Innovative Immunotherapy for Drug-Resistant Hematologic Cancers
Reviewing the possible cure of drug-resistant hematologic malignancies by innovative cell-mediated immunotherapy
Lashudaa Madura Madhavan¹, Hemavathy Nagarajan², Umashankar Vetrivel³, Madhavan Jagadeesan¹
- Dualhelix Genetic Diagnostics, Chennai, Tamil Nadu, India
- Centre for Bioinformatics, KIRVO, Vision Research Foundation, Chennai, Tamil Nadu, India
- ICMR-Department of Virology and Biotechnology / Bioinformatics Division, National Institute for Research in Tuberculosis, Chennai, Tamil Nadu, India
OPEN ACCESS
PUBLISHED: 30 November 2024
CITATION: Madhavan, LM., Nagarajan, H., et al., 2024. In Silico Characterization of Two Adjacent Novel Homozygous Nucleotide Variations in the PEX6 Gene Predict L898V Mutation to Enhance Infantile Refsum Disease and Heimler Syndrome. Medical Research Archives, [online] 12(11).
https://doi.org/10.18103/mra.v12i11.0000
COPYRIGHT: © 2024 European Society of Medicine. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
DOI https://doi.org/10.18103/mra.v12i11.0000
ISSN 2375-1924
ABSTRACT
Background: Zellweger spectrum disorders are autosomal recessive in origin due to defects in peroxisome biogenesis and with variable severity. The present work aims to characterize a South Asian Indian Zellweger spectrum disorder family with PEX6 mutation for a genotype-phenotype association.
Method: The affected and unaffected individuals in the family were evaluated. A comprehensive examination of the ocular, auditory, dental, integumentary, neuronal, hepato-renal, endocrine, skeletal, cardiac, and other systems was conducted. Investigations deemed fit for diagnosis and management were done. Karyotyping and molecular genetic screening of the peripheral venous blood were performed for aneuploidy and mutation detection. Retinal fundus photograph, optical coherence tomography, bilateral audiogram, magnetic resonance imaging of the brain, ultrasonography of the abdomen, electrocardiography, and echocardiogram were performed. Any non-synonymous nucleotide variations detected were analyzed using in silico methods.
Results: The proband was born of consanguinity and had retinitis pigmentosa in both eyes, bilateral sensorineural deafness, amelogenesis imperfecta, Beau’s line, and punctate leukonychia corresponding to Heimler syndrome. In addition, the proband had developmental nuclear cataracts, ichthyosis, developmental delay, cerebellar ataxia, cognitive deficit, peripheral neuropathy, and muscle movement disorder corresponding to inherited Refsum disease. The proband did not have anosmia. The brain’s magnetic resonance imaging, abdomen ultrasonography, electrocardiography, and echocardiogram were normal. The karyotype revealed homozygosity status. The molecular genetic screening detected adjacent homozygous non-synonymous nucleotide variations c.2691C>A (p.Ser897Arg) and c.2692C>G (p.Leu898Val) in the PEX6 gene. The pathogenic effect, structural destabilization effect, and functional impact of the variants were analyzed through in silico methods. The L898V variant was highly pathogenic compared to the non-conserved S897R variant of PEX6, leading to structural instability and loss of functionality. The molecular dynamics simulation studies also revealed the L898V variant to cause structural instability of PEX6 with higher backbone deviations and residue-wise fluctuations.
Conclusion: This study confirms the significance of L898V variant on the functionality of PEX6 that resulted in a severe disease phenotype. Genetic counseling, followed by multidisciplinary clinical evaluation was required to manage the patient with peroxisomal biogenesis disorder. A phytanic acid-restricted diet may be beneficial to control the infantile refsum disease severity.
Abbreviations:
IMAK, Immunotherapy by intentionally Mismatched Activated Killer cells; MRD, minimal residual disease; NK cells, natural killer cells; NKT cells, natural killer T cells; MDR, multi-drug-resistant cancer cells; SCT, allogeneic stem cell transplantation; DLI, donor lymphocyte infusion; GVHD, graft-vs-host disease; GvL, graft-versus-leukemia effects; RIC, reduced intensity conditioning; NST, non-myeloablative stem cell transplantation; ADCC, antibody-dependent cell-mediated cytotoxicity.
