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Current methods for detection of CNS involvement in lymphoma (CSF cytology, flow cytometry) have very limited sensitivity, particularly in cases of parenchymal brain involvement. Early detection is however critical to institute CNS-directed therapy that can avert dismal outcomes of overt CNS recurrence. Clonotype-specific cfDNA can be detected in the plasma of patients with lymphoma using next-generation sequencing (NGS), and residual cfDNA-based minimal residual disease (MRD) assay can predict impending recurrence (Roschewski et al., Lancet Oncol. 2015). cfDNA has not been systematically evaluated in the CSF, yet it may hold promise as a sensitive and specific method to detect CNS invasion. To evaluate the ability of an NGS-MRD assay in the CSF to detect CNS invasion, we examined CSF and plasma samples from patients with aggressive lymphomas who either had overt CNS disease, or who were without evident CNS invasion, but at high clinical risk. Genomic DNA from primary tumors was analyzed for tumor-specific clonotype using NGS of rearranged IGK, IGH (VJ or DJ), or IGL loci (Adaptive Biotechnologies; Carlson CS et al., Nat Commun. 2013). Tumor-specific clonotypes from each case were selected for subsequent tracking by NGS-MRD in CSF and plasma samples. Clonotype copy numbers are expressed per mL for acellular CSF, and clonotype frequency per all B-cells. NGS identified median 3 (range, 2-7) dominant immunoglobulin sequences in each primary lymphoma (N=16), with median dominant clonotype frequency 50.3% (range, 26.8-9.28%). In the CSF, the NGS-MRD assay detected the dominant clonotype in 9 out of 16 samples, including all (N=4) with overt CNS invasion (sensitivity=100%), of which 2 had parenchymal disease only with negative standard CSF cytology, flow cytometry, or IGH PCR. Median detectable cfDNA clonotype in the CSF was 1,077 copies per mL (range, 2-5,620), with median clonotype frequency of 28.4% (range, 0.1-98.5%). cfDNA copy counts were significantly higher in cases with positive CSF cytology than those with parenchymal or clinically occult disease (P=.0016). We observed no significant correlation between the red cell count in the CSF and the clonotype cfDNA concentration (P=0.73) or frequency (P=0.62) suggesting that the CSF cfDNA was not due to contamination by blood. There was also no evident correlation between cfDNA in plasma and CSF. Our results suggest that NGS-MRD assay for cfDNA in the CSF can identify intraparenchymal or leptomeningeal CNS invasion with high sensitivity, including cases not identifiable by traditional methods. Prognostic significance of detecting lymphoma-specific cfDNA in the CSF of some high-risk patients without overt CNS disease will be further explored in a larger sample. Pre-treatment NGS-MRD assay could be prospectively tested to predict the risk of CNS recurrence, with a future potential to enable more accurate selection of patients for CNS prophylaxis therapy. Citation Format: Adam J. Olszewski, Anna Chorzalska, Diana O. Treaba, John L. Reagan, Andrew Hsu, Pamela C. Egan, Habibe Kurt, James Butera, William Rafelson, Thomas Ollila, Rabin Niroula, Jordan Robison, Chelsea D. Mullins, Patrycja M. Dubielecka. Clonotypic cell-free DNA(cfDNA) in the cerebrospinal fluid (CSF) of patients with aggressive lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 733.

