Hematopoietic progenitor cell collection after autologous transplant for multiple myeloma: low platelet count predicts for poor collection and sole use of resulting graft enhances risk of myelodysplasia.

Collection of hematopoietic progenitor cells (HPC) after previous autologous hematopoietic progenitor cell transplant (aHCT) was studied in 221 patients with multiple myeloma (MM). With a total of 333 collections, the median number of CD34+ cells collected was 4.7 × 10(6) CD34+ cells/kg, and 74% of the patients collected ≥ 2.5 × 10(6) CD34+ cells/kg. Among 26 variables examined, the strongest predictor for poor collection was a platelet count <100 × 10(6)/l before mobilization (P<0.001). A subsequent aHCT was performed in 154 of the 221 patients. Sole use of HPC procured after aHCT in 86 patients was associated with delayed platelet recovery (P<0.001) and linked to development of myelodysplastic syndrome (MDS)-associated cytogenetic abnormalities (MDS-CA; P=0.027, odds ratio (OR) 10.34) and a tendency towards clinical MDS/acute myeloid leukemia (AML; P=0.091, OR 3.57). However, treatment-related mortality (P=0.766) and time to absolute neutrophil count recovery ≥0.5 × 10(9)/l (P=0.879) were similar to when a pre-aHCT graft was used. Indeed, adding HPC collected before any aHCT neutralized the risk of MDS-CA or MDS/AML. Therefore, we advise generous initial HPC collection to broaden the salvage armamentarium for patients with MM.

Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization.

This was an open-label, single-center, phase II study of 20 patients with multiple myeloma who were either proven poor mobilizers (n=10; group A) or predicted poor mobilizers (n=10; group B) and were planned for autologous hematopoietic SCT. The aim was to assess the safety and efficacy of plerixafor for stem cell mobilization and tumor cell contamination. The peripheral blood (PB) CD34+ cell count was generally very low pre- plerixafor and increased significantly post-plerixafor administration. Cumulative apheresis yields of > or =2 x 10(6) CD34+ cells/kg were observed in 7 of 10 patients (group A) and 8 of 10 patients (group B). Among the proven poor mobilizers, there was no evidence of tumor cell mobilization in the PB after G-CSF plus plerixafor treatment. Seventeen of 20 (85%) patients underwent transplantation. Neutrophil engraftment occurred at a median of 13 days for all patients. Platelet engraftment occurred at a median of 16 days and 19 days for all proven and predicted poor mobilizers, respectively. At 12 months, 12 of 17 patients had documented durable grafts, 3 of 17 patients died and 2 of 17 patients were lost to follow-up; but they had documented graft durability at the previous 3- and 6-month visit. The safety profile of plerixafor in all patients was consistent with previous reports.

UC blood infection with clinical strains of Mycobacterium tuberculosis: a novel model.

BACKGROUND: Use of unrelated cord blood transplantation (UCBT) is increasing, yet high rates of mortality secondary to infection remain a problem. We investigated the utility of using umbilical cord blood (UCB) as a model to study a naive cell population challenged by Mycobacterium tuberculosis. METHODS: Mononuclear cells were isolated from nine UCB samples and infected with each of four distinct strains of M. tuberculosis. The isolates used were two highly transmissible clinical strains, the virulent laboratory strain H37Rv and a unique strain isolated from only one case (i.e. non-virulent). CFU were assessed at 3 h post-infection (day 0) and at day 7 to generate growth curves. Viability of the mononuclear cells was assessed prior to infection, 3 h post-infection and at days 3, 5 and 7 post-infection. IFN-gamma and TNF-alpha levels were determined at 24 h post-infection. RESULTS: All three of the virulent strains demonstrated rapid growth in UCB cells that was significantly faster than the growth rate observed for the non-virulent unique isolate. There was no significant decrease in UCB cell viability after the 7-day incubation period regardless of infecting isolate. UCB cells secreted IFN-gamma in response to infection, with no significant difference related to infection with different isolates. However, there was a significant increase in the amount of TNF-alpha elicited following infection with the non-virulent isolate compared with the virulent isolates. DISCUSSION: These results show that UCB can be used as a model to study infection, hopefully leading to new therapies for neonates and UCBT recipients.

Recovery from neutropenia can be predicted by the immature reticulocyte fraction several days before neutrophil recovery in autologous stem cell transplant recipients.

