Polyclonal Pattern Research Articles

B-307 Comparative studies on serum quantitative immunoglobulin assay and serum protein electrophoresis for the diagnosis of multiple myeloma

Abstract Background Various clinical laboratory tests are valuable in enhancing the diagnosis of monoclonal gammopathy. The nephelometric-based serum quantitative immunoglobulin test provides great utility to understand the magnitude of antibody-mediated responses and is useful in interpreting Serum protein electrophoresis (SPEs). In clinical practice, several (SPEs) are ordered without a preliminary order of serum quantitative immunoglobulins but pathologists can and do order the test for comparison. However, there is seldom any data in the literature describing the correlative relationship between the two tests. It is therefore imperative to create an evidenced-based system of test-ordering for the laboratory investigation of the presence of monoclonal protein. The goal of the project is to compare the results of quantitative serum immunoglobulin assay and SPE in order to determine their concordance. Methods In this preliminary phase of the project, retrospective results of quantitative serum immunoglobulin assay and SPEs that were performed simultaneously were retrieved from the electronic medical records. The span of data extended to the past 1 year. If there is an additional immunofixation (IFE) order associated with the SPE, the IFE report was also retrieved for further comparison. Based on the etiology of multiple myeloma, results of calcium, red blood cell count and creatinine were also pooled and analyzed. Results of free light chain assay and associated kappa-lambda ratios were also obtained. All the data obtained were entered into and filtered in excel spreadsheet program and subsequently analyzed with GraphPad prism. Results 1000 patient results were analyzed, of which 404 were males and 596 were females. 200 of the patients had either SPE or quantitative immunoglobulin performed which had the following age demographic; 23 were between 18-39 years, 52 were 40-59 years and 125 were 60-89 years. This data suggests geriatric population are more likely to be seen for multiple myeloma suspicion. There were 60 concurrent orders. Of this, there was 65.5% agreement between the serum quantitative immunoglobulin assay and SPE or IFE while 34.5% showed a mismatch. 26% of the orders for quantitative immunoglobulins assay had no follow-up SPE. A high Kappa/lambda ratio was concordant with a positive SPE 83% of the time. But only 66% of normal kappa/lambda ratio matched a normal SPE. Conclusions Here, we have shown that in addition to the kappa/lambda ratio, the quantitative immunoglobulins test has a considerable agreement with SPE and may be an important preliminary test. The test could be useful to rule out monoclonal gammopathy, confirm polyclonal pattern or embolden a normal SPE interpretation. The present research will have a direct impact on the diagnosis and monitoring of monoclonal gammopathy and enhance appropriate test utilization. The research will improve the development of a better ordering policy for laboratory operations with respect to the work up of multiple myeloma. As more data is unraveled, better understanding will be gained into the relationship between quantitative immunoglobulin and SPE for better patient care.

Serratia marcescens Burden in a Neonatal Intensive Care Unit: Colonization Rate, Clinical Infections and Strain Relatedness

Background: Serratia marcescens (S. marcescens) is an environmentally associated organism known for causing healthcare associated infections and outbreaks in neonatal intensive care units (NICUs). The colonization or infection rates in NICU settings remain uncertain. This study aims to evaluate the rate of baseline colonization and clinical infection and relatedness of S. marcescens isolates. Methods: Prospective surveillance of rectal colonization and clinical infection of S. marcescens was conducted on patients admitted to the NICU at Mount Sinai Hospital in Toronto, Ontario, from March 1, 2023, to September 30, 2023. The NICU is a 57 bed unit with all private rooms. Monthly point prevalence assessments by rectal screening were performed, alongside active surveillance for clinical infections associated with S. marcesens. Isolates from screening or clinical samples underwent assessment for relatedness using pulse field gel electrophoresis (PFGE). Results: Over the 7 month study period, 12 different patients (5.4%) were colonized/infected with S. marcescens. Among these, 10 patients (4.5%) were identified through rectal screening (316 rectal swabs were collected from 224 patients) and two patients (0.9%) exhibited positive clinical specimens (urine and endotracheal aspirate) in association with pyelonephritis and ventilator-associated pneumonia, respectively. Of the two clinical cases, one case showed a negative preceding rectal swab and the other detected through a clinical sample before the point prevalence date. The age at which a positive S. marcescens swab or positive clinical specimen was identified ranged from 4 to 66 days (median=18 days, IQR 5-38.8) (Figure 1). Sixty seven infants had repeated screening. Three out of 67 (4.5%) were colonized with S. marcescens, the timing and sequence of positive and negative testing are presented in Figure 2. Females demonstrated a higher positivity rate compared to males [9.1% (9/99) vs 2.4% (3/125), p=0.04, respectively]. PFGE analysis of all 12 (100%) isolates revealed a polyclonal pattern. Most cases were detected from March to May in 9/12 cases (75%). Ten different strains were identified. Notably, two strains demonstrated clusters of two cases each, one during March and the other during May (Figure 3). No mortality was reported among the cases. Conclusions: The study highlights the polyclonal nature of S. marcesens and raises questions about the utility of point prevalence in anticipating clinical cases or patient-to-patient transmission, especially in patients with clinical infection where there were no preceding positive screening tests.