Introduction
Immunotherapy represents a most promising approach for control of hematologic malignancies otherwise resistant to available anti-cancer modalities. One of the most effective immunotherapy approaches available for treatment of patients with advanced and resistant hematologic malignancies may be accomplished by allogeneic stem cell transplantation (SCT) using fully matched related or haploidentical donor or fully matched unrelated donor following myeloablative or even non-myeloablative stem cell transplantation (NST) or reduced intensity conditioning (RIC).¹² The role of graft versus leukemia (GvL) as one of the most effective immunotherapy procedures for treatment of drug-resistant hematologic malignancies was supported by comparing the superior disease-free survival of patients undergoing similar myeloablative conditioning prior to syngeneic stem cells transplantation with no risk of graft-vs-host disease (GVHD).³ Also, higher relapse rates and improved disease-free survival were always observed among patients undergoing successful allogeneic SCT with no evidence of acute or chronic GVHD in comparison with patients with evidence of GVHD. Recognized that much of the therapeutic effects accomplished by SCT are induced primarily by (GvL) mediated by alloreactive donor lymphocytes, we could demonstrate that the hazardous myeloablative SCT could be replaced by much safer and better tolerated non-myeloablative stem cell transplantation (NST) or reduced intensity conditioning (RIC), confirming that the main role of engraftment of donor stem cells was induction of transplantation tolerance or unresponsiveness to donor’s alloantigens, thus allowing durable engraftment of donor’s alloreactive lymphocytes.¹²
Unfortunately, GvL effects induced following both NST or RIC were still accompanied by risky unavoidable acute and chronic graft-vs-host disease (GVHD) that could not be completely prevented despite the use of optimal immunosuppression anti-GVHD prophylaxis. Moreover, post grafting immunosuppressive treatment mandatory for prevention or treatment of GVHD also impaired the intensity and clinical efficacy of the anticipated GvL effects for treatment of resistant leukemia and higher incidence of leukemia relapse was observed when higher doses of cyclosporine A was used after allogeneic SCT.⁴ On the other hand, T cell depletion, the only effective procedure for consistent prevention of GVHD and the need for post grafting anti-GVHD prophylaxis is associated with both increased incidence of relapse and allograft rejection, confirming the important role of alloreactive T cells for elimination of residual malignant cells on the one hand, and residual patient’s hematopoietic stem cells on the other.⁵ Accordingly, the use of T cell depletion for prevention of GVHD had to be supported by more intensive immunosuppression conditioning.⁶⁷ We have pioneered the use of donor lymphocyte infusion (DLI) to maximize the GvL effects after allogeneic SCT and documented that GvL effects could be maximized by over activation of donor lymphocytes with interleukin 2 (IL-2).⁸⁹ More recently we have also documented that post-grafting DLI could be used for prevention of relapse in patients with high-risk disease.¹⁰ Much more disturbing was the fact that recurrent disease could occur despite development of severe acute and/or chronic GVHD and sometimes even after successful DLI, suggesting that the therapeutic potential of durable engraftment and maximally activated HLA compatible donor lymphocytes activated by GVHD may not be sufficient for complete elimination of all drug-resistant malignant cells and the much more resistant cancer stem cells even following successful SCT.
animal models²⁷,²⁸ and in several patients treated successfully with T cell depleted IL-2 activated CD56-positive NK cells that no GVHD was observed even among patients that were treated with activated NK and NKT cells following allogeneic SCT followed by durable engraftment of donor lymphocytes (unpublished observations).
Encouraged by the cure of the first patient that was successfully treated with IMAK, additional investigations were carried out to confirm feasibility, safety and potential efficacy of IMAK in a total of 40 consenting patients with very advanced metastatic solid tumors with heavy tumor burden.¹⁴ This pilot clinical trial confirmed that the conditioning was reasonable safe and that administration of fully mismatched killer cells could be safely accomplished with no clinically overt acute or chronic GVHD.¹⁴ Mild immunosuppressive conditioning administered prior to treatment with IMAK was intended to maintain the cytotoxic activity of infused donor killer cells, control regulatory T cells and facilitate homeostatic proliferation, and also to activate patient’s immune system to facilitate early rejection of mismatched donor lymphocytes. IMAK treatment was reasonably well tolerated with different WHO grade 1 to 3 adverse effects but no grade 4 toxicity was observed. Only 2 patients developed mild, self-limited skin rash possibly compatible with acute grade 1 GVHD.¹⁴
Our successful preliminary study that confirmed the feasibility and safety of IMAK in 40 patients with most advanced metastatic solid tumors with heavy tumor burden with only 4 with hematologic malignancies encouraged us to consider the use of IMAK for additional patients with different hematologic malignancies considered otherwise incurable. Accordingly, the next cohort of 33 patients with drug-resistant hematologic malignancies (14 with ALL, 9 with non-Hodgkin lymphoma, 8 with AML and 2 with multiple myeloma) were treated with IMAK on compassionate basis.¹⁷ The purpose of the immunosuppressive but non-myeloablative conditioning applied prior to infusion of killer cells was to combine several goals: (1) reduction of the number of host regulatory T cells; (2) optimize homeostatic proliferation of donor lymphocytes by establishing a “niche” for newly infused donor-derived killer cells; (3) eliminate unresponsiveness of host’s T cells and facilitate de novo proliferation of newly derived host T cells. Low dose IL-2 administration subcutaneously starting with cell infusion was accomplished as suggested by the preliminary investigations for continuous activation of circulating donor lymphocytes on the one hand and possibly also for activation of patient’s own T cells in order to facilitate early rejection of mismatched killer cells. Selective targeting of IMAK was accomplished in about half of the patients with CD20-positive malignant B cells, including ALL and NHL, but the number of patients treated with anti-CD20 monoclonal antibodies is too small for driving any meaningful conclusions about the role of targeting IMAK for treatment of patients with malignant B cells.