The duration of neutropenia (absolute neutrophil count (ANC) < or = 100/microl) identifies cancer patients at risk for infection. A test that precedes ANC > or = 100/microl would be of clinical value. The immature reticulocyte fraction (IRF) reflects erythroid engraftment and hence a recovering marrow. We evaluated the IRF as predictor of marrow recovery among 90 myeloma patients undergoing their first and second (75 patients) melphalan-based autologous stem cell transplantation (Mel-ASCT). The time to IRF doubling (IRF-D) preceded ANC > or = 100/microl in 99% of patients after the first Mel-ASCT by (mean+/-s.d.) 4.23+/-1.96 days and in 97% of the patients after the second Mel-ASCT by 4.11+/-1.95 days. We validated these findings in a group of 117 myeloma patients and 99 patients with various disorders undergoing ASCT with different conditioning regimens. We also compared the time to hypophosphatemia and to absolute monocyte count > or = 100/microl to the time to ANC > or = 100/microl. These markers were reached prior to this ANC end point in 55 and 25% of patients but were almost always preceded by IRF-D. We conclude that the IRF-D is a simple, inexpensive and widely available test that can predict marrow recovery several days before ANC> or = 100/microl.

Clonal cytogenetic changes and myeloma relapse after reduced intensity conditioning allogeneic transplantation.

To identify a correlation between metaphase cytogenetics and relapse after reduced intensity conditioning (RIC) allotransplant for patients with multiple myeloma, data on 60 patients (median age 52) who received grafts from a sibling (n = 49) or unrelated donor (n = 11) were analyzed. Fifty-three patients (88%) showed chromosomal abnormalities (CA) before the allotransplant, including 42 with abnormalities involving 13q (CA13). Twenty-two patients (41%) relapsed post-allotransplant at a median of 165 days. Of these, 11 patients showed abnormal cytogenetics at the time of post-allotransplant relapse at a median of 167 days. Of 54 patients who developed graft-versus-host disease, relapse occurred in 19 of 48 patients (43%) with CA present before RCI allotransplant, versus 1 of 6 without CA (17%) (P = 0.06). Loss of CA before RIC allotransplant and disease status > PR after RIC allotransplant were significantly associated with a lower risk of post-allotransplant relapse with cytogenetic abnormalities; 5.2 vs 36%, and 18 vs 53%, (both P < 0.05), respectively. The current data suggests that myeloma associated with persistent clonal cytogenetic abnormalities is an entity which most likely escapes the effects of a graft versus myeloma activity, maybe because of acquisition of resistance to immunologic manipulations.

Autologous stem cell transplantation followed by consolidation chemotherapy for patients with multiple myeloma.

Although high-dose therapy and autologous stem cell transplant (ASCT) is superior to conventional chemotherapy for treatment of myeloma, most patients relapse and the time to relapse depends upon the initial prognostic factors. The administration of non-cross-resistant chemotherapies during the post-transplant period may delay or prevent relapse. We prospectively studied the role of consolidation chemotherapy (CC) after single autologous peripheral blood stem cell transplant (auto-PBSCT) in 103 mostly newly diagnosed myeloma patients (67 patients were < or =6 months from the initial treatment). Patients received conditioning with BCNU, melphalan+/-gemcitabine and auto-PBSCT followed by two cycles of the DCEP+/-G regimen (dexamethasone, cyclophosphamide, etoposide, cisplatin+/-gemcitabine) at 3 and 9 months post-transplant and alternating with two cycles of DPP regimen (dexamethasone, cisplatin, paclitaxel) at 6 and 12 months post-transplant. With a median follow-up of 61.2 months, the median event-free survival (EFS) and overall survival (OS) are 26 and 54.1 months, respectively. The 5-year EFS and OS are 23.1 and 42.5%, respectively. Overall, 51 (49.5%) patients finished all CC, suggesting that a major limitation of this approach is an inability to deliver all planned treatments. In order to improve results following autotransplantation, novel agents or immunologic approaches should be studied in the post-transplant setting.

Clinical-grade myeloma Ag pre-loaded DC vaccines retain potency after cryopreservation.