Congophilic fibrils in the glomeruli with polyclonal immunoglobulin gamma staining - another cause for diagnostic overlap: A case report

BACKGROUND Glomerulopathy with fibrillary deposits is not uncommon in routine nephropathology practice, with amyloidosis and fibrillary glomerulonephritis being the two most frequently encountered entities. Renal amyloid heavy and light chain (AHL) is relatively uncommon and its biopsy diagnosis is usually limited to cases that show strong equivalent staining for a single immunoglobulin (Ig) heavy chain and a single light chain, further supported by mass spectrometry (MS) and serum studies for monoclonal protein. But polyclonal light chain staining can pose a challenge. CASE SUMMARY Herein we present a challenging case of renal AHL with polyclonal and polytypic Ig gamma (IgG) staining pattern by immunofluorescence. The patient is a 62-year-old Caucasian male who presented to an outside institution with a serum creatinine of up to 8.1 mg/dL and nephrotic range proteinuria. Despite the finding of a polyclonal and polytypic staining pattern on immunofluorescence, ultrastructural study of the renal biopsy demonstrated the presence of fibrils with a mean diameter of 10 nm. Congo red was positive while DNAJB9 was negative. MS suggested a diagnosis of amyloid AHL type with IgG and lambda, but kappa light chains were also present supporting the immunofluorescence staining results. Serum immunofixation studies demonstrated IgG lambda monoclonal spike. The patient was started on chemotherapy. The chronic renal injury however was quite advanced and he ended up needing dialysis shortly after. CONCLUSION Tissue diagnosis of AHL amyloid can be tricky. Thorough confirmation using other available diagnostic techniques is recommended in such cases.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

Molecular characterization and biofilm formation ability of Enterococcus faecium and Enterococcus faecalis bloodstream isolates from a Chinese tertiary hospital in Beijing

To investigate the molecular characteristics and biofilm-forming ability of 116 Enterococcus faecium (Efm) and 72 Enterococcus faecalis (Efs) isolates obtained from patients with bloodstream infections (BSI) at a Chinese hospital between July 2011 and March 2018. The presence of glycopeptide resistance genes and five virulence genes (esp, gelE, asa1, hyl, and cylA) was screened using two multiplex PCR. MLST was used to assess the clonality. Crystal violet staining was used to detect biofilms. Vancomycin resistance was detected in 30.1% of Efm and 2.8% of Efs isolates, respectively. All VRE strains carried the vanA gene. The esp, gelE, asa1, and cylA genes in 72 Efs strains were detected at 62.5%, 84.7%, 84.7%, and 69.4%, respectively. Among the 116 Efm isolates, 74.1% and 25.8% carried esp and hyl, respectively. The esp gene was significantly associated with vancomycin-resistant Efm (VREfm) compared to vancomycin-susceptible Efm (VSEfm). In total, 91.7% of Efs and 20.0% of Efm produced biofilms. Twenty-six STs were identified among the 72 Efs isolates, with ST4 (29.2%) being the predominant. In total, 116 Efm strains were grouped into 26 STs, with ST78 (46.6%) being the predominant. Both VREfm (41.7%) and VSEfm (48.8%) were dominant in ST78. There is no clear evidence suggesting that some STs are associated with vancomycin resistance or biofilm formation. Both Efm and Efs BSI isolates showed a polyclonal pattern with a dominant clone and many unique types, implying the coexistence of clonal dissemination and an influx of new clones. The horizontal transmission of resistance genes may play a more important role in VREfm prevalence than clonal expansion.

Immunologic Heterogeneity in 2 Cartilage-Hair Hypoplasia Patients With a Distinct Clinical Course.

Cartilage-hair hypoplasia (CHH) syndrome is a rare autosomal recessive syndrome associated with skeletal dysplasia, varying degrees of combined immunodeficiency (CID), short stature, hair hypoplasia, macrocytic anemia, increased risk of malignancies, and Hirschsprung disease. To provide clinical and immunological insights obtained from 2 unrelated patients who displayed clinical characteristics of CHH. Two patients with suspected CHH syndrome due to skeletal dysplasia and immunodeficiency underwent an immunological and genetic work-up using flow cytometry, next-generation sequencing (NGS) of the immune repertoire, and Sanger sequencing to identify the underlying defects. Patient 1 presented with low birth weight and skeletal dysplasia. Newborn screening was suggestive of T-cell immunodeficiency, as T-cell receptor excision circle levels were undetectable. Both the T-cell receptor (TCR) Vß and TCR-g (TRG) repertoires were restricted, with evidence of clonal expansion. Genetic analysis identified compound heterozygous RMRP variants inherited from both parents. Patient 2 presented with recurrent lung and gastrointestinal infections, skeletal dysplasia, failure to thrive, and hepatomegaly. The polyclonal pattern of the TCRß repertoire was normal, with only slight overexpression of TCR-ßV20 and restricted expression of Vßs. TRG expressed a normal diverse repertoire, similar to that of the healthy control sample. Genetic analysis identified biallelic novel regulatory variants in RMRP. Both parents are carriers of this mutation. Our findings demonstrate how the immunological work-up, supported by genetic findings, can dramatically change treatment and future outcome in patients with the same clinical syndrome.

