Purging in autologous and allogeneic bone marrow transplantation.

The success of hematoablative dosages of cytotoxic therapy followed by stem cell transplantation depends on the disease response to the intensive drug dose and the ability of the transplant to provide durable hematopoietic reconstitution. In autologous stem cell transplantation, patients' own bone marrow or peripheral blood is used for hematopoietic engraftment. In several diseases the bone marrow is involved, and in such situations, the success of autologous stem cell transplantation may improve if the hematopoietic elements are enriched (positive selection) or if the contaminating cancer cells are purged by negative selection. In patients undergoing allogeneic bone marrow transplantation, the T lymphocytes of the donor cells contribute to the development of graft-versus-host disease with significant morbidity and mortality. Selective purging of T lymphocytes has dramatically decreased the incidence of graft-versus-host disease. In this paper, various methods for purging hematopoietic stem cells are detailed with emphasis on treatment planning. Exciting new possibilities include growing stem cells from a small amount of bone marrow, enhancing immune surveillance, and decreasing drug resistance by genetic engineering.     

Transplantation of highly purified peripheral blood CD34+ cells from HLA-mismatched parental donors in 14 children: evaluation of early monitoring of engraftment.

HLA-mismatched family members may represent an important cell source for patients that require stem cell transplantation but lack both a matched sibling donor and a closely matched unrelated donor. We report the outcome of 19 transplantations from HLA two- or three- loci mismatched parental donors in which 14 pediatric patients with hematological malignancies or other disorders, received a median of 21.5 x 106 (range, 5.4-58) highly purified CD34+peripheral blood stem cells (PBSC), as well as 4.7 x 104 (range, 0.4-12) donor T cells per kg body weight. T cell depletion was performed using a two-step CD34-positive selection on two different magnetic beads devices. Ten of 14 patients presented with rapid myeloid engraftment. The four patients who presented with graft failure (two non-engraftments, two rejections) received a second stem cell graft and one a third. Graft rejection was detected early by polymerase chain reaction (PCR) analysis of FACS-sorted T cells. Eight of the 14 patients are still alive after a median observation period of 15. 6 months (range, 3-31.3) with full donor chimerism in all hematopoietic cell lineages. No acute organ graft-versus-host disease (GVHD) and no chronic GVHD have occurred. One patient experienced relapse of leukemia. We conclude that transplantation of allogeneic PBSC from haploidentical donors will open new perspectives for pediatric patients for whom an HLA-matched stem cell graft is not available. Close monitoring of recipient and donor hematopoiesis might be of clinical value, to recognize early engraftment or rejection.     

Haploidentical Transplantation for Leukemia

Hematopoietic stem cell transplantation from human leukocyte antigen (HLA)-haploidentical family members offers a potential cure for patients in need of allogeneic immunotherapy who have no immediate access to an HLA-matched donor. The use of ex vivo T-cell–depleted stem cells combined with immuno-myeloablative conditioning has enabled durable donor engraftment with a low incidence of acute graft-versus-host disease despite the HLA disparity. Moreover, additional transplant techniques involving in vivo T-cell depletion and reduced-intensity conditioning have further minimized the risks. However, a major drawback is delayed immune reconstitution leading to infections and high relapse rates, prompting significant research efforts focused on improving recovery in the post-transplant period. Infusions with donor lymphocytes are common, though newer manipulations with a focus on donor natural killer cells hold great promise, as do other modified donor T-cell infusions. Success of these new procedures will make haploidentical transplants safer and more effective, further broadening its appeal.     

