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Abstract 683

Allograft T cell depletion (TCD) reduces acute graft-vs. host disease (aGVHD) after myeloablative hematopoietic stem cell transplantation (HSCT). Lower incidences of aGVHD reported after reduced-intensity stem cell transplantation (RIST) may reflect delayed donor T cell engraftment. We compared the incidence of aGVHD following RIST with TCD (19 patients (pts) vs. T cell replete (TCR) allografts (20 pts)(Table). There was no difference in the incidence aGVHD, occurring in 71% of TCD recipients (median onset Day 47) and 70% of TCR recipients (median onset Day 25). After TCD, 100% of those who engrafted prior to any DLI developed aGVHD compared with 56% of those who engrafted after DLI, including two pts who developed “late-acute” aGVHD after Day 100, upon completion of donor T cell engraftment. T cell engraftment after TCD was uneven: engraftment kinetics were associated with residual host circulating CD8+ T cell counts after immune depletion; and aGVHD did not occur in the setting of mixed T cell chimerism (Figure). These observations demonstrate that aGVHD can occur with very low doses (105/kg) of allograft T cells after RIST. Protection from aGVHD conferred by mixed T cell chimerism may be lost with full donor T cell engraftment. With limited donor T cell numbers, host T cells appear to determine kinetics of engraftment and of aGVHD after RIST. Figure: Post-induction circulating CD8+ T cell counts in TCD recipients with delayed donor T cell engraftment. After TCD, all subjects who developed aGVHD did so at or after the establishment of T cell full donor T cell chimerism, and occasionally prior to any DLI. Shaded triangle represents the theoretical area in which values would fall if subjects developed aGVHD prior to complete donor T cell engraftment. Arrows: DLI. Patient and Transplant Characteristics and Outcomes Protocol TCD TCR Flu/Cy: Fludarabine and cyclophosphamide; EPOCH-F: Etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone and fludarabine; FDC: Full donor chimerism. Median (range) Median (range) Recipient Age 43 years (32 – 56) 44 (19 – 67) CMV Risk 14/19 16/20 Pretransplant Immune Depletion Flu/Cy EPOCH-F Conditioning Regimen Flu/Cy Flu/Cy Pre-conditioning Host Cell Counts: CD3 86 (1 – 701) 140 (21 – 441) CD4, p=0.017 44 (1 – 156) 71 (12 – 191) CD8 34 (0 – 555) 55 (2 – 309) NK 58 (0 – 376) 88 (3 – 467) Day 0 CD3 Cell Count 1 (1 – 6) 5 (0 – 42) Allograft CD34+ cells/kg 7.75 x 106 (5.1 – 12.9) 7.68 x 106 (4.6–18.4) Allograft CD3+ cells/kg 1.0 x 105 (preset) 3.63 x 108 (1.5 – 8.3) Donor T cell engraftment Day +70 (14 – 180) 14 (14 – 100) Figure Figure

Relapse of hematologic malignancy is a major problem after allogeneic hematopoietic stem cell transplantation (alloHSCT), with limited treatment options of proven benefit other than donor lymphocyte infusions (DLI). Additionally, toxicity from treatment with cytotoxic chemotherapy after alloHSCT may differ from conventional treatments, and could include graft-versus-host disease, allograft failure, or increased risk of infection. We retrospectively reviewed treatment outcomes of 25 patients who received cytotoxic chemotherapy as treatment for relapsed hematologic malignancies after reduced-intensity, matched-sibling donor alloHSCT. Combination therapies included: EPOCH+/− rituximab (n=12 patients); “7&3” AML induction (ida/ara-c) (n=2); FLAG (n=2); asparaginase/6-MP/vincristine (n=1); R-ICE (n=1); MIME (n=1); BVP (n=1); and bortezomib/doxorubicin/dexamethasone (n=1). Single-agent therapies included: bortezomib (n=6); gemcitabine (n=4), vinorelbine (n=3); vincristine (n=1); methotrexate (n=1); and intrathecal methotrexate and/or cytarabine (n=5). A total of 133 cycles or doses were administered, of which 20 were supported with donor stem cell boosts, and another 9 were followed by nonmobilized DLI. There were 52 Grade 3 or higher toxicity episodes recorded, most commonly: neutropenia (10 episodes); infection with neutropenia (5 episodes); thrombocytopenia (6 episodes); and anemia (3 episodes). There were 12 episodes of CMV reactivation in 8 of 15 patients at risk, including 2 cases of pneumonitis and 1 case of colitis. GVHD flares were considered definitely chemotherapy-induced (2), possibly chemotherapy-induced (7), and unlikely chemotherapy-induced (2). Unusual events after treatment included culture-negative sepsis-like illness after bortezomib (n=2), secondary (11q23) AML of donor origin after one cycle of EPOCH (n=1) and diffuse alveolar hemorrhage after EPOCH (n=1). Seven patients achieved CR with therapy for post-transplant relapse, including three durable remissions. Four patients achieved PR and seven achieved disease stabilization. Median survival after starting cytotoxic therapy was 263 days. Timing of relapse appeared to be associated with survival, with median overall survival of 95 days for patients requiring therapy before Day 100 vs. 508 days for patients after Day 100 (p=0.0008). Treatment of relapse with cytotoxic chemotherapy after alloSCT is feasible and can result in durable remissions in a minority of patients. However, administration of cytotoxic therapy after alloHSCT requires monitoring and support for hematologic toxicities, GVHD and CMV reactivation; selected patients may benefit from prophylactic stem cell boosts or immune suppression. Survival after Cytotoxic Therapy for Relapse after alloHSCT Survival after Cytotoxic Therapy for Relapse after alloHSCT

