Suprapubic percutaneous sclero-embolization of symptomatic female pelvic varicocele under local anesthesia.

PURPOSE: To evaluate the safety and feasibility of supra-pubic percutaneous sclero-embolization (SE) in the treatment of symptomatic female pelvic varicocele (FPV), performed under local anesthesia. MATERIALS AND METHODS: The authors selected 28 patients screened by transabdominal and transvaginal ultrasound, with venous Doppler signal. Clinicians performed SE by transfemoral catheterization, under local anesthesia, using of a mix of 2 ml of lauromacrogol 400 (Atossisclerol 3%, Chemische F. Kreussler, Wiesbaden, Germany) and 2 ml of air, in a mixed foam fashion. RESULTS: The total operative time for SE was 7.6 +/- 2.1 min. Intra-surgical blood loss was 40 +/- 14 ml. No migration of sclerosant material occurred and postoperative analgesic request during a 48 hr period occurred in 6 patients. Technical success was 100%. The Authors embolized 8 women bilaterally (28.5%), 18 on the left ovarian vein (OV) (64.2%) and 2 only in the right OV (7.1%): 7 women complained of transitory flank pain (25%), which disappeared in few minutes. The major complications in 10 days after SE were: fever (> 38 degrees C for two days) in 2 patients (7.1%) and pelvic pain for 3 days in eight patients (28.5%). After 30 days only 6 women suffered of FPV lower symptoms which disappeared in 180 days. A substantial reduction in size of pelvic varicosities was noted in all patients. CONCLUSIONS: SE is a safe and feasible procedure. It reduces significantly the mean time of scopies, the intensity of radiation emission, and it is performed under local anaesthesia. This minimally invasive procedure could be proposed to all women with supra-pubic FPV for its reproducibility and feasibility.

Off-label use of recombinant factor VIIa in patients following bone marrow transplantation.

Recombinant factor VIIa (rFVIIa, NovoSeven) is FDA-approved for the treatment of bleeding in patients with hemophilia A/B with inhibitors. A growing literature suggests that there may be expanded indications for the use of NovoSeven in patients with significant bleeding who do not have a known factor deficiency. Severe bleeding refractory to standard hematologic or hemostatic support is common in patients undergoing bone marrow transplantation (BMT). We review our experience with rFVIIa in three patients (8 years 8 months to 19 years, median 13 years) treated for pulmonary hemorrhage (n = 1), hemorrhagic cystitis (n = 3), and gastrointestinal bleeding (n = 2). Boluses of 90-270 microg/kg rVIIa with subsequent doses of 90 microg/kg every 4-24 h for 3-14 days were given, concurrent with maintaining platelet counts >50,000/mm(3). Transient clinical responses in gross hematuria (two patients) and in pulmonary hemorrhage were noted within several days of starting rFVIIa, but bleeding in a new site in two patients and renewed bleeding of the initial site in the third resulted in discontinuation of the drug. No toxicity or adverse events were observed while the patients were on rFVIIa treatment. Because of the substantial cost of this product, the lack of adequate monitoring methodology, and the variability of current dose and dosing intervals, large randomized studies are needed before definitive off-label use in the setting of BMT can be recommended.

Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients With leukemia or lymphoma.

