Sequencing-Based Minimal Residual Disease Assessment In T-Cell Malignancies: T-Cell Prolymphocytic Leukemia Case Study

Abstract Background The prognostic significance of minimal residual disease (MRD) quantification in the post-transplant setting has been demonstrated in multiple lymphoid malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Previous studies support the clinical utility of molecular MRD quantification of tumor burden after allogeneic hematopoietic cell transplantation (allo-HCT) (Logan et al, Leukemia 2013). We have developed the ClonoSIGHT™ test, which is based on the deep sequencing of immunoglobulin and T-cell receptor rearrangements and has a detection limit of one cancer cell per million leukocytes in peripheral blood or bone marrow (Faham et al, Blood 2012; Armand et al, Brit J Haematol 2013). In this report, we will discuss the technical performance of the ClonoSIGHT test for routine MRD quantification after allo-HCT and present a case study on a patient with T-cell prolymphocytic leukemia (T-PLL). Methods A 55 year old female presented with T-PLL including symptomatic CNS disease, received 12 weeks of Alemtuzumab therapy and then 12 weeks following her last Alemtuzumab treatment received an unrelated donor myeloablative allo-HCT using Fludarabine, BCNU and Melphalan conditioning with antithymoglobulin, Mycophenolate mofetil and cyclosporine primary immune prophylaxis. Peripheral blood samples were collected for MRD assessment before and serially after allo-HCT. Using universal primer sets, we amplified T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) variable, diversity, and joining gene segments from genomic DNA isolated from peripheral blood mononuclear cells (PBMC). Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination, and leukemia-specific clonotypes were identified based on their frequency within the T-cell repertoire (>5%). The leukemia-specific clonotype was then quantified in serial peripheral blood samples and reported as the absolute number of leukemic-specific clones among total leukocytes. Results A single clonal TRG gene rearrangement accounting for 26.1% WBC in the pre-transplant sample was identified and quantified in serial peripheral blood samples. A 4-log decline in MRD levels occurred post allo-HCT (Figure 1) thru 56 days following graft infusion; however, serial MRD monitoring demonstrated increasing levels of leukemia-specific clonotypes in the peripheral blood over time (Figure 1). Immunosuppression tapering strategies were employed in response to clinical events and MRD levels. Specifically, the patient developed an EBV+ post-transplant lymphoproliferative disease (PTLD) 60 days post allo-HCT, and cyclosporine was tapered in addition to instituting anti-CD20 rituximab treatment. As per institutional practice, a bone marrow biopsy 84 days post-HCT showed full donor engraftment with normal cellularity and no evidence of PLL was detected by flow cytometry when ClonoSIGHT detected 0.013% PLL in the patient's blood. Unfortunately, in the setting of immune suppression taper at 100 days post allo-HCT, the patient developed Grade II skin GVHD and was treated with 0.5mg/kg prednisone daily and tapered as indicated. At 160 days post allo-HCT, the patient presented with new skin papules suspected to be leukemia cutis. The PLL clonotype was detected in the skin biopsy; however, it was present at lower frequency in the TRG repertoire than in the blood, thus not supporting a diagnosis of leukemia cutis. In agreement, skin pathology revealed Verruca Vulgaris (warts). However, the patient's MRD continued to increase in the blood while immunosuppression was tapered and stopped completely 6 months post-HCT. Conclusions MRD assessment can be used to monitor a patient's disease progression after immune cellular therapy and aids immune suppression management following allo-HCT. Further, as presented in this case study, ClonoSIGHT detection of the leukemia clone in the blood compared with other tissues can sensitively and specifically assess extramedullary relapse. Disclosures: Kong: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay

Blood ◽

10.1182/blood.v122.21.627.627 ◽

2013 ◽

Vol 122 (21) ◽

pp. 627-627 ◽

Cited By ~ 2

Author(s):

Yasuhiro Oki ◽

Sattva S. Neelapu ◽

Michelle A. Fanale ◽

Larry W. Kwak ◽

Luis E Fayad ◽

...

Keyword(s):