BACKGROUND: The use of myeloma Ag-loaded mature DC vaccines, cryopreserved in single-use aliquots, is an attractive immunotherapeutic strategy. In this study we investigated the retention of phenotype, viability and potency of DC vaccines after freezing and thawing. METHODS: Plastic-adherent monocytes, derived from a steady-state leukapheresis, were cultured in serum-free media containing GM-CSF and IL-4. DC were loaded on day 6 with myeloma lysate (ML) or idiotype (Id) Ag and keyhole limpet hemocyanin (KLH), induced to mature on day 7 with CD40-ligand and cryopreserved on day 9. Seventeen clinical-scale cultures were evaluated for DC yield, recovery and immunophenotype after potency was validated with allogeneic mixed lymphocyte culture and Ag presentation assays. RESULTS: We produced 88 individual vaccines from 17 clinical-scale cultures. Median DC yield at harvest was 131 x 10(6) (range 37-375 x 10(6)) and median recovery of viable DC after thawing was 69% (range 11-100%). We confirmed viability (7AAD-), phenotype (CD14-, CD83+/CD40+, CD83+/CD80+, CD83+/CD86+, CD83+/CD54+, HLA-DR++) and the ability of the DC to present Ag and stimulate allogeneic T cells post-thawing. DISCUSSION: We have validated a serum-free culture system for the production of DC. Cryopreservation did not interfere with DC activity, allowed time for rigorous quality control (QC) and flexible scheduling of intranodal vaccination, and reduced the time to prepare multiple vaccines.

High-dose cyclophosphamide with or without etoposide for mobilization of peripheral blood progenitor cells in patients with multiple myeloma: efficacy and toxicity.

The purpose of the study was to examine the yield of CD34(+) cells, response rates, and toxicity of high-dose cyclophosphamide with or without etoposide in patients with multiple myeloma. In total, 77 myeloma patients received either cyclophosphamide 4.5 g/m(2) (n=28) alone or with etoposide 2 g/m(2) (n=49) in a nonrandomized manner, followed by G-CSF 10 microg/kg/day for the purpose of stem cell mobilization. The effects of various factors on CD34(+) cell yield, response rate and engraftment were explored. A median of 22.39 x 10(6) CD34(+) cells/kg were collected on the first day of leukapheresis (range 0.59-114.71 x 10(6)/kg) in 71 (92%) of patients. Greater marrow plasma cell infiltration (P=0.02) or prior radiation therapy (P=0.02) adversely affected CD34(+) cell yield. In total, 45% of patients receiving cyclophosphamide and 56% of those receiving cyclophosphamide/etoposide had at least a minimum response by EBMT criteria. In all, 25% of patients who received cyclophosphamide alone vs 75.5% of patients who received combined chemotherapy required hospitalization mainly for treatment of neutropenic fever. Cyclophosphamide alone is associated with impressive CD34(+) cell yields and clear antimyeloma activity. The addition of etoposide resulted in increased toxicity without significant improvement in CD34(+) cell yield or response rates.

Sweet syndrome in multiple myeloma: a series of six cases.

BACKGROUND: Sweet syndrome (SS), a paraneoplastic syndrome characterized by fever, neutrophilia, multiple erythematous painful plaques, and a dense dermal neutrophilic infiltration, has a known association with hematologic malignancies such as acute myelogenous leukemia. However, no clear association with multiple myeloma (MM) has been reported. MATERIALS AND METHODS: Pathology reports of the 2357 patients with multiple myeloma at the University of Arkansas for Medical Sciences were reviewed to retrieve cases who had developed SS. Cytogenetic studies and immunoglobulin secretory status were retrieved. Five cases of SS in MM and 25 cases of SS in patients without MM underwent syndecan-1 immunohistochemistry. OBSERVATIONS: Six cases of SS occurring in the setting of MM showed a predominance in patients secreting IgG paraprotein. Five of the six patients received granulocyte-colony stimulating factor while the sixth received granulocyte-monocyte-colony stimulating factor. Fifty percent showed a non-specific cytogenetic anomaly. CONCLUSIONS: There is no specific cytogenetic anomaly associated with SS in the setting of MM. This paraneoplastic syndrome may be secondary to elevated levels of granulocyte colony stimulating factor (G-CSF), possibly with a component of enhanced sensitivity to endogenous G-CSF. The immunoglobulin secretory status parallels that seen in MM with cutaneous involvement, but IgG secretors may be at an increased risk of developing SS compared with their counterparts who secrete other immunoglobulins.

Transplantation as salvage therapy for high-risk patients with myeloma in relapse.