Reduced-intensity conditioning is less potently tumoricidal than myeloablative regimens; thus, RIST relies more upon graft-versus-tumor (GVT) effects for disease eradication. However, variation in baseline host immune status contributes to inconsistent donor engraftment and may impede maximal GVT effects after RIST. We hypothesized that targeted immune depletion (TID) with conventional-dose chemotherapy before RIST might facilitate donor engraftment, thereby potentiating GVT effects. Thus, we evaluated dose-adjusted (DA-) EPOCH-F, a novel regimen of etoposide, prednisone, vincristine, cyclophosphamide (Cy), doxorubicin, and fludarabine (Flu), in a phase II manner in 83 pts with lymphoid malignancies undergoing RIST on 3 sequential, prospective clinical trials between 1999 and 2005. Pts received at least 1 and no more than 3 cycles of DA-EPOCH-F, targeting a peripheral blood CD4+ T cell count < 100 cells/µL. Pts achieving this CD4 count after only 1 or 2 cycles then proceeded to Flu/Cy conditioning and RIST from HLA-matched related donors. Pts otherwise proceeded to RIST after 3 cycles or if disease progression occurred during DA-EPOCH-F, regardless of CD4 count. After 2002, pts with CD20+ malignancies (n=28) also received rituximab (R) on day 1 of each DA-EPOCH-F cycle. Pt characteristics: median age 50 yrs; diagnoses Hodgkin lymphoma (14%), DLBCL (28%), other aggressive NHL (37%), follicular NHL (12%), CLL or other indolent NHL (8%); chemosensitive disease in 48%; median 3 prior regimens, prior autologous stem cell transplant in 28%; poor-risk IPI, FLIPI, IPF score, or Rai stage in 36%. Pts received 1 (n=26), 2 (n=20), or 3 (n=37) cycles of DA-EPOCH-F before RIST. Median CD4 count declined from 284 cells/µL at enrollment to 79 cells/µL after DA-EPOCH-F, which similarly depleted CD8+ T cells and B cells. DA-EPOCH-F toxicities were mainly hematologic and transient. Responses to DA-EPOCH-F were CR/CRu (11%), PR (27%), SD (35%), and PD (23%). Response rates differed by histology (P=0.006); DLBCL or other aggressive NHL were less likely to respond. Baseline CD4 counts were higher in DA-EPOCH-F responders than in non-responders (median 374 vs. 181 cells/µL; P=0.003), despite a similar extent of prior therapy. Full donor chimerism was present by day 14 after RIST in 85% of pts; no graft rejection occurred. The absolute lymphocyte count (ALC) after DA-EPOCH-F was more strongly associated with donor chimerism after RIST (P=0.02) than was CD4+ or other lymphocyte subsets. We retrospectively compared immune depletion in DA-EPOCH-F recipients with that observed in 17 previously untreated pts receiving 6 cycles of DA-EPOCH-R for mantle cell NHL. Although pts receiving DA-EPOCH-F before RIST had lower baseline ALC and all lymphocyte subsets except NKT cells, relative depletion of ALC, T and B cells from baseline levels was surprisingly similar with DA-EPOCH-F and DA-EPOCH-R. We conclude that DA-EPOCH-F effectively provides TID and disease control or stability in most pts with lymphoid malignancies prior to RIST. This strategy is associated with rapid conversion to full donor chimerism and may be useful to enhance potential GVT effects after RIST.