Overall, the protocol was reasonably well tolerated with no >grade 3 toxicity and no GVHD-like toxicity. Complete remission was accomplished in 22 patients (23 if the first one is included) and 6 observed for more than 5 years with no additional treatment could be considered cured.¹⁷ Other patients were not yet observed for >5 years at the time of reporting so theoretically, the cure rate may even be higher.
controlling the timing of circulation of non-engrafting donor lymphocytes, GVHD could be easily prevented by selective T cell depletion or positive selection of CD56-positive NK cells. Future confirmation of our preliminary results that could be further improved by maximizing cytokine-induced activation of donor killer cells combined with selective targeting donor’s killer cells against patient’s malignant cells by relevant monoclonal antibodies may represent an optimal immunotherapy procedure that should be considered for treatment of all patients with high-risk hematologic malignancies, preferably at an early stage of the disease following successful response to conventional chemotherapy. IMAK application against low tumor burden in patients with good performance status will certainly increase the option for cure of patients considered otherwise incurable. Since most patients with all types of hematologic malignancies respond initially to conventional chemotherapy, clinical application of IMAK at the stage of low tumor burden, ideally at the stage of MRD, could represent the optimal if not the only timing to accomplish cure.
In conclusion, cell-mediated immunotherapy based on the use of cytokine pre-activated intentionally mismatched killer cells including a mixture of T cells, NK cells and NKT cells could provide a most effective approach for immunotherapy of multi-drug-resistant hematologic malignancies. When applied against a low residual tumor load even one course of treatment could result in elimination of all drug-resistant malignant cells in patients considered otherwise incurable. Future prospective clinical trials should be recommended to confirm the safety and clinical efficacy of immunotherapy based on cytokine-induced activation of non-engrafting short circulation of mismatched killer cells. Whereas one treatment cycle may be sufficient for successful treatment of minimal residual disease, repeated treatment cycles may be indicated for treatment of patients with heavier tumor burden. Targeting pre-activated mismatched universal killer cells for selective anti-cancer immunotherapy using monoclonal antibodies against over-expressed cell-surface antigens may further optimize anti-cancer cytotoxicity and minimize the potential risks of GVHD for treatment of patients with otherwise incurable hematologic malignancies and possibly also for treatment of patients with drug-resistant metastatic solid tumors.³⁰,³¹
Acknowledgments:
I would like to acknowledge the devoted trust of all consenting patients and their supporting family member that agreed to consider treatment using IMAK, a new treatment that was never applied clinically before. In addition, I would like to thank the devoted assistance and support of the physicians, the nurses and technicians of my former Department of Stem Cell Transplantation & Cancer Immunotherapy at the Hadassah Medical Center in Jerusalem, and later on at the International Center for Cell Therapy & Cancer Immunotherapy at the Biotherapy International Clinic in Tel Aviv.
My special thanks to the generous support of Mrs. Margarita Louis Dreyfus in Honor of her late Mr. Robert Louis Dreyfus that made it possible to continue basic and clinical research toward continuous development of innovative anti-cancer immunotherapy.
References
1. Slavin S, Nagler A, Naparstek E et al. Non-myeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998;91:(3):756-763.
2. Giralt S, Estey E, Albitar M et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: Harnessing graft-vs-leukemia without myeloablative therapy. Blood 1997;89(12):4531-4536.
3. Weiden PL, Sullivan KM, Fluornoy N et al. Anti-leukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation. New Engl J Med 1981;304:1529-1533.
4. Bacigalupo A, Van Lint MT et al. Increased risk of leukemia relapse with high dose cyclosporine A after allogeneic marrow transplantation for acute leukemia. Blood 1991;77(7):1423-1428.
5. Waldmann H, Polliack A, Hale G et al. Elimination of graft-versus-host disease by in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1). Lancet 1984;2:483-485.