Severe Leukocyte Adhesion Deficiency-I (LAD-I) Lentiviral-Mediated Ex-Vivo Gene Therapy: Ongoing Phase 1/2 Study Results

BackgroundSevere leukocyte adhesion deficiency-I (LAD-I) is an autosomal recessive inborn error of immunity due to mutations in the ITGB2 gene that encodes the common CD18 subunit of β2-integrins, essential for neutrophil adhesion to the inflamed endothelium and transmigration into tissues. Severe LAD-I, defined as <2% of normal CD18 polymorphonucleocyte [PMN] expression, is characterized by frequent and often refractory bacterial and fungal infections, impaired wound healing, and significant pediatric mortality in the absence of allogeneic hematopoietic stem cell transplant (HSCT). HSCT is potentially curative but limited by donor availability, graft-versus-host disease, and graft failure. The gene therapy RP-L201-0318 (NCT03812263) employs autologous CD34+ cells transduced with a lentiviral vector carrying ITGB2 to restore CD18 expression. MethodsPatients ≥3 months old with severe LAD-I were enrolled. Hematopoietic stem cells (HSCs) were collected via apheresis after mobilization with granulocyte-colony stimulating factor and plerixafor and transduced ex-vivo with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning preceded RP-L201 infusion. Patients were followed for safety and efficacy measures, including survival to age two and ≥1-year post-infusion, peripheral blood [PB] PMN CD18 expression, PB vector copy number [VCN], neutrophilia improvement, decrease in infections/hospitalizations, and resolution of skin/periodontal abnormalities. ResultsNine patients (age five months to nine years) received RP-L201; follow-up was 3 to 24 months. RP-L201 cell doses ranged from 2.8 × 106 to 10 × 106 CD34+ cells/kg with a drug product VCN of 1.8 to 3.8. All nine patients demonstrated PMN CD18 restoration (median expression of 56.3%) with sustained, stable genetic markings (median PB mononuclear cell VCN of 1.53). At one year, the overall survival (OS) rate was 100% per Kaplan-Meier estimate. Pre-treatment leukocytosis improved uniformly. Hospitalizations and severe infections were significantly reduced following therapy. No RP-L201-related serious adverse events (SAEs) were reported. Insertion site analyses indicate highly polyclonal integration patterns across the entire cohort. ConclusionRP-L201 has a favorable safety profile and confers durable correction of the severe LAD-I phenotype with improved clinical course in all nine pediatric patients treated as demonstrated via all laboratory and clinical parameters.

Primary extranodal soft tissue Lennert lymphoma (lymphoepithelioid variant of peripheral T-cell lymphoma, unspecified): a case report and review of the literature

Lennert lymphoma (LeL) is a rare variant of peripheral T-cell lymphoma, not otherwise specified (PTCL/NOS) that is rich in epithelioid histiocytes. LeL may pose great diagnostic and therapeutic challenges to the pathologist and clinician. Primary extranodal soft tissue LeL is even rarer and has not been reported. Herein, we report a case of LeL arising from soft tissue.A 65-year-old male presented for evaluation of a painless mass in the subcutaneous soft tissue of the left forehead. There was no invasion of the bone and no ulceration on the surface of the skin. The surrounding skin was erythematous and swollen. Grossly, the tumor was gray-red and 30 mm × 20 mm × 10 mm in size.Microscopically, the demarcation between the lesion and surrounding tissues was unclear without a capsule. The tumor invaded the surrounding striated muscle and adipose tissue. The tumor had a diffuse proliferation of small-sized atypical lymphocytes and numerous large clusters of epithelioid histiocytes. Plasma cells, eosinophils, and Hodgkin-Reed-Sternberg (HRS) cells were not identified. Rare multinucleated histiocytes were noted, and well-formed granulomas were not present. Rare mitotic figures were noted, but no necrosis. The immunophenotypic features in this case were as follows: CD2+/CD3+/CD5low+/CD7+/CD4low+/ CD8+/CD30−/CD56− in neoplastic lymphocytes; CD163+/CD31+/CK(pan)− in epithelioid histiocytes; and CD20−/CD30−/TdT−/CD5−/ALK−/S-100−/CD1α−/CD21 + 23−/SSTR2− in neoplastic lymphocytes and epithelioid histiocytes. Epstein-Barr virus (EBV)-encoded RNA in situ hybridization (EBER-ISH) was negative. The Ki-67 index was elevated to 60%. PCR showed a polyclonal pattern for IgH and a monoclonal TCR γ-chain rearrangement.The final diagnosis was PTCL/NOS, lymphoepithelioid cell variant (LeL), which arose from soft tissue and had a rare double-positive CD4low+/CD8+ immunophenotype. The patient received four cycles of cyclophosphamide, doxorubicin liposomes, vincristine, and prednisone tablets (CHOP) and was followed for 20 months. Overall treatment efficacy was achieved without lymphadenopathy, and no other discomfort or illnesses were reported.

