longer cell cycle than the rest, the fixation of this altered clone is more likely. We also study the consequeces of the appearance of successive alterations with these characteristics and their fixations. This effect is purely due to inherent characteristics of the cell renewal dynamics and as time goes by it leads to a quiescence state for stem cells owing to the recurrent fixation of such altered cells. Therefore it would contribute to the aging process

Angioimmunoblastic T-Cell Lymphoma: Molecular Characterization of Clonal T and B-Cells and a Patient Derived Xenograft Model of Coexisting T and B-Cell Proliferations

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is a rare and aggressive type of lymphoma that accounts for about 20% of peripheral T-cell lymphomas with a 5 year overall survival rate of 30%. As most patients relapse after anthracycline-containing regimens and newer agents such as histone deacetylase inhibitors, other novel therapeutic approaches are needed. Signaling lymphocytic activation molecule F7 (SLAMF7), a molecule expressed on a subset of T-cells, activated B cells and myeloma cells, is an attractive target to explore based on our previous studies showing SLAMF7 expression in a subset of AITL cases. The association of AITL with Epstein Barr virus (EBV) positive B-cells is nearly always present and the efficacy of treatment in such patients with significant EBV viral load is not well-understood. In this study, we performed the molecular characterization of an aggressive EBV+ AITL case, established a patient-derived xenograft (PDX) AITL model of coexisting T and B-cell proliferations and evaluated novel therapeutic strategies. Methods: Primary tumor cells were injected into a NSG mouse. Flow cytometry, immunohistochemistry (IHC), CISH-EBER and BIOMED 2 PCR based clonality studies were used to confirm the engraftment and compared the consistency of engrafted tumor cells with the primary sample. Genomic DNA extracted from sorted T and B cells and from paired normal neutrophils of the original patient were subjected to Whole Exome sequencing (WES). In vivo AITL PDX model trials were tested for the efficacy of romidepsin (Rom), elotuzumab (Elo), rituximab (Rit) and in combinations of these drugs. Results: A 53 year old woman with AITL was treated with 6 cycles of CHOEP followed by autologous stem cell transplantation. 3 months after transplantation (9 months after diagnosis) she developed progressive fatigue and arthralgias. PET-CT scan showed new cervical, thoracic, abdominal and pelvic lymphadenopathy. A cervical lymph node biopsy was performed to confirm relapse. IHC staining showed atypical T cells expressing CD2, CD3, CD4, CD5, CD7, CD10, BCL6, PD1, SLAMF7 and CXCL13. Scattered CD20+/EBER+ B-immunoblasts were present with focal large clusters/small sheets. Primary tumor cells engrafted in NSG mouse via tail vein injection caused splenomegaly. Flow cytometry assay demonstrated the engraftment of tumor cells in peripheral blood, bone marrow and spleen tissue. CD3+CD19- cells dominated the engrafted cells in all three tissues. Histologic examination and immunophenotyping (IHC and EBER staining) of spleen were consistent with primary tumor tissue. Engrafted tumor cells were capable of serial passage in NSG mice with an increasing malignant B cell percentage that mimics the situation in which the B-cell component masks an underlying T-cell lymphoma in humans. T-cell receptor gene rearrangement assay confirmed the clonal identity of engrafted T-cells matched the primary relapsed tumor. A clonal IGH rearrangement of engrafted B-cells was also detected, while no monoclonal B-cell population was detected in the relapsed AITL sample, possibly due to the low number of EBV+ B-cells present in that biopsy. WES of sorted malignant T-cells showed 33 mutants in 31genes, including RhoA G17V, TET2,STAT3 and VAV1, previously described in AITL or other T-cell lymphomas. In parallel WES assay, 9 mutations were found in 9 genes from sorted EBV+ B immunoblasts. A PDX model using cells harvested from the second passage showed single agent, Elo or Rit, extended the survival of mice compared to the control group (p &amp;lt; 0.05). Rom alone had no such effect (p = 0.27). Combination of Rit with either Elo or Rom further improved survival compared to each single agent exposed cohort (p &amp;lt; 0.05). There was no significant difference between Rit/Elo and Rit/Rom (p = 0.067). PARP cleavage by IHC was higher in the Rit/Rom and Rit/Elo groups compared to other cohorts. Expression of SLAMF7 in a subset of engrafted T and B cells of the control mouse were confirmed via flow cytometry assay. Conclusions: To date, this is the first molecular characterization of AITL tumor cells in comparison with associated EBV+ B cells and use of such a PDX model for therapeutic evaluation of agents targeting both malignant T and B cells simultaneously. The in vivo data support further clinical investigation of applying elotuzumab or romidepsin in combination with rituximab in AITL containing EBV-positive B-cell proliferations. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Jazz: Consultancy; Cleveland Clinic&amp;Abbvie Biotherapeutics Inc: Patents &amp; Royalties: US8,603,477 B2.