PURPOSE: To improve the control of hyperuricemia in patients with leukemia or lymphoma, we tested a newly developed uricolytic agent, recombinant urate oxidase (SR29142; Rasburicase; Sanofi-Synthelabo, Inc, Paris, France), which catalyzes the oxidation of uric acid to allantoin, a highly water-soluble metabolite readily excreted by the kidneys. PATIENTS AND METHODS: We administered Rasburicase intravenously, at 0.15 or 0.20 mg/kg, for 5 to 7 consecutive days to 131 children, adolescents, and young adults with newly diagnosed leukemia or lymphoma, who either presented with abnormally high plasma uric acid concentrations or had large tumor cell burdens. Blood levels of uric acid, creatinine, phosphorus, and potassium were measured daily. The pharmacokinetics of Rasburicase, the urinary excretion rate of allantoin, and antibodies to Rasburicase were also studied. RESULTS: At either dosage, the recombinant enzyme produced a rapid and sharp decrease in plasma uric acid concentrations in all patients. The median level decreased by 4 hours after treatment, from 9.7 to 1 mg/dL (P =.0001), in the 65 patients who presented with hyperuricemia, and from 4.3 to 0.5 mg/dL (P =.0001) in the remaining 66 patients. Despite cytoreductive chemotherapy, plasma uric acid concentrations remained low throughout the treatment (daily median level, 0.5 mg/dL). The urinary excretion rate of allantoin increased during Rasburicase treatment, peaking on day 3. Serum phosphorus concentrations did not change significantly during the first 3 days of treatment, decreased significantly by day 4 in patients presenting with hyperuricemia (P =.0003), and fell within the normal range in all patients by 48 hours after treatment. Serum creatinine levels decreased significantly after 1 day of treatment in patients with or without hyperuricemia at diagnosis (P =.0003 and P =.02, respectively) and returned to normal range in all patients by day 6 of treatment. Toxicity was negligible, and none of the patients required dialysis. The mean plasma half-lives of the agent were 16.0 +/- 6.3 (SD) hours and 21.1 +/- 12.0 hours, respectively, in patients treated at dosages of 0.15 or 0.20 mg/kg. Seventeen of the 121 assessable patients developed antibodies to the enzyme. CONCLUSION: Rasburicase is safe and highly effective for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma.

Utility of obtaining blood cultures in febrile neutropenic patients undergoing bone marrow transplantation.

Infection remains an important cause of morbidity and mortality after bone marrow or stem cell transplantation. To evaluate the role of obtaining blood cultures for intermittent or persistent fever in neutropenic patients on antibiotic therapy, we performed a retrospective chart review of 196 consecutive patients admitted to the Bone Marrow Transplant Unit at the University of North Carolina Hospitals from 1995 to 1998. From the cohort of 196 patients, 154 patients developed neutropenic fever. The initial blood culture was positive in 16 of 145 patients during the first fever episode giving a prevalence of 11%. From the total of 109 patients that had blood cultures drawn after day 1 of fever, five patients had blood cultures positive for a pathogen, a prevalence of 4.6%. In only one patient, did blood cultures drawn after day 1 identify an organism not present on day 1 (prevalence 0.9%). After reviewing the results in the first 105 patients, we changed our timing of collection of blood cultures. Forty-nine patients were treated in this manner and we found that the mean number of blood cultures decreased from 9.2 to 4.7 per patient without a change in the frequency of infectious complications or length of hospitalization.

Comparison of granulocyte colony-stimulating factor (G-CSF)--mobilized peripheral blood progenitor cells and G-CSF--stimulated bone marrow as a source of stem cells in HLA-matched sibling transplantation.

HLA-identical bone marrow or stem cell transplantation from a sibling is the preferred treatment for patients with chronic myelogenous leukemia, bone marrow failure syndromes, relapsed acute leukemia, and specific inborn errors of metabolism. Several groups have shown that granulocyte colony-stimulating factor (G-CSF)--mobilized peripheral blood progenitor cells (PBPCs) obtained from HLA-matched siblings are effective in reconstitution of marrow function after marrow ablative conditioning therapy. To evaluate whether G-CSF treatment before bone marrow harvest leads to enhanced recovery of PBPC counts and recovery from limited graft-versus-host disease (GVHD), we assessed the outcome of a sequential cohort of patients treated identically and then given either G-CSF--mobilized PBPCs or G-CSF--stimulated bone marrow from HLA-identical siblings. We show that the time to neutrophil engraftment is identical in the 2 cohorts, whereas platelet engraftment is earlier with the use of PBPCs. The incidence of acute GVHD was decreased, and that of chronic GVHD significantly decreased, in the group receiving bone marrow. Overall survival was not different between the 2 groups. Thus, G-CSF--stimulated bone marrow offers a source of stem cells that allows for early neutrophil engraftment with a decreased risk of GVHD.