Comparing the Impact of NHEJ-CRISPR/Cas9 and Base Gene Editing on Rhesus Macaque Hematopoietic Stem Cell Clonal Behavior and Integrity

Despite recent transformative advancements in gene editing tools and hematopoietic stem and progenitor cell (HSPC) gene therapies, there are still unanswered questions about the impact of CRISPR/Cas9 and base editing technologies on HSPC clonal behavior, function, and integrity in vivo. Better understanding of the impact of available gene therapy modalities on HSCs in a relevant animal model should help safely move these therapies forward for clinical use and provide information regarding their relative advantages and risks. CRISPR/Cas9 editing generates double strand DNA breaks (DSBs) at targeted genomic locations and potentially at off-target sites, resulting in potential HSC genotoxicity and loss. To counter DSB-related consequences, base editors (BEs) have been developed with the ability to produce single-nucleotide conversion without DSBs, avoiding potentially deleterious DNA damage repair pathways. CD33 is expressed on all myeloid cells downstream of the common myeloid progenitor and is used as both a diagnostic marker and a therapeutic target for acute myeloid leukemia. Our past work demonstrated successful Cas9/NHEJ-mediated CD33 KO in rhesus macaque (RM) HSPCs with no functional impact (Kim et al, Cell, 2018). In the current study, we first genetically barcoded RM HSPCs, and then knocked out the CD33 locus with the intent of comparing the clonal impact of CRISPR/Cas9 editing via non-homologous end joining (NHEJ) to base editing in RM HSPCs in vitro by myeloid differentiation assays and in vivo following autologous HSPC transplantation. Highly diverse DNA barcodes were introduced into RM HSPCs via transduction with a lentiviral vector also expressing GFP. 24 hours later, the cells were electroporated with either Cas9/NHEJ or BE complexes targeted to disrupt CD33. Cells were further cultured in myeloid differentiation media for 8 days in vitro for differentiation assays (IVD). Cell viability and proliferation were analyzed over time during IVD. DNA was collected on day 8 for editing efficiency and barcode clonal analysis. In addition, GFP and CD33 expression were analyzed by flow cytometry to assess CD33 KO efficiency and examine whether the addition of gene editors impacted the clonality of the HSPCs as assessed by barcode retrieval, and whether the initial lentiviral transduction impacted editing efficiency. HSPC colony-forming unit (CFU) assays were carried out to assess differentiation potential of the transduced and/or edited HSPCs and check for biallelic versus monoallelic editing status at a clonal level. Throughout the 8 days of IVD, cell numbers increased overtime and maintained high cell viability (80-95%) in all test groups, which indicates barcoded lentiviral transduction and gene editing procedures had minimal impact on cell proliferation and viability. Base editing resulted in 80-85% CD33 KO efficiency, regardless of barcode lentivirus transduction prior to gene editing. However, Cas9/NHEJ was significantly less efficient, with CD33 KO efficiency of 10-20%, requiring additional optimization (Figure 1A). Variant allele frequency analysis confirmed the relative levels of CD33 locus editing by both editing methods. CFU assays indicated the lentiviral transduction and gene editing methods do not alter the HSPC differentiation potential as assessed in vitro. Barcode clonal analysis revealed both NHEJ and BE test groups demonstrated retention of high clonal diversity as assessed by Shannon Diversity Index, all > 8 regardless of gene editing methods (Figure 1B). In summary, lentiviral barcode transduction and editing procedures had no negative impact on cell viability and proliferation. Base editing allowed very high gene editing efficiency in HSPCs and can serve as a powerful editing tool. Importantly, both editing methods resulted highly diverse polyclonal clonal patterns in vitro long term, with no significant differences compared to the non-edited group. Combining genetic barcode technology and the BE or Cas9/NHEJ editing in an in vivo RM autologous HSPC transplantation model to track the hematopoietic clonal reconstitution of the edited HSPCs over time and compare the impact of BE or Cas9/NHEJ editing on engrafting HSPCs clonal behavior is ongoing. Therefore, these clinically relevant findings can determine which gene editing method is more suitable for developing human HSPC gene therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interim Results from an Ongoing Phase 1/2 Study of Lentiviral-mediated Ex-vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I)

Background: Leukocyte adhesion deficiency-I (LAD-I) results from mutations in ITGB2 encoding the β2-integrin CD18. Severe LAD-I (<2% CD18+ polymorphonucleocytes [PMNs]) causes severe and often refractory infections, impaired wound healing, and frequent childhood mortality. Allogeneic hematopoietic stem cell transplant is potentially curative but limited by donor availability, graft-versus-host disease, and graft failure. RP-L201-0318 (NCT03812263) employs autologous CD34+ cells transduced with a lentiviral vector carrying ITGB2. Methods: Patients ≥3 months old with severe LAD-I were enrolled. Hematopoietic stem and progenitor cells (HSPCs) were collected via apheresis after mobilization with granulocyte-colony stimulating factor and plerixafor and transduced ex-vivo with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning preceded RP-L201 infusion. Patients are followed for safety and efficacy including survival to age 2 and ≥1-year post-infusion, peripheral blood [PB] PMN CD18 expression, PB vector copy number [VCN], neutrophilia improvement, decrease in infections/hospitalizations, and resolution of skin/periodontal abnormalities. Results: Nine patients (ages 5 months to 9 years) received RP-L201, all with follow-up of 3 to 24 months. RP-L201 cell doses ranged from 2.8x106 to 10x106 CD34+ cells/kg with a drug product VCN of 1.8 to 3.8. All 9 patients demonstrated PMN CD18 restoration with median expression of 56.3% and sustained, stable genetic markings with median peripheral blood mononuclear cell VCN of 1.53. At 1 year, the overall survival (OS) rate is 100% per Kaplan-Meier estimate. Neutrophilia resolved uniformly. Hospitalizations and severe infections were significantly reduced following therapy. The safety profile has been highly favorable with no RP-L201-related serious adverse events (SAEs); insertion site analyses indicate polyclonal integration patterns. Conclusion: RP-L201 confers durable reversal of the severe LAD-I phenotype with improved clinical course in 9 of 9 pediatric patients.