Peripheral Blood ◽

High Throughput Sequencing ◽

Residual Disease ◽

Early Stage ◽

Lymph Node Biopsy ◽

Employment Equity ◽

Blood Samples ◽

Node Biopsy ◽

Stage Disease ◽

Equity Ownership

Abstract Background It was only a little more than a decade ago that the origin of the pathognomonic Reed Sternberg cells of Classical Hodgkin lymphoma (CHL) was established as B-cell malignancy characterized by a clonal expansion (Marafioti et al. Blood 2000). A previous report suggested that clonotypic B cells may be present in the blood of newly diagnosed CHL patients by virtue of identifying some light chain restricted populations by flow cytometry (Jones et al. Blood 2009). However, definitive investigation to assess whether CHL clones are detectable in the blood has been lacking. We developed the LymphoSIGHT™ platform, a high-throughput sequencing-based method, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease (MRD) after treatment (Faham et al. Blood 2012). This sequencing platform has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We conducted a pilot study to assess the ability of this platform to detect the lymphoma clone in peripheral blood samples from 13 CHL patients at the time of diagnosis or disease recurrence. Methods This study was IRB-approved and consent was obtained from all patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IGH) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell [PBMC] compartments) collected at initial diagnosis or at the time of recurrence. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in plasma and PBMC compartments from a blood sample obtained around the time of primary tissue biopsy. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. Results Clinical and histopathological characteristics are summarized in Table 1. We detected a high-frequency IGH clonal rearrangement in 8 of 13 (62%) lymph node biopsy samples. We observed a trend for a low rate of lymphoma clonotype identification in untreated patients with early stage disease (50%, 4 of 8 patients), and a higher rate of identification in patients with relapsed disease (80%, 4 of 5). In the 8 patients with an identified lymphoma specific clonotype in the biopsy sample, the clonotype was also detected in the plasma and/or PBMC compartment in 7 (88%) patients (Table 1). We detected a lymphoma clonotype more frequently in plasma (88%, 7 of 8) than in PBMC (33%, 2 of 6). Conclusions Our data is the first to show that circulating clonal tumor cells can be detected in the blood of patients with CHL, providing new opportunities to explore novel methods to detect minimal residual disease. Additional cases are currently being analyzed to determine the sensitivity and specificity in early stage versus advanced stage disease, and to determine whether the detection of MRD post therapy would correlate with clinical relapse. Disclosures: Carlton: Sequenta, Inc: Employment, Equity Ownership. Kong:sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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Variable Bruton Tyrosine Kinase (BTK) Resynthesis across Patients with Chronic Lymphocytic Leukemia (CLL) on Acalabrutinib Therapy Affect Target Occupancy and Reactivation of B-Cell Receptor (BCR) Signaling

Blood ◽

10.1182/blood-2018-99-110391 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4401-4401 ◽

Cited By ~ 2

Author(s):

Anfal A. Alsadhan ◽

Jean Cheung ◽

Michael Gulrajani ◽

Erika M. Cook ◽

Stefania Pittaluga ◽

...

Keyword(s):

Peripheral Blood ◽

De Novo ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Synthesis Rate ◽

De Novo Synthesis ◽

Employment Equity ◽

Blood Samples ◽

Bcr Signaling ◽

Equity Ownership

Abstract Inhibitors of B-cell receptor (BCR) signaling, in particular of Bruton tyrosine kinase (BTK), are effective in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a highly selective, covalent BTK inhibitor with rapid absorption and fast elimination (Barf et al., J Pharmacol Exp Ther 2017; 363:240-252). In a previous clinical trial of patients with relapsed/refractory CLL, acalabrutinib therapy was well tolerated and efficacious and had high target occupancy (Byrd et al., N Engl J Med 2016; 374:323-21). Here, we report on BTK occupancy, BTK resynthesis rates, and the relationship between BTK occupancy and BCR signaling in patients with CLL enrolled in a single center, phase 2 study of acalabrutinib monotherapy (NCT02337829). Patients were randomized to receive either 200 mg of acalabrutinib once daily (QD) or 100 mg twice daily (BID). The percentage of BTK bound by acalabrutinib (target occupancy) was measured with the aid of a biotin-tagged analogue probe. After 3 days of dosing, the median BTK occupancy in peripheral blood at peak (4 hours postdose) was 99% in both dosing groups (n=21 per group). However, at the drug trough time points (12 hours for BID; 24 hours for QD), median occupancy was greater in the BID than QD group (95% vs 87%, respectively; P<0.0001), with less interpatient variance. As acalabrutinib binds covalently to BTK, reactivation of the pathway requires de novo synthesis of BTK. We estimated the rate of BTK synthesis in vivo by measuring the accumulation of free BTK (% unbound by acalabrutinib) in serial blood samples taken 4, 12, 24, 36, and 48 hours postdose during a preplanned dose interruption on days 4 and 5. BTK was synthesized at a median rate of 13.4% per day, which was highly variable (fast vs slow) across patients (Figure). Interestingly, we found the median BTK de novo synthesis rate to be roughly twice as fast as observed in B cells from healthy volunteers (P<0.0001). BTK occupancy levels between matched peripheral blood and lymph node samples in individual patients were highly correlated (R=0.83; P<0.0001), with less than a 3% difference overall, suggesting a similar BTK synthesis rate in the lymph nodes and peripheral blood of patients with CLL. Similar results were observed for matched bone marrow and peripheral blood samples. We next correlated BTK occupancy levels and activity of BCR signaling in 10 representative patients. On Day 3, four hours after receiving the drug, significant reductions were observed in phospho-BTK (P=0.05), pNF-κB (P=0.03), and CD69 expression (P=0.001) compared with pretreatment. Next, we evaluated the change in BCR signaling from trough (for each dose group) to 36 (BID) or 48 (QD) hours after dosing (i.e., during the short window of drug withholding). A trend for increased signaling was driven by individual patients with low occupancy (≲70%). To mimic microenvironmental activation, we stimulated overnight with anti-IgM, all the peripheral blood samples collected during drug withholding (4 to 48 hours postdose). We found that upon BCR activation, B-CLL cells showed the ability to reactivate BCR signaling as measured by an increase in CD69 expression. Moreover, the extent of reactivation in BCR signaling positively correlated with the percentage of free BTK (R=0.73; P=0.0001). In conclusion, higher target coverage was achieved when acalabrutinib was administered 100 mg BID compared with 200 mg QD. Given that BTK de novo synthesis rates do vary across patients with CLL, and that reactivation of BCR signaling correlates with lower occupancy (higher free BTK), BID dosing provides the highest target coverage for a greater number of patients. This work was supported by the Intramural Research Program of National Heart, Lung, and Blood Institute, the National Institutes of Health, and Acerta Pharma. We thank our patients for donating blood and tissue samples to make this research possible. Figure. Figure. Disclosures Cheung: Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Davies-Hill:NIH/NCI: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-122787 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5603-5603 ◽