Treatment of refractory or recurrent malignancy with donor lymphocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (alloHSCT) is often not curative, with graft-vs-tumor (GVT) effects frequently accompanied by graft-versus-host disease (GVHD), requiring immune suppression that compromises efficacy. After alloHSCT, chimeric T lymphocytes infiltrating residual tumor (chimeric TIL) may provide enhanced antigen specificity and maintain tumor-specific homing. Compared with DLI, they may have a better GVT effect with less GVHD. Based on the success of autologous TIL therapy for melanoma, we tested the hypothesis that enhanced GVT with limited GVHD could be achieved through administration of ex-vivo activated chimeric TIL after alloHSCT. Preclinical TIL production carried out on several tumor types from non-transplanted patients demonstrated effective T cell isolation, expansion and activation using anti-CD3/CD28 bead co-stimulation, yielding a 10- to 30-fold expansion of CD3+cells. Clinical evaluation of chimeric TIL therapy was initiated with a 51 year-old woman for metastatic breast cancer whose disease progressed after a T cell-depleted reduced-intensity alloHSCT with delayed DLI from a 6/6 HLA-matched sibling donor and subsequent conventional therapy plus DLI. Two weeks after administration of unmanipulated DLI, two thoracic metastases were surgically removed. T cells were liberated from 9.4 cm of tumor using enzymatic digestion and mechanical dispersion, lymphocyte-enriched by density gradient separation, and expanded for 14 days through co-stimulation with anti-CD3/CD28-coated magnetic beads (3:1 bead-to-total nucleated cell ratio) and media containing IL-2 (100 or 1000 IU/mL). This process yielded 42.5 x 106cells, 33% expressing CD3, and generated 14.7 x 109chimeric TIL, 85% expressing CD3 (a 3.1-log T cell expansion). There was no tumor contamination of the T cell product by immunohistochemistry. Flow cytometry demonstrated that the CD4/CD8 T cell ratio increased from 1.3 to 1.9 after expansion. Three infusions of the chimeric TIL product were given in a dose-escalating manner (5, 25 and 100 x 106CD3+cells/kg). A fourth infusion was given in conjunction with low-dose IL-2 (6mu SQ per day x 3D). CT scan performed after each infusion monitored disease response, with progressive disease the indication for the next dose administration. No infusion-related or delayed toxicities were observed, except for rigors following IL-2 administration. The patient shows no evidence of GVHD, even after the highest dose of 108allogeneic T cells. Evaluation of the remaining thoracic lesion demonstrated progressive disease after the first two chimeric TIL dose-levels, transient disease stability one month after the third dose-level, and stable disease one month after the fourth dose with IL-2. This is the first clinical report of the application of chimeric TIL and represents a novel approach for developing new forms of adoptive immunotherapy in the setting of alloHSCT.

Allogeneic hematopoietic stem cells (HSC) generally engraft rapidly and completely after myeloablative conditioning. However, with reduced-intensity conditioning (RIC), mixed chimerism and graft failure are more common. Host immune status and HSC number are factors known to affect engraftment after reduced-intensity stem cell transplantation (RIST). In addition, donor T cells within the allograft may also influencethe kinetics of donor engraftment after RIST. To evaluate this, we performed a controlled comparison of engraftment outcomes among 3 groups undergoing RIST, varying by ex vivoT cell depletion (TCD) or in vivodepletion of activated T cells with methotrexate (MTX) to prevent graft-versus-host disease (GVHD). Group I (n = 50) received T cell replete (TCR) peripheral blood stem cells (PBSC) with cyclosporine (CSA) alone for GVHD prophylaxis. Group II (n = 17) received ex vivo TCD PBSC (positive/negative selection with T cell add-back to uniform dose of 1 x 105CD3+cells/kg) with CSA alone for GVHD prophylaxis. Group III (n = 31) received TCR PBSC with CSA plus MTX (5 mg/m2IV x 4 doses) for GVHD prophylaxis. The 3 groups were similarly immunosuppressed from prior therapy before RIST (median absolute lymphocyte counts 330/μL, 260/μL, and 307/μL for Groups I, II, and III, respectively), and received an identical RIC regimen (fludarabine/cyclophosphamide) plus comparable numbers of filgrastim-mobilized PBSC from HLA-matched sibling donors (median 7.9 x 106, 7.6 x 106, and 6.8 x 106CD34+cells/kg, respectively; median 3.6 x 108, 1.0 x 105, and 3.2 x 108CD3+cells/kg, respectively). Hematopoietic recovery was slowest in Group III, consistent with the myelosuppressive effects of MTX (Table). A greater proportion of patients in Group I achieved complete donor chimerism (≥ 95%) by day +28 than in Groups II or III (P < 0.025), and at day +100, mixed donor chimerism persisted more often in Groups II and III than in Group I patients (P < 0.01). Correspondingly, early (< day +42) occurrence of grade 3–4 acute GVHD, before initiation of planned sequential donor lymphocyte infusions (DLI) in Group II, was more frequent in Group I than in either Groups II or III (p=0.08).

