PD-1(hi)TIM-3(+) T cells associate with and predict leukemia relapse in AML patients post allogeneic stem cell transplantation.

Prognosis of leukemia relapse post allogeneic stem cell transplantation (alloSCT) is poor and effective new treatments are urgently needed. T cells are pivotal in eradicating leukemia through a graft versus leukemia (GVL) effect and leukemia relapse is considered a failure of GVL. T-cell exhaustion is a state of T-cell dysfunction mediated by inhibitory molecules including programmed cell death protein 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3). To evaluate whether T-cell exhaustion and inhibitory pathways are involved in leukemia relapse post alloSCT, we performed phenotypic and functional studies on T cells from peripheral blood of acute myeloid leukemia patients receiving alloSCT. Here we report that PD-1(hi)TIM-3(+) cells are strongly associated with leukemia relapse post transplantation. Consistent with exhaustion, PD-1(hi)TIM-3(+) T cells are functionally deficient manifested by reduced production of interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In addition, these cells demonstrate a phenotype consistent with exhausted antigen-experienced T cells by losing TN and TEMRA subsets. Importantly, increase of PD-1(hi)TIM-3(+) cells occurs before clinical diagnosis of leukemia relapse, suggesting their predictive value. Results of our study provide an early diagnostic approach and a therapeutic target for leukemia relapse post transplantation.

Spontaneous gingival bleeding in an otherwise asymptomatic patient.

This case is presented to challenge the reader to formulate a differential diagnosis for a patient who visits the dentist with spontaneous, continuous gingival bleeding. When this situation occurs, it is serious and requires immediate attention and a specific treatment plan to arrive at the underlying diagnosis and control the bleeding. The signs and symptoms of a patient with gingival bleeding are presented for diagnosis; the history and management are detailed, and may be useful in diagnosing and treating similar patients.

Tetraploidy in acute myeloid leukemia secondary to large cell lymphoma.

A 67 year old male developed a therapy related myelodysplastic process culminating in acute myeloid leukemia 16 years following initial treatment for a large cell lymphoma. A second relapse of this leukemia showed 12% blasts including numerous giant blasts. The presence of giant blasts suggested the possibility of relapsed malignant lymphoma, however, flow cytometry and immunohistochemistry identified them as myeloid and chromosomal analysis revealed a near-tetraploid cell line. No evidence of lymphoma was seen. Although remission was induced with chemotherapy he subsequently relapsed with marrow and/or CNS involvement and was maintained on palliative therapy until he developed sepsis and died, 13 months following the observation of tetraploidy and 33 months following the onset of acute leukemia.

Allogeneic peripheral blood stem cell transplant for myelodysplasia after chemotherapy for post-transplant lymphoma in a cardiac transplant recipient at 10 years.

A 32-year-old male received an allogeneic peripheral blood stem cell transplant (alloPBSCT) for myelodysplasia from his one HLA-A antigen mismatched brother. He is alive with trilineage engraftment and without active GVHD 200 days after transplant. In July 1986 he underwent orthotopic cardiac transplantation for viral cardiomyopathy and has received continuous immunosuppressive therapy. A post-transplant lymphoproliferative disorder with Hodgkin-like histopathology was diagnosed in August 1993 and was successfully treated with four cycles of MOPP chemotherapy. Due to persistent pancytopenia he underwent a bone marrow aspiration and biopsy in May 1996 which revealed monosomy 7 and morphologic changes compatible with myelodysplasia. This is the first report of a cardiac transplant recipient receiving an allogeneic hematopoietic stem cell transplant.

Relationship of CD34+ cell dose to early and late hematopoiesis following autologous peripheral blood stem cell transplantation.