Intra-oral Plasma Cell Granuloma: A Rare Clinico-pathological Entity-Report of Two Cases with Review

Plasma cell granulomas (PCGs) or inflammatory pseudotumors are non-neoplastic lesions that consist of predominantly antibody-producing plasma cells and innate immune cells such as neutrophils, macrophages, and eosinophils. Unlike in multiple myeloma, the plasma cells are polyclonal and proliferate in a fibroblast-rich stromal background. These lesions are predominantly reported in the lungs; however, they may involve other organs too. Very few cases are reported regarding the presence of PCG in the oral cavity and a confirmatory diagnosis is crucial for further management. A critical and careful examination from a pathological point of view is obviously necessary to rule out other plasma cell neoplasms. We present here two cases of intra-oral PCGs; one in the gingiva &amp; another within the jaw; both showing a polyclonal staining pattern in immunohistochemistry confirming a diagnosis of plasma cell granuloma. Keywords: Inflammatory pseudotumor, Plasma cell granuloma, Plasma cells, Polyclonal plasma cells, Lambda and kappa chain

Polyclonal evolution of Fanconi anemia to MDS and AML revealed at single cell resolution

BackgroundFanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive.MethodsHerein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage.ResultsOur finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage.ConclusionsWe thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.

Preclinical safety and efficacy of lentiviral-mediated gene therapy for leukocyte adhesion deficiency type I

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene, which encodes for the CD18 subunit of β2-integrins. Deficient expression of β2-integrins results in impaired neutrophil migration in response to bacterial and fungal infections. Using a lentiviral vector (LV) that mediates a preferential myeloid expression of human CD18 (Chim.hCD18-LV), we first demonstrated that gene therapy efficiently corrected the phenotype of mice with severe LAD-I. Next, we investigated if the ectopic hCD18 expression modified the phenotypic characteristics of human healthy donor hematopoietic stem cells and their progeny. Significantly, transduction of healthy CD34+ cells with the Chim.hCD18-LV did not modify the membrane expression of CD18 nor the adhesion of physiological ligands to transduced cells. Additionally, we observed that the repopulating properties of healthy CD34+ cells were preserved following transduction with the Chim.hCD18-LV, and that a safe polyclonal repopulation pattern was observed in transplanted immunodeficient NOD scid gamma (NSG) mice. In a final set of experiments, we demonstrated that transduction of CD34+ cells from a severe LAD-I patient with the Chim.hCD18-LV restores the expression of β2-integrins in these cells. These results offer additional preclinical safety and efficacy evidence supporting the gene therapy of patients with severe LAD-I.

Intraosseous delivery of platelet-targeted factor VIII lentiviral vector in humanized NBSGW mice

AbstractWe previously showed that intraosseous (IO) delivery of factor VIII (FVIII, gene F8) lentiviral vector (LV) driven by the megakaryocyte-specific promoter Gp1bα (G-F8-LV) partially corrected the bleeding phenotype in hemophilia A (HemA) mice for up to 5 months. In this study, we further characterized and confirmed the successful transduction of self-regenerating hematopoietic stem and progenitor cells (HSPCs) in treated mice. In addition, secondary transplant of HSPCs isolated from G-F8-LV–treated mice corrected the bleeding phenotype of the recipient HemA mice, indicating the potential of long-term transgene expression following IO-LV therapy. To facilitate the translation of this technology to human applications, we evaluated the safety and efficacy of this gene transfer therapy into human HSPCs. In vitro transduction of human HSPCs by the platelet-targeted G-F8-LV confirmed megakaryocyte-specific gene expression after preferential differentiation of HSPCs to megakaryocyte lineages. Lentiviral integration analysis detected a polyclonal integration pattern in G-F8-LV–transduced human cells, profiling the clinical safety of hemophilia treatment. Most importantly, IO delivery of G-F8-LV to humanized NBSGW mice produced persistent FVIII expression in human platelets after gene therapy, and the megakaryocytes differentiated from human CD34+ HSPCs isolated from LV-treated humanized mice showed up to 10.2% FVIII expression, indicating efficient transduction of self-regenerating human HSPCs. Collectively, these results indicate the long-term safety and efficacy of the IO-LV gene therapy strategy for HemA in a humanized model, adding further evidence to the feasibility of translating this method for clinical applications.

Morphologic Spectrum of Lymphadenopathy in Drug Reaction With Eosinophilia and Systemic Symptoms Syndrome.