Patients with myeloma relapsing after tandem transplant have a poor survival and treatment options are limited. The role of additional salvage transplant procedures for these patients is unknown. To evaluate the benefit and identify prognostic factors, the outcome of 76 consecutive patients with recurrent myeloma after tandem transplant receiving salvage transplants (ST) was analyzed. Prior to ST, 23 patients (30%) had shown chemosensitive response to preceding salvage chemotherapy: two complete remissions (CR); eight near CRs (nCR: only immunofixation positive); 13 partial remissions (PR >or=75% reduction in M protein). Fifty received an autologous transplant, 22 a sibling-matched allogeneic transplant, and four a matched-unrelated allogeneic transplant. Overall response after ST was 59%: eight CRs (11%); 14 nCRs (18%); 23 PRs (30%). Overall survival (OS) at 2 years was 19%; 2 year event-free survival rate (EFS) 7%. On univariate analysis for survival, only pre-transplant chemosensitive relapse (P < 0.05), serum albumin >3 g/dl (P = 0.001), normal LDH (P = 0.04), and long interval between the second transplant and relapse/progression were significant beneficial factors. In a Cox proportional hazard model, chemosensitive relapse, and albumin >3 g/dl were significant for better OS: hazard ratio (HR) 1.4, 1.7, respectively, while normal LDH, and absence of CA13 were significant for better EFS: HR 1.8, 1.7, respectively. Patients with albumin >3 g/dl who had chemosensitive disease before ST (n = 16) had a median survival of 16 months, compared to 7 months (n = 34) and 2 months (n = 26) for patients with only one (n = 34) or no favorable prognostic factors (n = 28), respectively (P < 0.001). Their survival at 2 years post-ST was 43%, 17% and 11%, respectively. Our study suggests further transplantation should only be considered in the setting of a clinical trial in patients with favorable prognostic factors.

Autotransplantation for advanced lymphoma and Hodgkin's disease followed by post-transplant rituxan/GM-CSF or radiotherapy and consolidation chemotherapy.

Disease relapse occurs in 50% or more of patients who are autografted for relapsed or refractory lymphoma (NHL) or Hodgkin's disease (HD). The administration of non-cross-resistant therapies during the post-transplant phase could possibly control residual disease and delay or prevent its progression. To test this approach, 55 patients with relapsed/refractory or high-risk NHL or relapsed/refractory HD were enrolled in the following protocol: stem cell mobilization: cyclophosphamide (4.5 g/m(2)) + etoposide (2.0 g/m(2)) followed by GM-CSF or G-CSF; high-dose therapy: gemcitabine (1.0 g/m(2)) on day -5, BCNU (300 mg/m(2)) + gemcitabine (1.0 g/m(2)) on day -2, melphalan (140 mg/m(2)) on day -1, blood stem cell infusion on day 0; post-transplant immunotherapy (B cell NHL): rituxan (375 mg/m(2)) weekly for 4 weeks + GM-CSF (250 microg thrice weekly) (weeks 4-8); post-transplant involved-field radiotherapy (HD): 30-40 Gy to pre-transplant areas of disease (weeks 4-8); post-transplant consolidation chemotherapy (all patients): dexamethasone (40 mg daily)/cyclophosphamide (300 mg/m(2)/day)/etoposide (30 mg/m(2)/day)/cisplatin (15 mg/m(2)/day) by continuous intravenous infusion for 4 days + gemcitabine (1.0 g/m(2), day 3) (months 3 + 9) alternating with dexamethasone/paclitaxel (135 mg/m(2))/cisplatin (75 mg/m(2)) (months 6 + 12). Of the 33 patients with B cell lymphoma, 14 had primary refractory disease (42%), 12 had relapsed disease (36%) and seven had high-risk disease in first CR (21%). For the entire group, the 2-year Kaplan-Meier event-free survival (EFS) and overall survival (OS) were 30% and 35%, respectively, while six of 33 patients (18%) died before day 100 from transplant-related complications. The rituxan/GM-CSF phase was well-tolerated by the 26 patients who were treated and led to radiographic responses in seven patients; an eighth patient with a blastic variant of mantle-cell lymphoma had clearance of marrow involvement after rituxan/GM-CSF. Of the 22 patients with relapsed/refractory HD (21 patients) or high-risk T cell lymphoblastic lymphoma (one patient), the 2-year Kaplan-Meier EFS and OS were 70% and 85%, respectively, while two of 22 patients (9%) died before day 100 from transplant-related complications. Eight patients received involved field radiation and seven had radiographic responses within the treatment fields. A total of 72 courses of post-transplant consolidation chemotherapy were administered to 26 of the 55 total patients. Transient grade 3-4 myelosuppression was common and one patient died from neutropenic sepsis, but no patients required an infusion of backup stem cells. After adjustment for known prognostic factors, the EFS for the cohort of HD patients was significantly better than the EFS for an historical cohort of HD patients autografted after BEAC (BCNU/etoposide/cytarabine/cyclophosphamide) without consolidation chemotherapy (P = 0.015). In conclusion, post-transplant consolidation therapy is feasible and well-tolerated for patients autografted for aggressive NHL and HD and may be associated with improved progression-free survival particularly for patients with HD.