Natural killer cell alloreactivity in allogeneic hematopoietic transplantation

PURPOSE OF REVIEW: This review will focus on the translation of natural killer cell recognition of missing self into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia. Allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen-matched sibling can cure leukemia but 75% of patients do not have a matched donor, one alternative source of stem cells includes full haplotype mismatched family members. As haploidentical transplantation must be extensively T cell depleted to prevent lethal graft-versus-host disease, it cannot rely on donor T cells for the graft-versus-leukemia effect. Mismatched transplantation, however, triggers alloreactivity mediated by natural killer cells which is based upon 'missing self recognition'. RECENT STUDIES: Recent studies using preclinical murine models of haploidentical transplantation demonstrated that conditioning with alloreactive natural killer cells ablates the recipient immune system and leukemia cells. In the clinical setting of mismatched hematopoietic stem cell transplantation, donor versus recipient natural killer cell alloreactivity has been associated with better outcome, particularly in patients with acute myeloid leukemia who are transplanted in remission. SUMMARY: Given the benefits of natural killer cell alloreactivity, it is expected that it will encourage greater use of haploidentical transplants for the large numbers of leukemia patients without matched donors.     

异基因造血干细胞移植相关研究进展

近年来,单倍体相合造血干细胞移植的发展使“人人都有移植供者”成为现实.因此,选择合适的供者以及解决移植相关并发症,如促进植入、降低移植后移植物抗宿主病的发生率和复发率成为改善移植预后的关键问题.文章介绍了异基因造血干细胞移植的相关研究进展.     

Clinical Studies in Hematologic Microtransplantation

The anti-tumor effects of allogeneic hematopoietic stem cell transplantation depend upon engraftment of donor cells followed by a graft-versus-tumor (GVT) effect. However, pre-clinical and clinical studies have established that under certain circumstances, anti-tumor responses can occur despite the absence of high levels of durable donor cell engraftment. Tumor response with little or no donor engraftment has been termed “microtransplantation.” It has been hard to define conditions leading to tumor responses without donor cell persistence in humans because the degree of engraftment depends very heavily upon many patient-specific factors, including immune status and degree of prior therapy. Likewise, it is unknown to what degree donor chimerism in the blood or tissue is required for an anti-tumor effect under conditions of microtransplantation. In this review, we summarize some key studies supporting the concept of microtransplantation and emphasize the importance of recent large studies of microtransplantation in patients with acute myelogenous leukemia (AML). These AML studies provide the first evidence of the efficacy of microtransplantation as a therapeutic strategy and lay the foundation for additional pre-clinical studies and clinical trials that will refine the understanding of the mechanisms involved and guide its further development as a treatment modality.     

Allogeneic hematopoietic cell transplantation for metastatic renal cell carcinoma after nonmyeloablative conditioning: toxicity, clinical response, and immunological response to minor histocompatibility antigens.

PURPOSE: This phase I trial assessed the safety, efficacy, and immunologic responses to minor histocompatibility antigens following nonmyeloablative allogeneic hematopoietic cell transplantation as treatment for metastatic renal cell carcinoma. EXPERIMENTAL DESIGN: Eight patients received conditioning with fludarabine and low-dose total body irradiation followed by hematopoietic cell transplantation from an HLA-matched sibling donor. Cyclosporine and mycophenolate mofetil were administered as posttransplant immunosuppression. Patients were monitored for donor engraftment of myeloid and lymphoid cells, for clinical response by serial imaging, and for immunologic response by in vitro isolation of donor-derived CD8(+) CTLs recognizing recipient minor histocompatibility (H) antigens. RESULTS: All patients achieved initial mixed hematopoietic chimerism with two patients rejecting their graft and recovering host hematopoiesis. Four patients developed acute, grade 2 to 3, graft-versus-host disease and four patients developed extensive chronic graft-versus-host disease. Five patients had progressive disease, two patients had stable disease, and one patient experienced a partial response after receiving donor lymphocyte infusions and IFN-alpha. CD8(+) CTL clones recognizing minor H antigens were isolated from five patients studied. Clones from three patients with a partial response or stable disease recognized antigens expressed on renal cell carcinoma tumor cells. CONCLUSIONS: Treatment of metastatic renal cell carcinoma with allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning with fludarabine/total body irradiation is feasible and may induce tumor regression or stabilization in some patients. CD8(+) CTL-recognizing minor H antigens on tumor cells can be isolated posttransplant and could contribute to the graft-versus-tumor effect. Such antigens may represent therapeutic targets for posttransplant vaccination or adoptive T-cell therapy to augment the antitumor effects of allogeneic hematopoietic cell transplantation.     