6. Slavin S, Or R, Naparstek E, Cividalli G et al. New approaches for prevention of rejection and graft vs host disease (GVHD) in clinical bone marrow transplantation (BMT). Is J Med Sci 1986;22:264-267.
7. Slavin S, Cividalli G, Brautbar C et al. The use of CAMPATH-1 for prevention of graft vs host disease (GVHD) and total lymphoid irradiation (TLI) for abrogation of host resistance to T-cell depleted allografts. J Cell Biochem 1986;suppl 10D:235.
8. Slavin S, Naparstek E, Nagler A et al. Allogeneic cell therapy for relapsed leukemia following bone marrow transplantation with donor peripheral blood lymphocytes. Exp Hematol 1995;23:1553-1562.
9. Slavin S, Naparstek E, Nagler A et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse post allogeneic bone marrow transplantation. Blood 1996;87(6):2195-2204.
10. Naparstek E, Or R, Nagler A et al. T-cell-depleted allogeneic bone marrow transplantation for acute leukaemia using Campath-1 antibodies and post-transplant administration of donor’s peripheral blood lymphocytes for prevention of relapse. Brit J Haematol 1995;89:506-515.
11. Lim WA, June CH. The principles of engineering immune cells to treat cancer. Cell 2017;168 (4):724-740.
12. Park JH, Riviere I, Gonen M et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. The New Eng J Med 2018;8(8):958-971.
13. Ludwig H, Terpos E, an de Dong N et al. Prevention and management of adverse events during treatment with bispecific antibodies and CAR T cells in multiple myeloma: a consensus report of the European Myeloma Network. Lancet Oncol 2023;24(6):e255-e269.
14. Slavin S, Ackerstein A, Or R et al. Immunotherapy in high-risk chemotherapy-resistant patients with metastatic solid tumors and hematological malignancies using intentionally mismatched donor lymphocytes activated with rIL-2: a phase I study. Cancer Immunol Immunother. 2010;59(10):1511-1519.
15. Pugatsch T, Oppenheim A, Slavin S. Improved single-step PCR assay for sex identification post-allogeneic sex-mismatched BMT. Bone Marrow Transplant 1996;17:273-275.
16. Slavin S. Allogeneic cell-mediated immunotherapy at the stage of minimal residual disease following high-dose chemotherapy supported by autologous stem cell transplantation. Acta Haematologica. 2005;114:214-220.
17. Slavin S. Immunotherapy with cure potential of multi-drug resistant hematologic malignancies using IL-2 preactivated intentionally mismatched donor lymphocytes J Cancer Res Clin Oncol. 2023;149(11):9277-9284.
18. Weiden PL, Fluornoy N, Sanders JE et al. Anti-leukemic effect of graft-versus-host disease contributes to /improved survival after allogeneic marrow transplantation. Transplantation 1981;13: 248-251.
19. Sullivan KM, Weiden PL, Storb R et al. Influence of acute and chronic graft-versus-host disease on relapse and survival after bone marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood 1989;73:1720-1726.
20. Horowitz M, Gale RP, Sondel PM et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990;75:555-562.
21. Higano CS, Brixey M, Bryant EM. Durable complete remission of acute non-lymphocytic leukemia associated with discontinuation of immunosuppression following relapse after allogeneic bone marrow transplantation. A case report of a probable graft-versus-leukemia effect. Transfusion 1990;50:175-178.
22. Kolb HJ, Mittermuller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990;76:2462-2465.
23. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients: European Group for blood and marrow transplantation. Blood 1995;86:2041-2050.
24. Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997;15:433-444.
25. Porter DL, Roth MS, McGarigle C et al. Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia. New Engl J Med 1994;330:100-106.
26. Mackinnon S, Papadopoulos EB, Carabassi MH et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 1995;86:1261-1268.
27. Weiss L, Weigensberg M, Morecki S et al. Characterization of effector cells of graft vs leukemia (GVL) following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia (BCL1). Cancer Immunol Immunother 1990;31:236-242.
28. Weiss L, Lubin I, Factorowich Y et al. Effective graft vs leukemia effects independent of graft vs host disease after T-Cell depleted allogeneic bone marrow transplantation in a murine model of B Cell leukemia/lymphoma. Role of cell therapy and rIL-2. J Immunol 1994;153(6):2562-2567.
29. Ljunggren HG, Karre K. In search of the “missing self’:MHC molecules and NK cell recognition. Immunology Today 1990;11:237-244.
30. Slavin S. Immunotherapy of cancer with alloreactive lymphocytes. Lancet Oncology 2001; 2:491-498.
31. Slavin S. Cancer immunotherapy with alloreactive lymphocytes. New Engl J Med 2000:343(11);802-803.