Fatal disseminated adenoviral infection associated with thrombotic thrombocytopenic purpura after allogeneic bone marrow transplantation.

Adenoviruses are increasingly recognized as a significant cause of morbidity and mortality in immunocompromised patients. We report on a patient who, approximately 4 weeks after allogeneic stem cell transplantation, developed fever, new liver lesions and thrombotic microangiopathy. Adenovirus type 2 was isolated from blood and urine samples. Liver biopsy showed parenchymal necrosis with intranuclear viral inclusion bodies. Immunohistochemistry was positive for adenovirus. In addition, on electron microscopy the morphologic pattern was highly suggestive of adenovirus. The patient died on post-transplant day 40. The relatively early post-transplant presentation of disseminated adenoviral disease and its possible association with a TTP-like picture are rather unusual after allogeneic transplantation.

Pulmonary toxicity syndrome following CDEP (cyclophosphamide, dexamethasone, etoposide, cisplatin) chemotherapy.

We report on three patients with multiple myeloma who developed drug-induced pneumonitis 1-2(1/2) months following maintenance (post autologous transplantation) chemotherapy with CDEP (cyclophosphamide, dexamethasone, etoposide, cisplatin) and 6-20 months after exposure to carmustine (BCNU) 300 mg/m(2), used in combination with melphalan 140 mg/m(2), as pre-transplant conditioning regimen. All patients had either a proven (two) or suspected (one) fungal pneumonia and were treated with liposomal amphotericin B. Dyspnea, fever and cough were the prominent clinical symptoms, while air-space disease with ground glass appearance was seen radiographically. Histologic features typical for drug-induced lung injury were detected. All patients had a dramatic, clinical and radiographic response to a brief course of corticosteroids. Although CDEP-induced pneumonitis appears to be a rare complication, its early recognition and prompt treatment, as well as its possible association with preceding fungal infection may have important clinical implications.

Pulmonary T cell repertoire in patients with graft-versus-host disease following blood and marrow transplantation.

Pulmonary inflammation is one of the risk factors associated with blood and marrow transplantation (BMT). To determine the potential role of T cells in pulmonary complications after transplantation, we analyzed the T-cell repertoire expressed in bronchoalveolar lavage fluids from eleven patients with graft-versus-host disease following BMT. A reverse transcriptase-polymerase chain reaction was used to amplify rearranged TCR transcripts in unfractionated, CD4+, and CD8+ T cells from bronchoalveolar lavage fluids. The relative expression of TCR variable (V) gene families and the diversity of junctional region lengths associated with different AV and BV gene families were analyzed. Nearly all TCR AV and BV gene families were detected in bronchoalveolar lavage cells from BMT recipients. Oligoclonal patterns of TCR junctional region lengths were observed in unfractionated, CD4+, and CD8+ bronchoalveolar T cells. The oligoclonal expansion of bronchoalveolar T cells in patients was confirmed by DNA sequencing. TCRV gene expression is almost completely restored in the lungs of BMT recipients as early as two weeks after transplantation. Increased oligoclonality among TCR gene families suggests either an incomplete restoration of TCR diversity or an antigen-driven expansion of T cells in the lungs of BMT recipients with graft-versus-host disease, not necessarily related to pulmonary infection.

High response rate in refractory and poor-risk multiple myeloma after allotransplantation using a nonmyeloablative conditioning regimen and donor lymphocyte infusions.

Standard allogeneic stem cell transplant (allo-SCT) regimens have been associated with a high transplant-related mortality (TRM) in multiple myeloma (MM). Nonmyeloablative therapy can establish stable engraftment after allo-SCT and maintain the antitumor effect with less toxicity, which is important in heavily pretreated and elderly patients. We report on 16 poor-risk MM patients receiving allo-SCT from an HLA-matched (n = 14) or mismatched (n = 2) sibling following conditioning with melphalan 100 mg/m(2) (MEL-100). Ten patients had refractory relapse, 4 responsive relapse, and 2 patients were in near complete remission (nCR) with poor-prognosis disease. Patients had received 1 (n = 9) or 2 (n = 7) prior autotransplants. Donor lymphocyte infusions (DLIs) were given to 14 patients with no clinical evidence of graft versus host disease (GVHD) either to attain full donor chimerism (n = 4) or to eradicate residual disease (n = 10). Fifteen patients showed myeloid engraftment, and 12 patients were full donor chimeras at day +21. No TRM was observed during the first 100 days. Acute GVHD developed in 10 patients; 1 had fatal grade IV GVHD. Seven progressed to chronic GVHD, limited in 3 and extensive in 4 patients. At a median follow-up of 1 year, 5 patients achieved and sustained CR, 3 nCR, and 4 partial remission. Of 4 patients progressing after transplantation, 3 achieved a remission following further chemotherapy and DLI. Remarkable graft versus myeloma responses were seen in chemotherapy-refractory patients. Two patients died of progressive disease, and 3 died of GVHD complications without active disease. GVHD remains a major problem with this procedure.