Histiocytic lesion mimicking intrinsic brainstem neoplasm. Case report.

This 10-year-old girl presented with a 1-month history of progressive bulbar palsy and a solitary enhancing mass originating within the floor of the fourth ventricle. Results of initial imaging studies and presentation were suggestive of neoplasia. Subtotal resection was performed and pathological examination revealed the mass to be a histiocytic lesion, with no evidence of a glioma. The patient had no other stigmata of histiocytosis and was treated with steroid medications, resulting in prolonged resolution of the lesion. This case demonstrates that for discrete brainstem lesions the differential diagnosis includes entities other than glioma for which treatment is available. Biopsy sampling should be considered when technically feasible.

Long-term engraftment failure after marrow ablation and autologous hematopoietic reconstitution: differences between peripheral blood stem cell and bone marrow recipients.

We infused peripheral blood stem cells (PBSC) into 51 patients with various malignant disorders, after myeloablative conditioning. Twenty-four patients also received autologous bone marrow (PBSC + BM). In a multivariate analysis, the only statistically significant predictors of neutrophil engraftment were log-dose CFU-GM (P < 0.001) and the number of prior chemotherapy regimens (P = 0.004). The factors predicting RBC and platelet engraftment were log-dose CFU-GM (P = 0.002), PBSC + BM infusion (P = 0.007) and the absence of neoplastic bone marrow involvement (P = 0.009). Seven patients remained platelet and/or red cell transfusion-dependent for 100 days or more post-transplant after good neutrophil recovery. Six of these seven long-term engraftment failures, as well as five additional patients, received < 10(5) CFU-GM/kg. Of the 11 patients who received < 10(5) CFU-GM/kg (low-dose patients), seven were PBSC recipients, of whom six were long-term engraftment failures. In contrast, there were no long-term engraftment failures among the four low-dose autologous marrow recipients. This difference in long-term engraftment failure rate was significant (P = 0.015). The low-dose PBSC patients all had a diagnosis of lymphoma with bone marrow involvement. The low-dose PBSC + BM group was more heterogeneous, but no patient had malignant involvement of the marrow. The low-dose PBSC patients had also received significantly more prior chemotherapy regimens than the low-dose PBSC + BM patients and a significantly higher proportion received total body irradiation (TBI) as part of their conditioning regimen. We conclude that marrow damage resulting from a combination of neoplastic infiltration, chemotherapy and TBI may result not only in low PBSC yields but also in an impaired capacity of the marrow microenvironment to support transplanted stem cells.

Storage of newborn stem cells for future use.

Human placental cord blood contains a large number of hematopoietic progenitor cells, which could be used as a source of stem cells for the treatment of hematologic disorders and malignancies. Advantages of cord blood use include noninvasive collection, low risk for viral infection, and immunologic naivete. Cord blood may be used as stem-cell rescue in all applications of bone marrow transplantation, including gene therapy. Given this potential utility, questions are raised regarding the regulation of cord-blood collection, storage, and use. Should the prospective parent(s) donate the infant's cord blood for treatment of unrelated recipients, or should they invest in cord-blood storage as biologic "life insurance" for the child's later use? This issue presents many conflicts for families and their health care providers.

DNA ploidy analysis of pediatric germ cell tumors.