Cited By ~ 1

Author(s):

Cherie Tracy Ng ◽

Jeanette Ampudia ◽

Robert J. Soiffer ◽

Jerome Ritz ◽

Stephen Connelly

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Gvhd ◽

Vehicle Control ◽

Control Group ◽

Employment Equity ◽

Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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Detection of Diffuse Large B-Cell Lymphoma in Peripheral Blood Using High-Throughput Sequencing Assay.

Blood ◽

10.1182/blood.v120.21.2666.2666 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2666-2666

Author(s):

Yasuhiro Oki ◽

Malek Faham ◽

Victoria Carlton ◽

Sattva S. Neelapu ◽

Anas Younes

Keyword(s):

B Cell ◽

Peripheral Blood ◽

High Throughput Sequencing ◽

B Cell Lymphoma ◽

Lymph Node Biopsy ◽

Employment Equity ◽

Tumor Biopsy ◽

Blood Samples ◽

Large B Cell Lymphoma ◽

Equity Ownership

Abstract Abstract 2666 Background: In patients with diffuse large B-cell lymphoma (DLBCL), circulating lymphoma cells in the bloodstream are rarely detected by conventional morphology or flow cytometry evaluation. We developed a high-throughput sequencing based platform, LymphoSIGHT, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease after treatment. This sequencing method has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We herein report the results of our pilot study assessing the ability of this method to detect the lymphoma clone in peripheral blood samples from 5 DLBCL patients at the time of diagnosis. Methods: This study has been approved by IRB and consent has been obtained from patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell (PBMC) compartments) collected at initial diagnosis. Amplified products were sequenced to obtain >1 million reads (>10× sequencing coverage per IgH molecule), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in cell-free and PBMC compartments from the diagnostic blood sample. A quantitative and standardized measure of clone level among all leukocytes in the diagnostic sample was determined using internal reference DNA. Results: We detected a high-frequency IgH clonal rearrangement in all 5 lymph node biopsy samples. The lymphoma clonotype that was identified in the tumor biopsy was also detected in the plasma and/or PBMC compartment in all 5 patients at diagnosis. Specifically, the lymphoma clonotype was detected in the plasma compartment in 4 patients, while 3 patients demonstrated the presence of the lymphoma clonotype in the PBMC compartment (Table 1). We hypothesize that the positive lymphoma clone in the plasma is due to rapid proliferation and necrosis of the primary tumor, releasing the degraded component of lymphoma into the blood stream. However, in this small sample size, we did not observe an obvious correlation between the level of detection (PBMC or plasma) and clinical parameters (LDH, stage, size of tumor, tumor Ki67, cell-of-origin). All patients achieved complete response after initial treatment and four are being followed. We plan to analyze blood specimens while they are in remission. Conclusions: IgH clonal rearrangements were detected by sequencing in all tumor biopsy samples. Importantly, all peripheral blood samples showed signs of circulating lymphoma material in either the plasma or PBMC compartment at diagnosis. Analysis of diagnostic and post-therapy samples from additional DLBCL patients is ongoing. These data will determine whether the sequencing assay is a strong indicator for response to therapy and relapse monitoring. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

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Annotation Of Pseudogenous Gene Segments By Massively Parallel Sequencing Of Rearranged Lymphocyte Receptor Loci

Blood ◽

10.1182/blood.v122.21.3480.3480 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3480-3480

Author(s):

Ryan O Emerson ◽

Anna Sherwood ◽

Harlan Robins ◽

Christopher S Carlson ◽

Mark Rieder

Keyword(s):