Cytomegalovirus infection and non-neutropenic fever after autologous stem cell transplantation: high rates of reactivation in patients with multiple myeloma and lymphoma.

In a retrospective study, we examined the association between cytomegalovirus (CMV) infection and non-neutropenic fever immediately following autologous peripheral blood stem cell transplantation for a variety of haematological malignancies and solid tumours. Sixty non-neutropenic febrile episodes (41 in CMV-seropositive and 19 in CMV-seronegative patients) were evaluated. CMV reactivation, documented by CMV antigenaemia, was detected in 16 out of 41 (39%) seropositive patients compared with 0 out of 19 seronegative patients. In 12 of these 16 patients, CMV infection was considered the sole cause of fever. Thirteen patients had maximum antigenaemia levels > 5 cells/slide. Specific antiviral treatment led to the resolution of the fever in all, but two, patients, who developed fatal CMV pneumonia. Patients with multiple myeloma and lymphoma, possibly owing to a combination of disease-related characteristics and prior immunosuppressive treatment, had high rates of CMV reactivation and may require more frequent diagnostic evaluation and prompt therapeutic intervention.

Tamoxifen-based treatment induces clinically meaningful responses in multiple myeloma patients with relapsing disease after autotransplantation.

Tamoxifen has been shown to induce apoptosis in multiple myeloma cell lines and primary myeloma cells. Daily tamoxifen was given to 12 consecutive multiple myeloma patients who either relapsed following autologous stem cell transplantation (11) or had progressive disease on conventional chemotherapy (1). Methotrexate was also given biweekly to enhance the antiangiogenetic effect. Two patients achieved complete remission lasting 8 and 18 months. Two additional patients had stable disease (SD) for 7 and 11 months. All responders were men and the earliest signs of response were seen after approximately 6-8 weeks of treatment. The regimen was very well tolerated. Speculations about a possible mechanism of action of tamoxifen in multiple myeloma are discussed.

In situ hybridization for detection of HIV RNA.

In HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright-field, dark-field, specular reflectance, or laser-scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides.

Measurement of in situ hybridization.

Hybridization of labeled specific molecular probes to nucleic acids in tissues allows geometric and functional location of gene expression or of foreign genome sequences. Estimates of amounts and location of target nucleic acid sequence can be made with phosphor storage imaging and molecular controls.

Encouraging preliminary results in 12 patients with high-risk haematological malignancies by omitting graft-versus-host disease prophylaxis after allogeneic transplantation.

Immunosuppressive therapy, routinely given after allogeneic transplantation to modulate graft-versus-host disease (GVHD) may have an adverse effect on the graft-versus-tumour (GVT) effect. Twelve patients with high-risk haematological malignancies were given cyclophosphamide, total body irradiation and antithymocyte globulin followed by peripheral blood stem cell grafts from HLA-identical siblings without prophylactic immunosuppression. At the earliest clinical evidence of GVHD, patients were treated with high-dose solumedrol and tacrolimus. Prompt haematological recovery [absolute neutrophil count (ANC) > 1.0 x 109/l] was observed (median time 9 d). All patients developed grade III-IV GVHD (median onset 9 d), involving the skin (11), intestine (five) and liver (three). Of nine evaluable patients, seven developed chronic GVHD [extensive (six), limited (one)]. Six patients died 1-6.5 months after transplantation. Three patients died from treatment-related complications, two from acute GVHD and one from relapsing disease. The remaining six patients are alive 5-26 months after transplantation, five in complete remission and one myeloma patient in very good partial remission. In conclusion, omission of post-transplantation GVHD prophylaxis is feasible, does not lead to graft failure or a high incidence of uncontrollable GVHD and appears to be associated with encouraging clinical responses in a group of patients with high-risk disease features.