To determine the usefulness of DNA ploidy analysis in the biological assessment of pediatric germ cell tumors (GCTs), paraffin-embedded tissues from primary, recurrent, and metastatic neoplasms in 32 patients under age 18 were analyzed using static image cytometry. Primary sites included testis (eight), ovary (nine), sacrococcygeal region (10), central nervous system (three), and other extragonadal sites (two). The majority of the tumors were endodermal sinus tumors (ESTs) and teratomas. Of 13 teratomas, eight were diploid and five aneuploid. Immature ovarian teratomas of up to Grade II were diploid, whereas all of three Grade III immature teratomas were aneuploid. All sacrococcygeal teratomas, regardless of grade, were diploid; the development of EST in two sacrococcygeal teratomas was associated with the development of aneuploidy. Of 17 endodermal sinus tumors, five were diploid, five aneuploid, and seven tetraploid. All diploid ESTs occurred in the testis or extragonadally in patients 24 mo of age or younger. Infantile testicular ESTs were either tetraploid (one) or diploid (two), and ovarian ESTs were either aneuploid (two) or tetraploid (three). Comparing our data with that available for adult GCTs, we suggest that some pediatric GCTs may be pathogenetically distinct from adult GCTs. Adult testicular GCTs have been shown to be aneuploid, whereas infantile testicular ESTs are often tetraploid or diploid. This suggests a different pathogenesis for these groups of tumors and may explain their biological differences as well. The transformation of a sacrococcygeal teratoma into a malignant EST is poorly understood. Ploidy analysis presented suggests genetic alterations may accompany this transformation.

"Recall" pneumonitis: adriamycin potentiation of radiation pneumonitis in two children.

The radiographic findings in two children with acute "recall" pneumonitis, associated with administration of Adriamycin (doxorubicin hydrochloride) and actinomycin D at variable intervals after local radiation therapy, were presented to emphasize the unique radiographic appearance and clinical course. A 10-year-old girl underwent radiation therapy 9 weeks after completing an initial cycle of chemotherapy. Within hours of the resumption of chemotherapy, she was in clinical respiratory distress. Chest radiography showed a well-defined area of alveolar consolidation in the periphery of the right lung corresponding to the area of radiation. Shortness of breath and right chest rales developed in a 15-year-old boy within 12 hours of the resumption of chemotherapy 6 weeks after radiation therapy was completed. Chest radiography showed an alveolar infiltrate extending from the apex to the base of the right lung corresponding to the area of radiation. Symptoms may be confused with an infectious pathogenesis; thus, knowledge of the history of radiation therapy and the radiation port is important in initiating treatment with steroids rather than antibiotics.

Treatment of recurrent and refractory pediatric solid tumors with high-dose busulfan and cyclophosphamide followed by autologous bone marrow rescue.

PURPOSE: The purpose of this study was to determine the toxicities of and responses to high-dose busulfan and cyclophosphamide with autologous bone marrow transplant (ABMT) in patients with recurrent or refractory pediatric solid tumors. PATIENTS AND METHODS: We treated 18 patients (ages, 2 to 38 years; median, 14) who had tumors that were resistant to conventional chemotherapy and radiotherapy with busulfan 16 mg/kg and cyclophosphamide 200 mg/kg. Seventeen patients received bone marrow purged with 4-hydroperoxycyclophosphamide; one received unpurged marrow. RESULTS: Despite extensive prior treatment, including radiotherapy in 16 patients, toxicity generally was acceptable. For seven patients with measurable disease, there were three partial responses of 2, 10, and 20 months' duration, three patients with stable disease (SD), and one early, toxic death. Of the 11 patients with no measurable disease at the time of transplantation, one patient with osteosarcoma continues in remission at 57+ months and one third of the patients survived for at least 16 months. Mucositis was the predominant nonhematopoietic toxicity. CONCLUSION: Although the high-dose busulfan and cyclophosphamide combination showed modest activity, changes in the preparative regimen should be considered to improve the response rate in refractory tumors.

A simple method of estimating cerebrospinal fluid pressure during lumbar puncture.