Immune System ◽

T Cell ◽

B Cells ◽

Gene Segment ◽

Antigen Receptor ◽

Cell Receptor ◽

T And B Cells ◽

Employment Equity ◽

Immune Receptor ◽

Equity Ownership

Abstract The adaptive immune system generates a remarkable breadth of antigen-specific T cell receptors (TCRs) and B cell receptor (BCRs) by combinatoric shuffling of gene segments, enabling the immune system to recognize a diverse and unpredictable set of antigens. To generate this enormous diversity, lymphocytes undergo somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) region gene segments, which collectively encode the CDR3 region along with non-templated deletion or insertion of nucleotides at the V-D, and D-J junctions. Many of the V, D, and J gene segments at immune receptor loci are annotated as non-functional due to defects in primary sequence, motifs necessary for rearrangement, or chromosome position. However, full annotation of functional, pseudogene, or ORF, has proven elusive due to the random and unpredictable nature of non-templated deletions and insertions, the huge space of potential receptors, and the large V and J gene families. We amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged immunoglobulin heavy chain (IGH) and T cell receptor beta (TCRB) receptor locus. Deep DNA sequencing allowed us to characterize the TCRB and IGH immune repertoire from mature T and B cells in a large cohort of healthy adults. Random chance predicts that less than one-third of somatic rearrangements at the TCRB and IGH loci will lead to transcripts with key motifs in-frame and no premature stops (i.e. a productive antigen receptor). Selection during lymphocyte maturation ensures that all mature T and B cells carry at least one rearrangement coding for a productive receptor, with a second allele rearranging in some cells that can be out of frame or include a V, D or J pseudogene segment (i.e. non-productive receptor). We classified each V, D, and J gene segment as functional or pseudogene based on the proportion of in-frame rearrangements and on 3-nt periodicity in the length of the CDR3 hypervariable coding region. Based on these data we were able to conclusively annotate the functional status of each gene segment in the complex TCRB and IGH immune receptor loci. In TCRB, we found 2 presumed-functional gene segments that are clearly pseudogenes; in IgH we found 3 presumed-functional genes that are actually pseudogenes and 1 annotated pseudogene that is clearly functional. In both loci we identified numerous cases of gene segment functional/pseudogene status segregating among healthy subjects, indicating that genotype at the TCR and IGH loci may be a source of substantial differences in the naive antigen receptor repertoire between individuals. These results have helped us to more accurately characterize the genetic landscape underpinning somatic rearrangement of antigen receptors, a seminal event in the generation of robust antigen-specific immune responses. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership.

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Characterization of Allogeneic T Cells Expressing Inducible Caspase-9 Following Adoptive Transfer in Children Receiving an HLA-Haploidentical Hematopoietic Stem Cell Transplant for the Treatment of Myeloid Malignancies

Blood ◽

10.1182/blood-2018-99-119779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4534-4534

Author(s):

Joanne Shaw ◽

Xiaoou Zhou ◽

An Lu ◽

Valentina Bertaina ◽

Daria Pagliara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Adoptive Transfer ◽