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DRESS) is a drug-induced, adverse T-cell-mediated hypersensitivity reaction that most often involves skin. The pathologic findings of DRESS-related lymphadenopathy have been described infrequently in the literature. To present a case series of DRESS-related lymphadenopathy with an emphasis on the morphologic spectrum. We describe detailed clinical and pathologic findings along with the literature review. We focus on the differential diagnosis between DRESS lymphadenopathy and angioimmunoblastic T-cell lymphoma (AITL). There were 4 men and 1 woman with a mean age of 41 years (range, 23-59 years). One patient (20%) died. Three lymph node biopsy specimens showed a pattern reminiscent of AITL (AITL-like pattern) and 2 cases showed necrotizing lymphadenitis (Kikuchi-like pattern), associated with vasculitis in 1 case. The AITL-like morphology of DRESS-related lymphadenopathy may be difficult to distinguish from genuine AITL. The clinical information is important for differential diagnosis, including history of drug exposure, age, and the rarity or absence of AITL-associated manifestations such as hemolytic anemia and hypergammaglobulinemia. Molecular analysis of the T-cell receptor genes is helpful, typically revealing a polyclonal pattern in DRESS-related lymphadenopathy. In the literature, 4 histologic patterns of DRESS lymphadenopathy have been described: reactive lymphoid hyperplasia, necrotizing lymphadenitis, Hodgkin lymphoma-like, and AITL-like. These patterns, particularly those that resemble lymphoma, highlight the importance of correct diagnosis to avoid unnecessary therapies.

Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors

We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMTP140K-based in vivo selection approach, γ-globin+ RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin+ RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin+ RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.

Prevalence of Allelic Variants and Clonality of IGHV1-69 Expressing B-Cells in Patients with Different Severity of COVID 19 Disease

Introduction: Since the first months of the COVID-19 pandemic, efforts have been made to understand the importance of broadly neutralising natural antibodies in determining the response to SARS-CoV-2. Previous studies have shown that allelic variants of the IGHV1-69 gene play a dominant role in protective natural antibody responses to several other viral pathogens, including influenza virus, hepatitis C virus, human immunodeficiency virus and, most notably, the SARS-CoV-2-related viruses SARS-CoV and MERS-CoV. These allelic variants are commonly known as 51p1-related and differ from the other IGHV1-69 alleles (known as hv1263-related) in the presence of a Phe54 residue in the CDR2 region. Importantly, crystallographic studies have shown that the Phe54 residue is critical for the binding of IGHV1-69 antibodies to the SARS-CoV and MERS-CoV spike proteins. In this study, we evaluated the prevalence of 51p1 and hv1263 alleles and the clonality of 51p1- and hv1263-expressing B cells in a large cohort of healthy individuals and COVID-19 patients and correlated the findings with the severity of the disease.Мaterials and methods: A total of 419 samples were included in the study, of which 78 asymptomatic/mildly symptomatic individuals, 200 hospitalized patients with severe disease, 94 critically ill patients and 47 healthy donors. Peripheral blood was collected 8-20 days after the onset of symptoms and total cellular RNA was extracted from whole blood using an automated procedure. Аllelle-specific Ig-gene fingerprinting of IgM heavy chain transcripts was used to simultaneously analyse the clonality of the IgM+ B-cell population and the clonality of the 51p1- and hv1263-expressing B cell populations. The significance of the differences in the prevalence of clonal B-cell populations between healthy donors and patients and between patients with different severity of the disease was calculated with the Chi-Square test.Results: Analysis of the clonality of the IgM+ B-cell population showed a polyclonal pattern in most of the investigated healthy individuals (33/47, 70%) but in only 20% of all SARS-CoV-2 infected individuals (75/372, p<0.001). A significant difference was also observed between mildly affected and severely/critically ill patients [31/78 (39.7%) vs. 44/294 (15%), respectively) (p<0.001)], but not between severely and critically ill patients [28/200 (14,%) vs. 16/94 (17,1%), (p=n.s.)]. No 51p1 transcripts were detected in 74/372 (19.9%) of SARS-CoV-2 infected individuals and in 14/47 (29,8%) of the control group (p>0,01), while hv1263 transcripts were not detected in 155/289 (53,6%) and in 27/47 (68,6%) tasted patients and controls, respectively (p>0,05). We did not find a statistically significant difference in the prevalence of 51p1 and hv1263 alleles between patients with different disease severity. However, a significantly higher number of patients displayed clonal expansions of 51p1- or hv1263-expressing B cells (219/372(58.9%) and 118/244 (48,4%), respectively in comparison to healthy donors [5/47(10.6%) and 7/47(14.9%), respectively]. There was no statistically significant difference between mildly affected and severely/critically ill patients in the clonallity status of 51p1- 38/61 (62,3%) and 182/237 (76,7%) respectively or between hv1263- expressing B cells in the same two groups of patients [20/25 (80%) and 98/109 (89,9%), p>0.05].Conclusions: Our results show that SARS-CoV-2 infection stimulates clonal expansions of IGHV1-69 -expressing B-cells, but this is independent of the severity of the disease. In addition, no difference in the prevalence of IGHV1-69 alleles was observed between patients at different stages of the disease, indicating that natural neutralizing antibodies encoded by this gene are not an important determinant of COVID-19 severity and progression. DisclosuresNo relevant conflicts of interest to declare.