It is often difficult to measure cerebrospinal fluid (CSF) pressure in children. CSF flow through a spinal needle is described by the equation: Flow = pressure/(needle constant x relative viscosity). Thus, CSF flow rate during lumbar puncture can be used to estimate CSF pressure. Because the viscosity of CSF is approximately the same as that of normal saline, 0.9% NaCl was used to model CSF flow in vitro. Flow of saline through various spinal needles was measured as pressure and temperature were varied to determine needle constants and variation in viscosity with temperature. Counting periods for which the number of drops counted equals the pressure (in centimeters of H2O) then were determined for each needle size. At patient temperatures less than 40 degrees C, counting periods were calculated at 21, 39, and 12 seconds, for 22-gauge 1.5-inch, 22-gauge 3.5-inch, and 20-gauge 3.5-inch spinal needles, respectively. Viscosity decreased slightly above 40 degrees C, and counting periods became 20, 37, and 11 seconds. Finally, the method was tested prospectively in 12 patients by comparing drop count (over the calculated counting period) to manometric pressure measurement. Drop counts were within 15% of manometric pressure in all patients. This method allows simple and rapid estimation of CSF pressure during lumbar puncture.

Discrete pigmentation after chemotherapy.

Discrete areas of cutaneous hyperpigmentation were seen in two children with metastatic sarcoma who received chemotherapeutic bone marrow ablation with cyclophosphamide, etoposide, and carboplatin prior to autologous bone marrow transplantation. The hyperpigmented patches occurred only in areas of skin occluded by tape, electrocardiogram pads, or elastic bandages. Identical skin findings were reported in five adult women who received intravenous thiotepa and cyclophosphamide. Measurable levels of thiotepa were detected in these patients' serum, skin, sweat, and occluded gauze, suggesting that the chemical was excreted onto the skin surface in sweat and accumulated under occlusive dressing, thus producing some toxic effect on the epidermis or melanocytes resulting in abnormal pigmentation. We suspect that a similar mechanism was operative in our patients to produce the unusual patterned hyperpigmentation, and suggest that this complication may be prevented by minimizing sweat accumulation in areas occluded by adhesive materials.

Analysis of factors predicting speed of hematologic recovery after transplantation with 4-hydroperoxycyclophosphamide-purged autologous bone marrow grafts.

We previously described the predictive value of graft colony-forming units granulocyte macrophage (CFU-GM) content after 4-hydroperoxycyclophosphamide (4-HC) purging for the duration of aplasia after autologous bone marrow transplantation. Despite the uniform 4-HC concentration, we observed heterogeneity in CFU-GM survival and the kinetics of engraftment. We have now analysed patient and graft characteristics for 154 patients undergoing autologous transplantation with 4-HC purged grafts to further define this heterogeneity. Patients transplanted for the treatment of malignant lymphoma reached a peripheral blood granulocyte count of greater than 0.5 x 10(9)/l (median, 20 versus 40 days; p less than 0.001) and platelet transfusion independence (median, 30 versus 70 days; p less than 0.001) significantly faster than patients transplanted for acute non-lymphoblastic leukemia. Other diagnostic groups were intermediate. These differences were independent of graft CFU-GM content. Multiple other patient and graft factors including patient age, peripheral blood counts on day of harvest, and amounts of other hematopoietic progenitors also predicted the kinetics of engraftment in univariate and multivariate analysis. Cytomegalovirus infection during the aplastic period predicted a delay in granulocyte (p = 0.024) but not platelet recovery (p = 0.174). This analysis demonstrates that multiple patient, graft, and post-transplant factors predict the engraftment capacity of autografts, and the kinetics of engraftment with 4-HC purged grafts. The multiple predictive factors explain a significant portion of the variability in engraftment kinetics observed after transplantation with 4-HC purged autografts.

Predictive value of colony-forming unit assays for engraftment and leukemia-free survival after transplantation of chemopurged syngeneic bone marrow in rats.