Large Granular Lymphocytosis and Its Impact on Long Term Clinical Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation: 14-Year Follow-up Data

Large Granular Lymphocytosis and Its Impact on Long Term Clinical Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation: 14-Year Follow-up Data

Mechanistic Insights into the Inhibition of T Regulatory Cells By Dasatinib May Predict Immunostimulatory Effects in CML Patients

Background Dasatinib potently inhibits the Src family kinase Lck at therapeutic concentrations. Lck plays a critical role in signalling from the T cell receptor with immediate downstream targets including ZAP70 and LAT. The cell surface scaffolding protein LAT has been previously shown to play a central role in normal Treg development. STAT5, the downstream target of IL-2, also plays a critical role in Treg differentiation and maintenance of FOXP3 expression through its binding of the promoter region of the FOXP3 gene. The proportion of Tregs has been shown to inversely correlate with improved molecular response in patients on dasatinib and a subset of patients taking dasatinib will develop a large granular lymphocytosis (LGL) which is associated with inflammatory toxicity and improved outcome (Mustjoki et al. Leukaemia 2009). We hypothesised that a reduction in Treg frequency and function would correlate with the expansion of a clonal LGL population in certain patients taking dasatinib. To investigate this, we performed ex-vivo analysis of the phosphorylation of key cell signalling proteins and intracellular cytokine production in Tregs and T effectors from CML patients and healthy controls. Methods We first performed phosphoflow cytometry in Tregs and T effectors to assess the effect of dasatinib on signalling downstream from the TCR, including activated/phosphorylated ZAP70 (pZAP70), LAT (pLAT) and STAT5 (pSTAT5). 11-colour flow cytometry was performed after cells were activated with H2O2 for 15 minutes, due to its activity as a potent phosphatase inhibitor. A gating strategy of CD3+/CD4+/CD25+/FOXP3+/CD127lo cells was used for identification of Tregs. We then performed 10-colour intracellular flow cytometry assessing the impact of dasatinib on cellular cytokine production including TNF, IFN gamma, IL-2, IL-4 and IL-10 after stimulation with OKT3. Results 8 patients with CML and 2 healthy controls were recruited. Of the CML patients 5 were taking dasatinib, 100mg daily, two were taking imatinib and one nilotinib. Patients on dasatinib had lower proportion of Tregs compared with the non dasatinib group with a mean proportion of CD3+ cells of 1.4 vs 2.1. Patients on dasatinib had significantly reduced phosphorylation of ZAP70 compared with the non dasatinib group in CD3+ cells, CD4+ cells and Tregs, following H2O2 stimulation. The mean increase in median fluorescence intensity (MFI) of pZAP70 was 1.7 vs 3.6 in CD3+ cells, 1.4 vs 3.5 in CD4+ cells and 1.5 vs 2.8 in Tregs (p=0.006, p=0.004, p=0.045). Similarly, pLAT showed a lower increase in phosphorylation in the dasatinib group in all T cell subsets evaluated, with mean increase in MFI of 4.3 vs 11.9 in CD3+ cells, 4 vs 12.3 in CD4+ cells and 4.3 vs 8.2 in Tregs. STAT5 also showed reduced phosphorylation in the dasatinib group with a mean increase in MFI of 6.4 vs 21.2 in CD3+ cells, 5.7 vs 20.3 in CD4+ cells and 5.9 vs 19.7 in Tregs (p=0.007, p=0.003, p=0.01). Two patients on dasatinib had reversal of normal CD4:CD8 ratio, with one of these also having absolute lymphocytosis, in keeping with likely development of clonal LGL populations. These patients had lower increase in MFI of pZAP70, pLAT and pSTAT5 following stimulation within isolated Tregs compared to patients on dasatinib with normal CD4:CD8 (Figure 1A). Within CD4+ cells the mean proportional increase in IL-2 production was lower in the dasatinib group at 0.4 vs 5 (p=0.003). Using an additional panel, a single patient was also evaluated and shown to have strikingly reduced production of TNF within isolated Tregs and Il-2 within CD4+cells compared to control (Figure 1B). Conclusion Dasatinib inhibits phosphorylation of ZAP70, LAT and STAT5 within T cell subsets including Tregs, with the strongest effect seen against STAT5. In addition, dasatinib causes a reduction in pro-inflammatory cytokine production within CD4+ cells, with the most significant inhibitory effect seen against IL-2. Tregs have abundant expression of the IL-2 receptor on the cell surface and binding leads to STAT5 signalling resulting in transcription of FOXP3. A more pronounced inhibition of phosphorylation of ZAP70, LAT and STAT5 is seen in two patients on dasatinib with reversal of CD4:CD8 when compared with other patients on dasatinib, suggestive of a more potent reduction of Treg function. Reduction in number and function of Tregs might explain the immunostimulatory effects seen in patients who develop a monoclonal LGL population. Disclosures Harrington: Bristol-Myers Squibb: Research Funding. Dillon:TEVA: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Rousselot:Incyte: Research Funding; Pfizer: Research Funding. Radia:Blueprint Medicines: Consultancy; Novartis: Consultancy, Speakers Bureau. Harrison:Promedior: Honoraria; Gilead: Speakers Bureau; Sierra Oncology: Honoraria; Roche: Honoraria; Celgene: Honoraria, Speakers Bureau; CTI: Speakers Bureau; Shire: Speakers Bureau; AOP: Honoraria; Janssen: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy. De Lavallade:Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding.