Allogeneic stem cell transplantation for renal cell carcinoma

Allogeneic hematopoietic stem cell transplantation from a compatible donor has been utilized as adoptive immunotherapy in metastatic, cytokine-refractory renal cell carcinoma (RCC). Since the year 2000, several investigators have established that RCC is susceptible to a graft-versus-tumor effect: they reported that patients with renal cancer may have partial or complete disease responses, in the 20-40% range, after allogeneic transplantation following a reduced-intensity regimen. However, transplant-related mortality is still high in the 10-20% range, and responses are rarely durable. Experimental evidence suggests that donor-derived T cells and natural killer cells are the main mediators of the graft-versus-RCC effect upon allogeneic hematopoietic stem-cell transplantation. Isolation of CD8(+) cytotoxic T lymphocyte clones recognizing several target antigens of graft-versus-RCC effect (minor histocompatibility antigens on RCC cells; a peptide epitope derived from human endogenous retrovirus type E; the tumor-associated antigen encoded by the Wilms' tumor 1 gene) has increased our knowledge of the disease and has opened up the possibility of antigen-specific adoptive cell therapy. The introduction in the clinic of molecularly targeted agents that interfere with neoangiogenesis, both monoclonal antibodies and small tyrosine-kinase inhibitor molecules (e.g., sunitinib, sorafenib and bevacizumab), has decreased the use of allogeneic transplantation. Although not curative, novel targeted agents may be combined with allogeneic transplantation or with adoptive cell therapy in order to maximize the chances of cure.     

Allogeneic stem cell transplantation for renal cell carcinoma

Allogeneic hematopoietic stem cell transplantation from a compatible donor has been utilized as adoptive immunotherapy in metastatic, cytokine-refractory renal cell carcinoma (RCC). Since the year 2000, several investigators have established that RCC is susceptible to a graft-versus-tumor effect: they reported that patients with renal cancer may have partial or complete disease responses, in the 20–40% range, after allogeneic transplantation following a reduced-intensity regimen. However, transplant-related mortality is still high in the 10–20% range, and responses are rarely durable. Experimental evidence suggests that donor-derived T cells and natural killer cells are the main mediators of the graft-versus-RCC effect upon allogeneic hematopoietic stem-cell transplantation. Isolation of CD8⁺ cytotoxic T lymphocyte clones recognizing several target antigens of graft-versus-RCC effect (minor histocompatibility antigens on RCC cells; a peptide epitope derived from human endogenous retrovirus type E; the tumor-associated antigen encoded by the Wilms’ tumor 1 gene) has increased our knowledge of the disease and has opened up the possibility of antigen-specific adoptive cell therapy. The introduction in the clinic of molecularly targeted agents that interfere with neoangiogenesis, both monoclonal antibodies and small tyrosine-kinase inhibitor molecules (e.g., sunitinib, sorafenib and bevacizumab), has decreased the use of allogeneic transplantation. Although not curative, novel targeted agents may be combined with allogeneic transplantation or with adoptive cell therapy in order to maximize the chances of cure.     

Nonmyeloablative stem cell transplantation for chronic myeloid leukemia

Conventional myeloablative hematopoietic stem cell transplantation carries risks of morbidity and mortality from regimen-related toxicities that have restricted its use to relatively young patients in good medical condition. In nonmyeloablative allogeneic hematopoietic stem cell transplantation, enhanced immunosuppression rather than myeloablative cytotoxic conditioning has allowed the engraftment of allogeneic stem cells, with lower early transplant related mortality and morbidity. This approach shifts tumor eradication to the graft-versus-leukemia effect mediated by donor T lymphocytes. The development of nonmyeloablative transplantation has allowed the application of a potentially curative procedure to elderly or medically infirm patients who would not be able to tolerate high-dose conditioning regimens.     