Peripheral Blood ◽

Employment Equity ◽

Advisory Committees ◽

Myeloid Malignancies ◽

Donor T Cells ◽

Equity Ownership

Abstract Background: Adoptive transfer of allogeneic donor T cells can be an effective treatment for hematological malignancies through recognition of leukemia-associated antigens (LAAs) on tumor cells or through alloreactivity. However, alloreactive T cells can also cause graft-versus-host disease (GvHD) limiting their use as an immunotherapy. To leverage the anti-tumor effects of allogeneic polyclonal T cells while minimizing GvHD, we have genetically modified donor T cells with the inducible caspase-9 (iC9) safety switch, which induces apoptosis following exposure to the small molecule ligand rimiducid. Here we show that iC9-modified allogeneic T cells (BPX-501) persist, expand and contain functional LAA-specific T cells in children receiving an alpha/beta TCR and CD19-depleted HLA-haploidentical hematopoietic stem cell transplant (haplo-HSCT) for the treatment of myeloid malignancies. Methods: Pre-infusion products (BPX-501: donor T cells modified with the bicistronic retroviral vector encoding iC9 and truncated CD19 (ΔCD19)) and patient peripheral blood mononuclear cells (PBMCs) were analyzed from twelve patients (AML (10), MDS (1), JMML (1)) receiving BPX-501 (1x106 cells/kg) following an alpha/beta T cell and CD19 B cell-depleted haplo-HSCT (BP-004U: NCT03301168). Engraftment and persistence were measured by coexpression of CD3 and CD19 by flow-cytometry. Endogenous and gene-modified T cells were also phenotyped for CD4:CD8 ratios, memory cell composition (TN, TCM, TEM, TEMRA; CD45RA and CD62L) and T cell receptor Vβ diversity. BPX-501 products and post-treatment samples were characterized for LAA-specific T cells using IFN-γ ELISpot against peptide pools (15 aa overlapping by 5 aa) derived from WT1, PRAME, MAGE (A1, C1, C3), NE and PR3, with and without exposure to 10 nM rimiducid to determine the anti-leukemic contribution of BPX-501. Results: BPX-501 was infused at a median time of 22.5 days after HSCT (range 12-34, one patient was infused at day 89 and one patient was infused at day 147). BPX-501 cells (CD3+CD19+) were detectable in the peripheral blood at 1-2 weeks after infusion in all 12 patients, reaching a peak expansion frequency of a median of 24% ± 17% of total CD3+ T cells, and an absolute cell number of 66.9 ± 112 cells/µl at 2 months post-infusion and could be detected for up to 24 months. BPX-501 T cells showed a CD8-skewed phenotype whereas endogenous T cells exhibited a more balanced CD4:CD8 ratio. BPX-501 were predominantly CD45RA-CD62L+ and CD45RA-CD62L- central and effector memory T cells, respectively. In BPX-501 products, we detected LAA-specific T cells by ELISpot using overlapping peptide pools to WT1, PRAME, MAGE, NE and PR3, and in peripheral blood samples obtained 2 to 5 months post-T cell infusion. Importantly, LAA-reactivity was greatly diminished with exposure to iC9-activating rimiducid. Further, we measured the TCR Vβ usage and observed highly-skewed TCR repertoire in BPX-501 T cells compared to endogenous T cells in 6 months after HSCT indicating selection and expansion of TCR clones. Three patients engrafted BPX-501 were treated with rimiducid to control GvHD resulting in a rapid decrease (62% ± 12%) of CD3+CD19+ T cells in the peripheral blood. In patients treated with rimiducid, CD3+CD19+ T cells recover without further instances of GvHD suggestive of in vivo depletion of alloreactive T cell clones using iC9. Summary: Allogeneic T cells engineered with the iC9 safety switch engraft, expand and demonstrate long-term persistence following adoptive transfer into patients receiving a haplo-HSCT. LAA-specific T cells and alloreactive T cells within the BPX-501 product are detectable in the peripheral blood following infusion and likely contribute to elimination of myeloid malignancies. Disclosures Shaw: Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhou:Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu:Bellicum Pharmaceuticals: Employment, Equity Ownership. Aldinger:Bellicum Pharmaceuticals, Inc.: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Bellicum: Employment, Equity Ownership.

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A Circulating T Cell Clone in Early Mycosis Fungoides Is Not a Negative Prognostic Factor When the Disease Is Treated by a Combination of PUVA + Interferon α.

Blood ◽

10.1182/blood.v104.11.3269.3269 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3269-3269

Author(s):

Serena Rupoli ◽

Gaia Goteri ◽

Renzo Ranaldi ◽

Capretti Roberta ◽

Anna Rita Scortechini ◽

...

Keyword(s):

T Cell ◽

Peripheral Blood ◽

Gene Rearrangement ◽

Cell Clone ◽

Cell Receptor ◽

Disease Recurrence ◽

Pcr Analysis ◽

Blood Samples ◽

T Cell Clone ◽

Mean Time

Abstract In patients with early Mycosis Fungoides (MF), a dominant T-cell receptor (TCR) gene rearrangement can be detected in the skin in 50–75% of cases, while in 15–40%, an identical T-cell clone is detectable also in the peripheral blood This may indicate an unfavourable subset of patients. We observed on omogeneus group of early MF patients all receiving the some protocol. The purpuose of this study were: - T-cell receptor gene rearrangement analisys of peropheral blood samples; - evaluation of the prognostic impact of T-cell monoclonality on CR and relapse rate. 44 patients diagnosed as having MF at early stage (25 M, 19 F; mean age 58.7 yrs, range 34–77; 11 in stage IA, 28 in stage IB, 5 in stage IIB) and showing a dominant T cell clone in the skin lesions at diagnosis, were included in the present study. Peripheral blood samples were collected for DNA extraction at diagnosis. PCR amplification for TCRγ gene was performed as previously reported by Ashton-Key et al. (1997) in all 44 cases both in peripheral blood and skin and reduplicated: amplification products were visualised by 10% polyacrylamide gel electrophoresis. Peripheral blood samples were considered positive for a circulating T cell clone only if the monoclonal signals in peripheral blood and skin were reproducible and overlapping. All patients received the same treatment, consisting of a combination protocol with low-dose IFNα + PUVA for 14 months, and then followed up. Clinical response to the therapy and further disease recurrences were registered. After a mean time of 6.02 months (range, 1–21), 7 patients failed to respond to combination therapy, while 37 obtained a clinical complete remission (CCR). Among them, 15 experienced a disease recurrence during the follow-up (mean time, 29.8 months, range, 1–77 months). PCR analysis of TCRγ gene showed in the peripheral blood the same T-cell clone detected in the skin in 16 cases (36.4%). Failure to obtain a CCR was found in 3 out of 28 cases without a T cell clone in peripheral blood (10.7%) and in 4 out 16 cases with a circulating T cell clone (25%; c2 test: P=0.41, NS). Disease recurrence was observed in 9 out of 25 cases without a T cell clone in peripheral blood (36%) and in 6 out of 12 cases with a circulating T cell clone (50%; c2 test: P=0.65, NS). Disease-free survival curves plotted by Kaplan-Meier method showed that patients with and without a circulating T cell clone did not behave differently and that half of the patients would have experienced a relapse after a similar period of time (34 and 36 months, respectively, for patients with TCRg+ and − peripheral blood; log rank test, P=0.79). PCR analysis of TCRγ gene rearrangement analysis has allowed us to detect monoclonality in the peripheral blood in 36% of early MF cases with a documented monoclonality in the skin. At this stage of the disease a circulating T cell clone could indicate a subset of patients in which skin-directed therapies are more likely to fail to completely eradicate the malignant cells. Interestingly, our data seems to show that the negative influence of a circulating clone can be bypassed by the combination of a skin-directed therapy like PUVA and the systemic immunoregulatory effects of IFNα.