Introduction: In the treatment of acute leukemia, much of the therapeutic effect of allogeneic stem cell transplantation is centered on engraftment of donor cells into the host, with a resultant graft versus leukemia effect. Work previously done by our group and others has shown the infusion of haploidentical stem cells in patients with acute leukemia results in a cytokine storm and exhibits an anti-tumor effect in the absence of engraftment. Whether the underlying clinical response was due to this cytokine storm or secondary to host or donor effector cell interactions was not determined. We present in vitro data collected from leukemia patients in which patient CD3+ cells are stimulated with randomly selected unrelated normal donor peripheral blood mononuclear cells (PBMC). The stimulated patient CD3+ cells are then collected and tested for their ability to lyse leukemia cells from the patient. Methods: New leukemia patients were identified through standard pathologic diagnostic criteria. Patient samples were collected and separated into CD3+ and CD3- fractions. Patient CD3+ cells were then placed in a mixed lymphocyte culture and stimulated with mitomycin C treated unrelated normal donor PBMC. Cell proliferation was measured on Day 5. On Day 7, the stimulated patient CD3+ cells were collected and then cultured with Chromium 51 labeled CD3- cells, the bulk of which were leukemic blast cells. Cytolytic activity was measured through Cr51 release assays. Cell lysis was recorded as lytic units/million cells (LU/106), which is inversely related to the effector to target ratio at which 30% cell lysis occurs. Patient demographic data including sex and age in addition to leukemia type, cytogenetics, and molecular markers were recorded. Results: 10 total patients were enrolled in the study; 8 AML, 1 CML, and 1 CMML. 9 patients underwent ex vivo CD3+ stimulation with 2 separate donor PBMC samples while 1 sample underwent only 1 donor stimulation for 19 total assays. The average white blood cell count was 57,640 cells/uL (range 7,100-336,500 cells/ul) with 4-80% peripheral blasts. 7 out of 19 (37%) assays showed leukemia cell cytolytic activity greater than 1 lytic unit. All 7 successful assays were derived from patients with AML (Table 1). Conclusions: Patient CD3+ cells stimulated with random unrelated donor PBMC are able to generate a cytolytic anti-leukemia response in a donor dependant fashion. This cytolytic activity appears to be unrelated to host lymphocyte proliferation upon donor antigenic stimulation. Interestingly, of the seven assays with cytolytic activity greater than one lytic unit, four were performed in the two patients with known FLT3 ITD positive leukemia, which has a notoriously poor prognosis. Further studies to elucidate a mechanism of action for cellular therapy are warranted. Citation Format: John L. Reagan, Loren D. Fast, Martha Nevola, Peter J. Quesenberry. Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2013-1252

Previous work done by our group had found that infusion of G-CSF mobilized haploidentical donor white blood cells at a dose of 1 – 2 x 108 CD3+ cells/kg into refractory cancer patients after 100 cGy total body irradiation resulted in a number of complete and partial responses in those patients with hematological malignancies. These responses occurred despite elimination of the donor cells by 2 weeks after infusion. The effector mechanism(s) responsible for these anti-cancer responses were not determined although it was noted that there was a cytokine storm appearing within a day following infusion of the cells. Alloreactive responses could be driving the anti-cancer responses using a variety of mechanisms. One possible mechanism is that the alloreactive response overrides inhibitory mechanisms present in the cancer patient allowing the existing cancer reactive effector cells to become activated and lyse cancer cells. Alternatively the alloreactive cytolytic T cells could be cross-reacting with the antigens expressed on the tumor cells. To test these possibilities, peripheral blood mononuclear cells (PBMNC) or the CD3+ T cells isolated from the PBMNC of newly diagnosed leukemic patients were mixed with mitomycin C treated fully allogeneic stimulator PBMNC (from 1 or 2 different normal controls chosen at random) or the CD3- cells from the leukemic patients. Their ability to proliferate in response to the allogeneic stimulation was tested on day five and their ability to lyse autologous leukemia cells after stimulation was tested on day seven. Ten total patients (8 AML, 1 CML, and 1 CMML) were enrolled in the study. The average white blood cell count was 57,640 cells/uL (range 7,100 – 336,500 cells/uL) with 4 -80% peripheral blasts. The results indicated that in 16/20 combinations, leukemic T cells were able to proliferate in response to allogeneic stimulators, however anti-cancer cytolytic activity was only detected in 9/19 evaluable combinations. These results demonstrated that alloreactive responses are able to induce anti-leukemic cytolytic responses by T cells from a subset of leukemic patients. One explanation for the inability of some combinations to generate anti-leukemic cytolytic activity could be the presence of inhibitory mechanisms which could not be overcome by the alloreactive responses. Immunophenotypic analysis of the leukemic T cells did reveal an increased number of T regulatory cells and increases in the expression of inhibitory receptors on the lymphocytes from some of the patients. Alternatively, it may be that the leukemic T cells had not been stimulated with the appropriate allogeneic antigens. An allogeneic stimulator pool comprised of cells from ten different normal donors was prepared and will be used to test whether increasing the number of allogeneic disparities in culture would enhance the ability of the leukemic T cells to generate anti-leukemic responses. These studies have suggested that cancer-reactive cells can be generated from leukemic patient T cells using alloreactive responses. To further test the role of these effector cells and identify additional effector cells, blood samples will be obtained from patients enrolled in the clinical protocol which is in the process of being restarted, having received FDA approval. Clearer definition and characterization of the anti-cancer effector cells should permit alterations to the protocol that would lead to enhanced anti-cancer responses in an increased number of refractory cancer patients. Citation Format: Loren D. Fast, John Reagan, Martha Nevola, Peter Quesenberry. Antitumor responses driven by alloreactivity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B22.