Generation of cytotoxic donor CD8+ T cells against relapsing leukemic cells following allogeneic transplantation by stimulation with leukemic cell- or leukemic lysate pulsed donor cell-derived dendritic cells

To treat leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the possibility of immunotherapy using donor CD8+ T cells that were generated by stimulating leukemic cell-derived dendritic cells (leukemic-DCs) or leukemic cell lysate pulsed donor cell-derived DCs (donor-DCs). Leukemic- and donor-DCs were generated from mononuclear cells of patients and CD14+ cells of HLA-matched donors, respectively. The expression of CD80, CD83, CD86, CD1a, and CD40 on leukemic-DCs was significantly lower than that on donor-DCs. Donor-DCs exhibited a higher capacity to stimulate allogeneic T cells compared with leukemic-DCs. Donor CD8+ T cells stimulated by leukemic- or donor-DCs were more cytotoxic than unprimed CD8+ T cells, and slightly higher cytotoxicity was observed with donor-DCs compared to leukemic-DCs. This study indicates that leukemic- or donor-DCs pulsed with leukemic cell lysates can effectively prime donor cytotoxic T cells in vitro, and that they may be used as a potential alternative tool for treating leukemic patients who relapse after allogeneic HSCT.     

异基因造血干细胞移植治疗首例自体移植复发霍奇金淋巴瘤的临床分析

目的探讨霍奇金淋巴瘤自体移植复发后行异基因造血干细胞2次移植的可能性和安全性。方法对1例10年前行自体造血干细胞移植复发的霍奇金淋巴瘤患者,行异基因造血干细胞移植,供者为患者母亲,采用外周血干细胞移植,预处理方案采用氟达拉滨+马法兰+兔抗人淋巴细胞免疫球蛋白,预防移植物抗宿主病采用环孢素A、霉酚酸酯、甲氨蝶呤,输注单个核细胞数14.03×108/kg,CD34+细胞6.57×106/kg。结果 2次移植后移植物成功植入,形成完全供者来源造血,移植后第20天骨髓初步植活,造血功能恢复后患者出现皮肤植物抗宿主病,FISH嵌合状态供者细胞植入率为100%,随访至今一直长期无病生存。结论异基因造血干细胞移植,可有效治疗自体移植复发的霍奇金淋巴瘤,是安全有效的挽救治疗措施。     

The role of total body irradiation in the conditioning of patients receiving haploidentical stem cell transplantation

BACKGROUND: Nearly 40% of patients requiring a hematopoietic stem cell transplant lack a suitable donor. However, virtually all these patients have a potential family donor with whom they share one HLA haplotype. METHODS: We report the rationale for making hematopoietic stem cell transplantation from haploidentical related donors feasible, as well as the method followed to achieve this. Two studies are reported, designed to overcome the problem of rejection and graft-versus-host disease after haploidentical stem cell transplantation. We describe how our total body irradiation-based, highly immuno- and myelosuppressive conditioning regimens were developed and how they have been modified over the years in an attempt to improve the clinical outcome of high-risk acute leukemia patients receiving large numbers of extensively T-cell-depleted hematopoietic stem cell transplantations from full-haplotype mismatched family donors. RESULTS: A high engraftment rate and an extremely low incidence of graft-versus-host disease were obtained. Modifications of the pretransplant schedules allowed the reduction of transplant-related toxicity. CONCLUSIONS: The main obstacles that limited the use of haploidentical stem cell transplantation have been overcome. The procedure is now a reality that should be recommended in high-risk acute leukemia patients who do not have a suitable matched donor.     