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Minimal Residual Disease Measurement By Deep Sequencing Reflects Changes In Disease Load During Therapy In Diffuse Large B Cell Lymphoma Patients

Blood ◽

10.1182/blood.v122.21.1785.1785 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1785-1785

Author(s):

Nina Wagner-Johnston ◽

Nancy L. Bartlett ◽

Kenneth R. Carson ◽

Mary Anderson ◽

Li Weng ◽

...

Keyword(s):

B Cell ◽

Peripheral Blood ◽

Treatment Time ◽

Residual Disease ◽

B Cell Lymphoma ◽

Employment Equity ◽

End Of Treatment ◽

Equity Ownership ◽

Pre Treatment ◽

Time Point

Abstract Background Assessment of prognosis in patients (pts) with diffuse large B cell lymphoma (DLBCL) is suboptimal. Pre-treatment clinical evaluations such as the International Prognostic Index (IPI) score are not useful in assessing response or tailoring therapy. Interim PET/CT, as an early indicator of response has a low positive predictive value and current guidelines recommend against its use, underscoring the need for improved predictive markers for early response assessment. The LymphoSIGHT™ platform is a high-throughput sequencing-based method for detecting lymphoid malignancies in peripheral blood that could potentially be used for detecting minimal residual disease (MRD) during and after treatment (Faham et al. Blood 2012). LymphoSIGHT™ has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood and can be used to identify circulating tumor DNA in DLBCL (Armand et al., Brit J Haematol 2013). Here we assessed the ability of the sequencing method to detect lymphoma clones in pre-treatment tumor biopsy (bx) and peripheral blood samples obtained during and post-therapy from 33 DLBCL pts. Methods Fifty pts with DLBCL undergoing chemotherapy with curative intent were prospectively enrolled in a study to evaluate for clonal immunoglobulin (Ig) DNA in baseline tumor and peripheral blood samples collected pre-treatment, day 8, week (wk) 4, wk 7 and end of treatment. Using universal primer sets, we amplified Ig heavy chain (IGH) variable, diversity, and joining and Ig kappa chain (IGK) gene segments from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each pt based on their high-frequency within the B-cell repertoire in the lymph node (LN) bx sample. The presence of the tumor-specific clonotype was then quantitated in plasma and serum samples obtained at pre-treatment time points. A quantitative and standardized measure of clone level per million leukocytes in each follow-up sample was determined using internal reference DNA. Results Paired tumor/plasma specimens were available from 41 pts. Of 33 LN bx samples screened, 30 had adequate DNA, and we detected a high-frequency clonal rearrangement in 25/30 (83%, CI 70-97%). In pts with an identified lymphoma specific clonotype in the bx sample, the clonotype was also detected in the plasma and/or serum compartment in 17 of 24 (71%) pts at the pre-treatment time point. We observed high qualitative and quantitative correlation (r2 = 0.89) between MRD levels measured in the matched plasma (median 0; average 53,036) and serum (median 0; average 111,496) samples. Three qualitative discordances were observed, in which the serum was negative and the plasma was positive at the same time point, all of which can be attributed to low amounts of lymphoma clone molecules that were detected in the positive samples. Pts who later relapsed tended to have higher levels of lymphoma-specific clones in their pretreatment samples compared to pts in sustained CR (t-test p=0.10).We also monitored MRD kinetics in response to therapy and observed a marked decline in MRD positivity over the course of treatment. Specifically, MRD positivity was observed in 4/5 (80%) pts at Day 8, in 6/19 (32%) at wk 4, in 1/21 (5%) at wk 7 and in 1/20 (5%) at the end of treatment (Figure 1). The average MRD level per million leukocytes was 261,681 at pre-treatment, 14,698 at wk 4, 0.35 at wk 7, and 74 at the end of treatment time point (Figure 1, horizontal lines). Thus, MRD levels demonstrated a substantial decline with treatment and reflected changes in disease load. Of note, the single pt that was MRD positive at the end of treatment was the only pt with refractory disease. Of 5/18 pts with a positive interim PET/CT following 2-3 cycles of chemotherapy, only 2 have relapsed. Three of the 5 pts (including one that relapsed) were tested for detectable clonal DNA at 7 wks, and none of the three was positive. Results of all 41 pts will be presented. Conclusions Our data demonstrate that circulating clonal tumor DNA can be detected in the blood of pts with DLBCL, and that MRD levels decline consistent with tumor burden. Further studies are warranted to better characterize the predictive value of this novel sequencing platform. Disclosures: Weng: Sequenta, Inc: Employment, Equity Ownership. Klinger:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership.