Interim Results from a Phase I/II Clinical Gene Therapy Study for Newly Diagnosed Infants with X-Linked Severe Combined Immunodeficiency Using a Safety-Modified Lentiviral Vector and Targeted Reduced Exposure to Busulfan

▪Early trials of gene therapy for X-linked Severe Combined Immunodeficiency (XSCID) restored T cell immunity in most cases, but did not correct B cell function and carried a high risk of iatrogenic leukemia. The subsequent development of self-inactivating γ-retroviral vectors has enhanced safety, but has not restored B cell function to date. We developed a new approach for XSCID gene therapy that utilizes a safety modified lentiviral (LV) vector (CL20-i4-EF1α-hGC-OPT) together with reduced exposure busulfan (Bu) conditioning for newly diagnosed infants with XSCID. Recently, we reported that the combination of reduced dose Bu used together with our LV vector restored T and B cell function in older XSCID children with declining immune function following haploidentical transplantation (De Ravin SS et al, Sci Transl Med, 2016). Here we report initial results of LVXSCID-ND; a multi-center, phase I/ II safety and efficacy study using our LV vector and dose-adjusted Bu for the first time in newly diagnosed XSCID infants. We enrolled six subjects over the last 12 months (median age of 4.5 months, range: 2-12 months). Purified bone marrow (BM) CD34+ cells were transduced with the CL20-i4-EF1α-hGC-OPT vector generated by a stable producer cell line and then cryopreserved to facilitate central manufacturing for multiple study sites and evaluation of release criteria prior to conditioning. Busulfan was given as two single daily doses to target a total cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (60-90 mg*hr/L = myeloablative cAUC). The median dose of transduced CD34+ cells was 8.06 x 106 cells/kg (range: 4.6 - 11 x 106) and the median vector copy number (VCN) in graft CFU-C was 0.40 copies /cell (range: 0.16 - 0.97). An average Bu cAUC of 22.9 mg*hr/L (range: 20.0 to 24.2) was achieved, which was within 10% of the intended cAUC in all patients. As of July 2017, no severe adverse events related to BM harvest, Bu exposure, or cell infusion have been observed. In the first 5 evaluable cases, complete hematopoietic recovery occurred by 3-4 weeks without any blood product support. Follow-up data from the oldest patient who presented with high levels of maternal T cell engraftment, severe neutropenia requiring G-CSF therapy, CMV viral infection, with a graft VCN of 0.16, and who is now 12 months post therapy, demonstrated delayed and partial T cell reconstitution. Cases 2 and 3 have been followed for nine and six months, respectively and have significantly higher VCNs in peripheral blood (PB) subsets (CD14/15+myeloid cells 0.65, 0.30 copies/cell: CD3+ T cells 2.78, 2.71; CD19+ B cells 1.01, 0.78; and CD56+ NK cells 2.72, 2.11 respectively). Bone marrow aspirates on week 16 yielded VCNs in sorted CD34+ cells of 0.56, 0.50; and BM myeloid CFU-GM of 0.61, 0.24. Rapid T cell reconstitution in cases 2 and 3 resulted in normal numbers of CD3+, CD4+, CD4 naïve, and CD8+ cells with TRECs 626 and 1170 copies per ug DNA 16-20 weeks post therapy. In both cases at 16 weeks, T cell proliferation was 86% and 81% of control, and V-β spectratype scores were normal at 194 and 155. At nine months, case 2 had a normal isohemagglutinin anti-A titer of 1:32 and normal 4-week trough serum immunoglobulins levels (IgG 713 mg/dl, IgM 54 mg/dl, and IgA 16 mg/dl). IVIG supplementation has been discontinued for approximately 3 months before vaccination responses are assessed. Case 4 has been followed for 12 weeks with PB VCNs for CD3+ cells of 1.36 copies/cell, CD19+ 0.58, CD56+ 1.38 and CD14/15+ 0.02. Flow cytometry analysis of PB at 12 weeks shows 4.5% of PB leukocytes are CD3+, with 61% CD4+, 11% CD8+, and a significant fraction expressing a CD45RA+, RO- naive phenotype at this early time point. Cases 5 and 6 are still too early to evaluate for immune phenotype and function. Preliminary vector insertion site analysis shows highly polyclonal marking patterns in case 2 with 11,000 insertion sites in CD3+ cells, 5049 sites in CD19+ cells, 756 sites in BM myeloid CFU-C without any evidence of clonal dominance. In summary, gene therapy for newly diagnosed XSCID patients using a LV vector with targeted reduced exposure Bu conditioning is well tolerated and results in rapid T cell reconstitution in most cases. Efficient vector marking in bone marrow CD34+ cells, myeloid cells, and B cells indicate that this approach will likely provide broad immune reconstitution rather than restricted T cell correction seen in past trials using γ-retroviral vectors with no Bu. DisclosuresLong-Boyle:InsightRX: Consultancy. Puck:UpToDate: Patents & Royalties: Recieve royalties to write and edit entries on primary immune disorders; InVitae, a clinical DNA sequencing company: Other: Spouse employment and stock options. Cowan:Homology Medicine: Consultancy.