单倍体造血干细胞联合脐血输注治疗白血病的临床研究

目的 分析单倍体造血干细胞联合第三方脐血输注治疗白血病的疗效及安全性.方法 对苏州大学附属第一医院2011年1月至2012年5月44例采用单倍体造血干细胞联合第三方脐血输注治疗的白血病患者进行回顾性分析,其中急性淋巴细胞白血病（ALL） 16例、急性髓系白血病（AML）24例、慢性粒细胞白血病（CML）3例、T淋巴母细胞淋巴瘤1例.结果 44例白血病患者回输的单倍体供体移植物中CD34＋细胞中位数为3.14×106/kg（1.68×106/kg ～ 8.32× 106/kg）,回输的第三方脐血中CD34＋细胞中位数为1.03×105/kg（0.31×105/kg～5.32×105/kg）.42例（95.45％）患者造血重建,中性粒细胞造血重建时间为14d（10～29 d）,血小板造血重建时间为23d（11～89d）.对42例植入成功的患者长期监测供受体嵌合率（STR）,除5例（11.90％）患者在移植后3～13个月复发,其余37例（88.10％）患者STR均≥95％.17例（40.47％）患者发生不同程度的急性移植物抗宿主病（aGVHD）,发生Ⅱ～Ⅳ度aGVHD的患者共8例（19.05％）,发生Ⅲ～Ⅳ度aGVHD的患者为4例（9.52％）.23例（54.76％）发生慢性移植物抗宿主病（cGVHD）,其中局限型17例（40.47％）,广泛型6例（14.29％）.1年总生存（OS）率为70.84％,1年无事件生存（EFS）率为63.34％.结论 初步研究证实单倍体造血于细胞联合第三方脐血输注治疗白血病安全有效,从而为异基因造血干细胞的移植提供了新的方法.     

Patient‐individualized CD8+ cytolytic T‐cell therapy effectively combats minimal residual leukemia in immunodeficient mice

Adoptive transfer of donor‐derived cytolytic T‐lymphocytes (CTL) has evolved as a promising strategy to improve graft‐versus‐leukemia (GvL) effects in allogeneic hematopoietic stem‐cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)‐reactive CD8⁺ CTL with central and effector memory phenotype in a new allogeneic donor‐patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA⁺ donor CD8⁺ T cells with either single HLA antigen‐mismatched or HLA‐matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine‐optimized short to intermediate (i.e., 2–8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg^null mice when engraftment with patient AML reached bone marrow infiltration of 1–5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA‐mismatched and strong reduction of HLA‐matched AML infiltration, respectively. Most importantly, mice receiving AML‐reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML‐reactivity ex vivo. Moreover, injections with human IL‐15 clearly promoted CTL persistence. In summary, we show that naive donor‐derived CD8⁺ CTL effectively combat patient AML blasts in immunodeficient mice. The donor‐patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML‐reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T‐cell receptors for redirecting immunity to leukemias even in a patient‐individualized manner.     

Allogeneic stem-cell transplantation of renal cell cancer after nonmyeloablative chemotherapy: feasibility, engraftment, and clinical results.

PURPOSE: To evaluate the feasibility and safety of nonmyeloablative allogeneic stem-cell transplantation in patients with metastatic renal cell cancer (RCC) and to evaluate efficacy with respect to engraftment and tumor regression. PATIENTS AND METHODS: Between February 1999 and June 2001, patients with refractory, metastatic RCC were screened for enrollment. A fludarabine and cyclophosphamide-based conditioning regimen was used. Patients received granulocyte-macrophage colony-stimulating factor-mobilized, unmanipulated stem cells from a 6/6 HLA-matched sibling donor. Prophylaxis against graft rejection and graft-versus-host disease (GVHD) included tacrolimus and mycophenolate mofetil. RESULTS: A total of 284 patients with metastatic RCC were seen during this time period. Eighty-four patients who had siblings available for HLA typing were actively screened for enrollment, and 15 patients have undergone treatment. Durable donor engraftment was achieved in one of the first four patients treated. Patients no. 5 through 15 received a more immunosuppressive conditioning regimen, and all have achieved sustained donor engraftment. In the 12 patients with at least 180 days of follow-up, acute GVHD has occurred in two patients and chronic GVHD in six patients, with four transplant-related mortalities. Four partial responses have been observed (response rate, 33% in all patients; 44% in the nine patients with sustained donor engraftment). CONCLUSION: Nonmyeloablative allogeneic stem-cell transplantation is feasible for a minority of patients with metastatic RCC. Adequately immunosuppressive conditioning is required for sustained donor engraftment, which is required for an antitumor response. Acute and chronic GVHD are the major causes of substantial morbidity and mortality. Metastatic RCC is susceptible to a graft-versus-tumor effect promoted by allogeneic stem-cell transplantation.     