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Minimal Residual Disease Monitoring In Acute Lymphoblastic Leukemia Patients Using Immune Receptor Sequencing

Blood ◽

10.1182/blood.v122.21.1381.1381 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1381-1381

Author(s):

Malek Faham ◽

Maddalena Paganin ◽

Giulia Fabbri ◽

Elisa Magrin ◽

Emanuela Giarin ◽

...

Keyword(s):

T Cell Receptor ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Clinical Relapse ◽

Employment Equity ◽

Childhood All ◽

Lymphoid Malignancies ◽

Equity Ownership ◽

Minimal Residual

Abstract Background The assessment of residual tumor cells persisting after therapy, or minimal residual disease (MRD), is a central component of accurate disease prognosis in acute lymphoblastic leukemia (ALL) (Pui et al, JCO 2011). MRD assessment has recently been shown to be useful for monitoring disease before and after stem cell transplantation and during salvage therapy for early detection of an imminent relapse (Bruggemann et al., SemOncol 2012). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We recently demonstrated the utility of sequencing-based MRD assessment in lymphoid malignancies (Faham et al., Blood 2012). This quantitative approach, termed the LymphoSIGHT™ platform, relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection techniques. This sequencing platform has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood and bone marrow samples. In this retrospective study, we evaluated the ability of the sequencing and ASO-PCR methods to detect MRD prior to clinical relapse in 17 patients with childhood ALL. Methods Using the sequencing assay, we analyzed bone marrow and/or peripheral blood samples collected at the diagnostic and relapse time points from 17 childhood ALL patients. Diagnostic and relapse samples were assessed for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. Following identification of leukemia specific clonotype(s), we measured the corresponding MRD levels in 66 follow-up samples that were collected prior to clinical relapse. We analyzed the time from MRD positivity to clinical relapse for each patient using the sequencing and ASO-PCR methods. Results Sequencing detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 17 childhood ALL patients; all patients had at least 2 calibrating receptors at diagnosis and/or relapse, 15 patients had at least 3 calibrating receptors, and 6 patients had 4 or more. The IGH-VDJ and TRG assays were the most frequent gene rearrangement: at least one IGH-VDJ and/or TRG clonal rearrangement was detected in 13 ALL patients. TRD was the third most informative receptor, with clonal rearrangements being detected in 11 patients. In the majority of patients, most leukemic clones identified at diagnosis were also present at high levels in the relapse sample. We analyzed the time from MRD positivity to clinical relapse in the patient cohort. In 2 patients (Figure 1, Patients G and P), we observed MRD positivity by sequencing 16 and 6.5 months prior to relapse, respectively. ASO-PCR also detected MRD positivity in Patient P at the 6.5 month time point. In 6 patients there was a sample collected within 1-3 months prior to relapse, and sequencing detected MRD in 4 of these patients (67%, Figure 1 Patients B, A, R, I), while ASO-PCR detected MRD in 2 of the 6 patients (33%, Figure 1 Patients B, R). Conclusions The clinical value of monitoring MRD by ASO-PCR for assessment of treatment response and outcome has been established in multiple lymphoid malignancies. This preliminary study provides further support for the sequencing-based MRD monitoring in ALL patients. This new approach offers improvements over ASO-PCR in the ability to monitor multiple clonal sequences and in the sensitivity to detect the leukemic cell. Thus, the sequencing method represents a new potential approach for MRD monitoring. However a larger number of patients must be analyzed to support its clinical application. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Pepin:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership.