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

In Reply.—Dr Caceres raises several concerns about AIDS patient data reported to the CDC and the ways in which these data are presented. The CDC has long recognized that some AIDS patients have multiple risk factors.1-3 The patient classification system most commonly used is hierarchical2-5 because it is both simple and straightforward. It will not always accurately reflect how each individual acquired HTLV-III/lymphadenopathy-associated virus (LAV) infection and AIDS, but it does reflect the most likely route of transmission.6 For example, a homosexual/bisexual man who had received a blood transfusion may have acquired HTLV-III/LAV infection in a nonsexual way, but for surveillance purposes, would be classified in the homosexual/bisexual group. However, if 5% of the adult population are gay men and if they have the same risk of transfusionassociated AIDS as the rest of the US adult population,6 then the number of transfusion-associated cases may actually

An important aspect of the epidemic of acquired immunodeficiency syndrome (AIDS) is its impact on health care provision in the United States. Because of the severe and disabling nature of AIDS, patients with the disease often require specialized medical care and psychosocial support. As AIDS becomes increasingly common, detailed information on health care needs is urgently required for long-term planning.One approach to quantifying such needs is to estimate the cost of health care for AIDS patients. Such estimates allow comparisons with costs for other diseases and are useful in determining the efficiency of various systems of health care provision. Although the cost of caring for patients with AIDS is generally recognized to be high, few attempts have been made to quantify these costs. Our earlier report1 estimated health care expenditures for the first 10 000 AIDS patients in the United States and pointed out the paucity of relevant

Fourteen of 156 renal-transplant recipients treated with cyclosporine and steroids developed Pneumocystis carinii-related pneumonia (PCP) over a 19-month period. This was a significant change from past experience with this disease in renal-transplant patients receiving azathioprine and steroids (six cases among 179 patients from 1977 to 1981). Epidemiological investigation failed to implicate either person-to-person or nosocomial spread of infection. Cases of PCP occurred more frequently in males. Twelve patients (86%) had onset of disease in the third or fourth months after transplantation. Comparison of cases to matched controls revealed that the cases had received lower doses of steroids and had a higher incidence of cytomegalovirus infection. This suggested that the cases may have been more effectively immunosuppressed than the controls. After institution of prophylaxis with trimethoprim-sulfamethoxazole, no further cases of PCP developed.

The pyridines (VI), which are substituted by six‐membered heterocyclic rings, are prepared from the chloromethylpyridine hydrochloride (I) or the pyridine aldehyde (VII) and transformed into the thioamide derivatives (XII).

In order to increase the efficiency of use of automated DNA synthesisers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotide sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.

For the microscopic examination of fur fibers two colorless media (synthetic plastics) have been found useful for permanent mounts, and two liquids for temporary mounts. Study of the pigmentation or of the cuticular scales may be favored by choice of refractive indices. Microscopic examination of prime silver fox and of mink pelts led to the conclusion that three main types of fibers contribute materially to the quality of furs. These are the underfur fibers, the regular guard hairs, and the “single” guard hairs. The three types of hairs were studied for general characteristics, such as relative lengths, form, and color, as well as specific characteristics, such as the features of their surface structure and relations to each other in the follicles. Specialized hairs are present in the tail gland and in a tuft near the navel. Impressions of the surface structure of the fibers were made on thermoplastic films by a modification of the original method. This modification was necessary to prevent the crushing effect on the thicker fibers. These impressions reveal features of surface structure in certain parts of the hairs that are characteristic for certain types of fibers and for different species of animals.