Molecular persistence of chronic myeloid leukemia caused by donor T cells specific for lineage-restricted maturation antigens not recognizing immature progenitor-cells.

Although donor lymphocyte infusion (DLI) induces complete remissions in 70% of patients with relapsed chronic myeloid leukemia (CML) after allogeneic stem-cell transplantation (SCT), some patients are refractory to DLI by showing disease persistence. In a patient who received DLI for relapsed CML, we observed persisting molecular disease despite a hematological and cytogenetic remission in the absence of graft-versus-host disease (GVHD). To determine the nature of this immune response, we isolated leukemia-reactive donor T-cell clones from the bone marrow (BM) of the patient at the time of clinical response. Four different types of CD8+ HLA class I restricted T-cell clones were obtained that were cytotoxic against Ebstein-Barr virus-transformed B-cell lines (EBV-LCL) of the patient, but not the donor, indicating recognition of minor histocompatibility antigens (mHags). By using survival studies with CFSE labelled BM cells populations, a hematopoietic progenitor cell inhibition assay and direct morphological examination we showed that the T-cell clones recognized mature monocytic and myeloid cells, whereas immature BM progenitor cells were insufficiently lysed. This patient's refractoriness for DLI appears to be caused by inadequate lysis of progenitor cells by these cytotoxic T cells. These findings support the hypothesis that for eradication of CML a cytotoxic T-cell response against leukemic progenitor cells is essential.     

Long‐term follow‐up of haploidentical hematopoietic stem cell transplantation without in vitro T cell depletion for the treatment of leukemia

BACKGROUND:

Many patients who require allogeneic hematopoietic stem cell transplantation (allo‐HSCT) lack a human leukocyte antigen (HLA)‐matched donor. Recently, a new strategy was developed for HLA‐mismatched/haploidentical transplantation from family donors without in vitro T cell depletion (TCD).

METHODS:

Over the past 9 years, 756 patients underwent haploidentical transplantation using a protocol developed by the authors, which combines granulocyte‐colony stimulating factor–primed bone marrow (G‐BM) and peripheral blood stem cells without in vitro TCD. The long‐term outcome with this treatment modality was reported, and a risk‐factor analysis was provided.

RESULTS:

Of these patients, 752 (99%) achieved sustained, full donor chimerism. The incidence of grades 2 through 4 acute graft‐versus‐host disease (GVHD) was 43%, and the 2‐year cumulative incidence of total chronic GVHD was 53%. The 3‐year cumulative incidence of nonrelapse mortality was 18%. The 2‐year cumulative incidences of relapse were 15% and 26% in the standard‐risk and high‐risk groups, respectively. Of the 756 patients, 480 survived throughout the follow‐up period of 1154 days (range: 335‐3511 days) with the 3‐year leukemia‐free survival rates of 68% and 49% in the standard‐risk and high‐risk groups, respectively. Lower leukemia‐free survival was associated with high‐risk disease status (P = .001), chronic myelogenous leukemia disease type (P = .004), neutrophil engraftment beyond 13 days after transplant (P = .012), and the occurrence of grades 2 through 4 acute GVHD (P = .019).

CONCLUSIONS:

The results from the authors' 9‐year experience showed that G‐BM combined with peripheral blood stem cells from haploidentical donors, without in vitro TCD, is a reliable source of stem cells for transplantation by using the protocol developed by the authors. Cancer 2013. © 2012 American Cancer Society.