TCR γδ Large Granular Lymphocyte Leukemia (LGLL) Displays Features between CD8+ and CD4+ TCR αβ LGLL

BackgroundT-large granular lymphocyte leukemia (T-LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Usually, leukemic cells of this T-cell neoplasia express the αβ T-cell receptor (TCR); only a small subset of cases expresses the γδ TCR denoting the TCRγδ-LGLL. Currently, among the different LGL diseases, TCRγδ-LGLL remains less studied and several clinical and laboratory data already described in TCRαβ-LGLL have not yet been explored in TCRγδ-LGLL. In particular, data are still missing about STAT3 and STAT5b mutations, frequently observed in TCRαβ-LGLL, and about T-LGL clonotypes discovered to be private to TCRαβ-LGLL as compared to healthy controls.AimsThis work was aimed to perform the analysis on: STAT3 and STAT5b mutations, LGL immunophenotype, TCRγ clonotype repertoire and clinical presentations in a group of TCRγδ-LGLL patients. The objective was to reveal common and different features of this rare kind of LGLL with the more frequent TCRαβ-LGLL.MethodsThe cohort of study included 11 patients affected by TCRγδ-LGLL. Sanger sequencing was used for mutational analysis on hot-spot regions of the 2 genes more frequently mutated in LGL disorders, STAT3 and STAT5b . Immunophenotype of LGL clone was defined by flow cytometry analysis. CDR3 repertoire and frequency distribution of TCR gamma gene rearrangements were obtained by Next-Generation Sequencing (NGS).ResultsTCRγδ-LGLL showed a higher occurrence of STAT mutations (82%) as compared to TCRαβ-LGLL (43%). In detail, 9 out of 11 TCRγδ LGLL patients were characterized by STAT3 or STAT5b mutations in a mutually exclusive pattern. Being characterized by both STAT3 and STAT5b mutations, TCRγδ LGLL resembles both CD8+ TCRαβ-LGLL and CD4+ TCRαβ-LGLL, the first being mostly associated to STAT3 mutations and the latter to STAT5b . Similarly, TCRγδ-LGLL showed the same relationship observed between immunophenotype and kind of mutation in TCRαβ-LGLL: TCRγδ-LGLL patients with CD56- LGL immunophenotype were characterized by STAT3 mutations (as in CD8+ TCRαβ-LGLL), while TCRγδ-LGLL patients with CD56+ LGL immunophenotype by STAT5b mutations (as in CD4+ TCRαβ-LGLL). Anyway, in TCRγδ-LGLL no correlation was found between mutations and clinical course as observed in CD8+ TCRαβ-LGLL patients carrying STAT mutations, rather, we observed that patients with γδLGLs positive for Vδ2 showed frequently indolent course, while Vδ1 was associated to a more symptomatic disease (4 out of 5 symptomatic patients were Vδ1+). By NGS we obtained the TCR gamma repertoire of each patient and we revealed that all patients were clonal with the exception of 2 showing a polyclonal pattern. The 2 polyclonal cases were the only 2 patients without STAT mutations. Among the remaining STAT3 mutated patients (n=4), 3 were polyclonal and one biclonal, while STAT5b mutated patients (n=5) were more frequently monoclonal (4/5 monoclonal and 1/5 biclonal). Considering the CDR3 repertoire of the immunodominant clones, as observed in TCRαβ-LGLL, common clonotype sequences were present with low frequency in almost all TCRγδ-LGLL patients. Interestingly, 2 different CDR3 sequences were found shared in different patients at frequency >10% of the total rearrangements. In terms of clonal rearrangements, Vγ3-Jγ1/2, Vγ9-JγP and Vγ8-Jγ1/2 were the combination usages most frequently detected, showing a limited combinatorial diversity of TCR genes similarly to CD4+ and differently from CD8+ TCRαβ-LGLL, this last having larger variability. Analysis about the clonotype repertoire in a 2 years-follow up showed that patients maintain their clonal pattern along the time.ConclusionsOur results highlight common and different characteristics of TCRγδ-LGLL as compared to TCRαβ-LGLL. The data suggest that TCRγδ-LGLL can be considered the intersection of the 2 types of TCRαβ-LGLL, sharing CD8+ or CD4+ T-LGLL mutational and phenotype features. Similarly to TCRαβ-LGLL, also TCRγδ disease showed a decreased diversity of TCR repertoire, with common TCR sequences shared among patients and with limited number of Vγ-Jγ combinations. A distinctive feature of TCRγδ-LGLL is that STAT mutations do not correlate with a symptomatic clinical behavior, rather, Vδ1 expression appears to be indicative for symptomatic disease. DisclosuresGalimberti:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau.

Combined Mutational and Clonality Analyses Support the Existence of Intra-Tumor Heterogeneity in Papillary Thyroid Cancer.

Despite its potential clinical impact, intra-tumor genetic heterogeneity (ITH) has been scantly investigated in papillary thyroid cancer (PTC). We studied ITH in PTC by combining, for the first time, data derived from the evaluation of the normalized allelic frequencies (NAF) of the mutation/s, using a customized MassARRAY panel, and those obtained by the HUMARA clonality assay. Among tumors with a single mutation, 80% of cases with NAF 50 ± 5% were clonal, consistent with the presence of a single mutated clone, while 20% of cases showed a polyclonal pattern, suggesting the presence of the same mutation in two or more clones. Differently, all cases with NAF < 45% were polyclonal. Among tumors with double mutation, cases with both mutations showing NAF 50 ± 5% were monoclonal, consistent with the presence of a single clone harboring both mutations. On the other hand, all cases with double mutation at NAF < 45% were polyclonal, indicating the presence of two clones with different mutations. Finally, no significant differences in the clinico-pathological characteristics were found between monoclonal and polyclonal tumors. In conclusion, the present study adds insights into the concept of ITH in PTC, which warrants attention because the occurrence of this phenomenon is likely to affect the response to targeted drugs.