We evaluated early and late hematopoietic reconstitution in 27 patients with advanced lymphoma, Hodgkin's disease, and breast or ovarian cancer after treatment using high-dose/myeloablative conditioning regimens and autologous peripheral blood stem cell PBSC) transplantation. Eighteen patients (67%) received G-CSF 5 micrograms/kg/day following chemotherapy and nine (33%) were mobilized using G-CSF alone. Each patient had 7 x 10(8) mononuclear cells (MNC) per kg collected. G-CSF was administered post-PBSC infusion. While all patients showed prompt granulocyte recovery by day 14, platelet recovery failed to occur in our (15%) heavily pretreated patients with non-Hodgkin's lymphoma. Retrospective analysis in 17 patients revealed that the infused number of CD34 surface antigen-positive cells correlated with time to granulocyte (r = 0.59, P = 0.012) and platelet (r = 0.58, P = 0.021) recovery. Patients receiving the higher numbers of CD34+ cells had consistently better hematologic parameters at 11 times examined. At 180 days post-transplant, the median Hb level was 124 g/l vs 88 g/l (P = 0.004); platelet count was 202 x 10(9)/l vs 25 x 10(9)/l (P = 0.004); and neutrophil count was 3100 x 10(6)/l vs 1400 x 10(6)/l (P = 0.15). Hemoglobin strongly correlated with the CD34+ cell dose at 360 days (r = 0.90, P = 0.01). We conclude that graft CD34+ cell content appears to be an indicator of the quality of late as well as early hematopoietic function.

Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation.

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.

High-dose cyclophosphamide, carboplatin, and etoposide with autologous stem cell rescue in patients with breast cancer.

This study was designed to establish the toxicity and response rates o observed with a combination of high-dose cyclophosphamide, carboplatin, and etoposide with stem cell rescue in patients with breast carcinoma. Eligibility criteria included metastatic or locally advanced breast carcinoma ; aged < or equal to 60 years; performance status Eastern Cooperative Oncology Group (ECOG) 0-1; and creatinine clearance > or equal to 65 ml/min. Chemotherapy consisted of cyclophosphamide 25 mg/kg i.v. X 4 days, etoposide 400 mg/m(2) i.v. X 4 days, and carboplatin 375 mg/m(2) X 4 days. Bone marrow or peripheral blood stem cells were reinfused 48 h after completion of chemotherapy. Seventeen patients were treated in this study. The major toxicity was gastrointestinal (grades I and II). Fevers associated with neutropenia were observed in all the patients, but no episodes of bacteremia were documented. Hematopoietic toxicities were acceptable. No toxic deaths were observed. Six patients had chemotherapy-sensitive disease at time of transplant, nine had refractory disease, and two were untested. A response rate of 62% with 18% complete response (CR) was achieved. Two patients are free of disease at +7 and +9 months after transplantation. The combination of high-dose cyclophosphamide, carboplatin, and etoposide is well tolerated with a response rate comparable to previously reported high-dose chemotherapy regimens. However, in a poor prognostic risk group, namely patients with chemoinsensitive disease, this therapeutic approach seems to be of no advantage over standard chemotherapy.

Toxicity of busulfan and cyclophosphamide (BU/CY2) in patients with hematologic malignancies.

Between July 1991 and January 1994, 52 patients with hematologic malignancies underwent BMT using BU/CY2 as conditioning regimen. Median patient age was 38 years. Eleven patients underwent autologous BMT, 22 HLA-identical allogeneic BMT, and 19 patients underwent a MUD or an allogeneic mismatched BMT. GVHD prophylaxis was with cyclosporine/methylprednisone in 26 patients; T cell depletion was used in 15 patients. VOD was observed in 7.5% of patients, IP in 12%, seizures in 4%. The overall incidence of grade II-IV acute GVHD was 35%. Delayed platelet engraftment was observed in seven of 11 patients who underwent autologous BMT. Graft failure was seen in seven of 19 (37%) patients who underwent MUD or allogeneic mismatched BMT. Six of the seven patients received T cell depletion as GVHD prophylaxis. BU/CY2 transplantation from an unrelated or family-mismatched donor with T cell depletion is associated with a high incidence of graft failure.

Late graft failure due to dual bone marrow infection with variants A and B of human herpesvirus-6.