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Cellular Kinetics and Anti-Therapeutic Antibody in Relapsed/Refractory B-NHL Patients Treated with JWCAR029

Blood ◽

10.1182/blood-2019-126408 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4083-4083

Author(s):

Zhitao Ying ◽

Pengpeng Xu ◽

Ming Hao ◽

Li Wang ◽

Shu Cheng ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Phase I ◽

Employment Equity ◽

Blood Samples ◽

Car T Cells ◽

Best Response ◽

Car T ◽

Equity Ownership

Background: JWCAR029 is a CD19-directed 4-1 BB CAR T cell product, of which CD4 and CD8 CAR T cells are produced together and transfused in non-fixed ratio. A phase I, single-arm, open label study was conducted to evaluate the safety and efficacy of JWCAR029 in patients (pts) with relapsed or refractory B-NHL. Previously, preliminary data in six pts (Yan et al, Blood 2018 132:4187) showed high response rates and favorable safety profiles of JWCAR029. Herein, we presented the data of the Phase I trial of JWCAR029 (NCT03344367 and NCT03355859) in 29 pts with pharmacokinetics (PK), pharmacodynamics (PD), and anti-therapeutic antibody (ATA) evaluations. Methods: Eligible pts received lymphodepletion, with 25mg/m2 flu and 250mg/m2 cy, followed by a single dose of JWCAR029 at one of four dose levels (DL1, 25×106 cells; DL2, 50×106 cells; DL3, 100×106 cells; DL4, 150×106 cells). Blood samples were collected and analyzed for PK, PD, and ATA at a central lab per protocol defined time points. The existence and duration of CAR T cells (PK) were measured by validated flow cytometry and qPCR assays. CD4 and CD8 subpopulation of CAR+ T cells were detected by cetuximab targeting EGFRt as a marker co-expressed with CAR transgene in fresh peripheral blood. In parallel, batched frozen blood samples collected from each pt were detected for integrated CAR transgene by qPCR at the same protocol defined time points. Plasma ATA against murine CD19 scFv (FMC63) was measured with a validated electrochemiluminescent (ECL) assay. Results: As of July 5, 2019, blood samples from 29 pts who received JWCAR029 treatment with a minimum follow-up of 6 M (median, 6 M) were evaluable in the analysis. From DL1 to DL4, median Cmax, Tmax and AUC0-28 for JWCAR029 transgene detected by qPCR did not differ among dose levels (Table 1). CD4/CD8 ratio (range, 0.23-5.50) at cryopreserved drug product of JWCAR029 was not associated with best response of CR/PR at 6 M. Greater in vivo expansion was detected by both qPCR and flow cytometry in pts with best response of CR/PR than those with SD/PD at 6 M (Table 1). Higher concentration of CD8+CAR+T cells than CD4+CAR+T cells were detected in PB by flow cytometry for all treated pts (Cmax median= 30.6 vs 5.64). At 3 M, 81.5% (22/27) and 48.2% (13/27) pts had detectable CD8+ and CD4+ CAR+ T cells, respectively. Of those pts with detectable CAR+ T cells at 3 M, 70% (14/20) and 35% (7/20) had detectable CD8+ and CD4+ CAR+ cells at 6 M, respectively. Significantly higher Cmax and AUC0-28 were observed in patients with ≥ Grade 1 CRS (Cmax median= 85004 vs 16328, P<0.01; AUC median=536543 vs 141731, P<0.01). And relatively higher Cmax and AUC0-28 were found in patients with NT (Cmax median= 116112 vs 40391; AUC median=711306 vs 301035). 27.5% of pts (8/29) had detectable ATA in plasma, of which 25% (2/8) pts had pre-existing antibodies before CAR T cell infusion. 6 pts developed antibodies without pre-existing antibodies and were considered treatment-induced. The median time for treatment-induced antibody development was 6 M (range, 3-12). Increasing level of antibodies were detected at median time of 6 M (range, 6-6) for pts who had pre-existing antibodies and were considered treatment-boosted. No significant differences in PK profiles of JWCAR029 transgene levels were found between ATA negative group and treatment-induced ATA positive group (Cmax median= 44497 vs 50032; AUC median= 420635 vs 313654; Fig.1). Although the sample size of the treatment-boosted subgroup was small, there was a trend for lower expansion of CAR T cells in pts who had pre-existing ATA than pts who did not develop ATA (Cmax median= 3051 vs 44497; AUC median = 16437 vs 420635;Fig.1). In ATA positive subgroup, 100% (8/8) pts responded with CR rate of 75% (6/8). 6 M-response rate was 65.5% (5/8) for ATA positive subgroup and 57.1% (12/21) for ATA negative subgroup. Incidence of ≥ Grade 1 CRS or NT was indistinguishable between ATA positive and negative subgroups, 50% (4/8) in ATA positive vs 57.1% (12/21) negative. Conclusion: Preliminary data from JWCAR029 Phase I study has demonstrated that pts with best response of CR/PR at 6 months had a relatively higher CAR T cell expansion. Current data suggested that the prevalence of pre-existing ATA may compromise CAR+T PK profile. No association of the presence or boost of ATA with efficacy or safety of JWCAR029 was observed. Further exploration of ATA and clinical outcomes will be studied in the ongoing pivotal Phase 2 study in 70 pts with B-NHL. Disclosures Hao: JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zhou:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Yang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Lam:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Li:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zheng:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership.

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