Chronic lymphocytic leukemia with central nervous system involvement: Report of three cases

Central nervous system (CNS) involvement is a rare complication of chronic lymphocytic leukemia (CLL). Optic neuritis (ON) as well as myelitis are rarer. We report 3 cases of CNS involvement during CLL revealed by ON. We included patients with CLL who had revealing or subsequent neurological symptoms with monoclonal population of lymphocytes demonstrated on cerebrospinal fluid (CSF) study or imaging findings suggestive of CNS process. Clinical course, diagnostic work-up and treatment were determined. Three patients were included (sex ratio: 0.5). They had 56, 48 and 64 years old. Neurological symptoms were inaugural in one patient. They were concomitant to a relapse of CLL in one patient. All patients developed optic neuritis as a first neurological symptom. One patient developed later a paraparesis with evidence of myelitis in MRI of the spine. Brain MRI showed multiple T2 and Flair hypersignals in the periventricular and subcortical regions in one patient. It was normal in the 2 other patients. CSF study showed predominantly lymphocytic pleocytosis in 2 patients. Flow cytometry couldn’t be performed (small number of lymphocytes). In one patient, immunophenotyping report indicated the presence of monoclonal proliferation of B cells in an axillary adenopathy and in CSF. These B cells express the same surface markers, thus confirming CNS infiltration. Intrathecal chemotherapy was indicated in 2 patients with favorable outcome in one of them. Involvement of the CNS during CLL although rare, should be considered as a differential diagnosis of inflammatory disease of the CNS. Inaugural neurological symptoms make the diagnosis challenging.

Limited Utility of Flow Cytometry Immunophenotyping for Assessment of Plasma Cells in Hematopoietic Progenitor Cell Apheresis Product From Patients With Multiple Myeloma

Abstract Aim Flow cytometric immunophenotyping (FCI) is routinely performed in our laboratory to assess the clonality of plasma cells in hematopoietic progenitor cell apheresis (HPC-A) products as part of the quality control for multiple myeloma patients undergoing autologous stem cell transplantation. The purpose of this project is to investigate whether FCI is indicated for HPC-A when FCI is also performed on a bone marrow biopsy sample obtained before or after HPC-A collection. Materials and Methods The FCI results of HPC-A samples were retrieved from the laboratory database and analyzed. Relevant FCI results of the corresponding bone marrow biopsy samples were also retrieved and analyzed. All FCI was performed using BD FACSCalibur/FACSCanto with a four-color antibody panel, and the listmode data were analyzed by BD FACSDiva. Results FCI was performed on a total of 1,621 HPC-A samples in our laboratory from 02/01/2012 to 02/02/2018. A total of 58 HPC-A samples were positive for a monoclonal plasma cell population (3.8% of the total sample). Among those positive samples, 55 had bone marrow biopsy done before or after HPC-A collection: 38 within a month, 8 between 1 and 2 months, and 9 between 3 and 6 months. Fifty-four of 55 bone marrow samples were positive for a monoclonal plasma cell population by FCI. Conclusion The utility of FCI in the quality assessment of HPC-A products from patients with multiple myeloma is a very limited when FCI is performed on a bone marrow biopsy obtained within 6 months of HPC-A collection.