Hematopoietic effects of human Herpesvirus-6 (HHV-6) infection following bone marrow transplantation (BMT) include delayed engraftment and early myelosuppression. Variant A has not been isolated after BMT. A case of graft failure is reported following an HLA-identical BMT for chronic myelogenous leukemia (CML) in chronic phase. Evaluation of bone marrow during the period of graft failure revealed variants A and B of HHV-6 by culture, immunofluorescence, polymerase chain reaction (PCR), and immunohistochemistry. Evidence for other cases of graft failure, including cytomegalovirus (CMV), could not be found. A hypothesis is proposed that late graft failure in this case was due to variant A of HHV-6.

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period.

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.

Hematopoietic progenitor cell content of vertebral body marrow used for combined solid organ and bone marrow transplantation.

While cadaveric vertebral bodies (VB) have long been proposed as a suitable source of bone marrow (BM) for transplantation (BMT), they have rarely been used for this purpose. We have infused VB BM immediately following whole organ (WO) transplantation to augment donor cell chimerism. We quantified the hematopoietic progenitor cell (HPC) content of VB BM as well as BM obtained from the iliac crests (IC) of normal allogenic donors (ALLO) and from patients with malignancy undergoing autologous marrow harvest (AUTO). Patients undergoing WO/BM transplantation also had AUTO BM harvested in the event that subsequent lymphohematopoietic reconstitution was required. Twenty-four VB BM, 24 IC BM-ALLO, 31 IC AUTO, and 24 IC WO-AUTO were harvested. VB BM was tested 12 to 72 hr after procurement and infused after completion of WO grafting. IC BM was tested and then used or cryopreserved immediately. HPC were quantified by clonal assay measuring CFU-GM, BFU-E, and CFU-GEMM, and by flow cytometry for CD34+ progenitor cells. On an average, 9 VB were processed during each harvest, and despite an extended processing time the number of viable nucleated cells obtained was significantly higher than that from IC. Furthermore, by HPC content, VB BM was equivalent to IC BM, which is routinely used for BMT. We conclude that VB BM is a clinically valuable source of BM for allogeneic transplantation.

A unique population of CD34+ cells in cord blood.

Human umbilical cord blood (CB) is a rich source of hematopoietic stem cells for both research and stem cell transplantation. In clinical studies, it appears that recovery from myeloablative therapy using CB requires significantly fewer cells than a typical allogeneic marrow transplant. This suggests that CB may be enriched for early hematopoietic progenitors. The present studies were undertaken to determine the presence of CD34+ cells in CB with the phenotypic characteristics of multipotential stem cells. In 22 CB harvests, the average percentage of CD34+ cells was 1.33 +/- 0.21% (SE), a value similar to that in adult normal bone marrows (BM). However, the distribution of CD34+ cells was distinctly different from either BM or granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell harvests. CB contained a defined population of brightly staining CD34+ cells with low side scatter. These CD34 (bright) cells comprised a mean of 14.5 +/- 2.5% of the CB CD34+ cells, whereas < 1% of BM CD34+ cells has been shown to be CD34- bright. Eighty-five to ninety percent were negative for three antigens expressed at an early stage of stem cell maturation: CD38, HLA-DR and LFA-1. Fifty-five percent of these CD34 (bright) cells did not express the CD45RA isoform, an additional marker of immaturity. The antigen-bright cells also lacked lineage-specific antigens including CD33, CD56, CD19, CD10 and CD7 as well as CD71. Approximately 46% were Thy-1+, and 40% expressed c-kit receptors. These data suggest that, by phenotypic criteria, CB may be a particularly enriched source of primitive hematopoietic precursors.

Reversible cyclosporine-induced cortical blindness in allogeneic bone marrow transplant recipients.

We report the occurrence of reversible cyclosporine-induced cortical blindness in three allogeneic bone marrow transplant recipients. Possible mechanisms involved in this rare complication, as well as the associated radiographic and pathologic findings, are discussed.

Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy.

The CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC-labeled anti-CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE-labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < 6 microns in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG-1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+lin- cells. Duplicate samples varied by < 10%. Initial studies indicate that this procedure can be reliably used to measure CD34+lin- cells in blood, pheresis products, and bone marrow harvests. This CD34 enumeration procedure should result in increased consistency in enumerating this stem cell population.