We evaluated the efficacy of in vitro clonogenic assays for acute myeloid leukemia (AML) (CFU-Leuk) and granulocyte-macrophage progenitor cells derived from normal bone marrow (BM) (CFU-GM) to predict hematopoietic engraftment, median survival time (MST) and leukemia-free survival (LFS) in LBN rats that received injections of untreated or drug-treated AML and/or normal BM cells. Injection of untreated AML cells resulted in a log-linear relationship between AML cell dose and time of death from leukemia; LBN rats given 10(6) cells died with AML (MST, 24 days; range, 19-28) after injection. A minimum of 0.5-1.0 X 10(6) untreated normal BM cells was needed to insure satisfactory hematopoietic reconstitution in at least 50% of lethally irradiated LBN rats. After ex vivo incubation with graded concentrations of 4-hydroperoxycyclophosphamide (4HC) or bleomycin (BLEO), LBN AML or normal BM cells were cultured for CFU-Leuk or CFU-GM and injected into untreated or lethally irradiated syngeneic recipients. Over a variety of drug concentrations (4HC, 3-30 micrograms/ml; BLEO, 100-10,000 mU/ml) and cell doses (10(6)-10(7)/animal) examined, the log-kill estimates derived from in vitro CFU-Leuk assays correlated with the observed MST or LFS. Recovery of greater than 1% CFU-GM from 4HC- or BLEO-treated suspensions of normal BM was associated with satisfactory engraftment in lethally irradiated LBN rats. Clonogenic assays also predicted for engraftment and LFS in animals that received mixtures of AML and normal BM cells (1:10) treated with 4HC and/or BLEO. We conclude that CFU-Leuk and CFU-GM assays are useful screening techniques to develop and evaluate strategies for ex vivo purging with chemotherapeutic agents in this preclinical model of autologous marrow transplantation for AML.

Selection of normal human hematopoietic stem cells for bone marrow transplantation using immunomagnetic microspheres and CD34 antibody.

Complete yet nontoxic removal of tumor cells from autologous marrow grafts has proved difficult. New methods for separating normal stem cells from tumor cells are needed. The CD34+ cells in bone marrow, 1-2% of the low-density leukocytes, include precursors of all lymphohematopoietic lineages and probably also the primitive cells responsible for engraftment. A nontoxic, inexpensive, reproducible, and clinically applicable method for positive selection of CD34+ cells was developed. Paramagnetic microspheres coated with goat anti-mouse IgG1 are used to partition the cells; brief incubation with chymopapain is used to release them from the beads. Chymopapain exposure does not injury colony-forming cells or delay engraftment in rodents. Clinical volumes of bone marrow can be processed rapidly. In pilot experiments, the resulting grafts have a purity of 85-99% CD34+ cells and 40% median recovery of the assayable colony-forming cells. These studies form the background for a Phase I trial of autologous BMT using CD34+ stem cells.

Development and characterization of a cyclophosphamide-resistant subline of acute myeloid leukemia in the Lewis x Brown Norway hybrid rat.

Preclinical studies of resistance to alkylating agents in the Lewis x Brown Norway hybrid (LBN) rat model of acute myeloid leukemia (AML) have hitherto been limited by the sensitivity of LBN AML cells to cyclophosphamide (CY). We developed a CY-resistant subline of LBN AML by serial intravenous (IV) passage of AML cells followed by in vivo exposure to CY (100 mg/kg) 14 days later. After 18 and subsequent passages, CY-treated AML cells remained viable despite ex vivo incubation with 70 to 100 mumol/L 4-hydroperoxycyclophosphamide (4HC) or in vivo exposure to 100 to 300 mg/kg of CY. Once established, resistance to incubation with 4HC was stable in LBN AML cells after at least six serial in vivo passages without exposure to CY. Nevertheless, both control and CY-treated AML cells demonstrated similar dose-dependent sensitivity to 100 to 500 mumol/L phosphoramide mustard (PhM), the active alkylating end-product of CY activation in vivo. Levels of aldehyde dehydrogenase (ALDH), which inactivates CY by prevention of formation of PhM, were significantly elevated in these CY-resistant AML cells: cytosolic and particulate ALDH fractions from these cells were 11 to 13 times control with NAD cofactor and propanal substrate and three to four times control with NADP cofactor and benzaldehyde substrate. Further studies with this animal model of AML, in which resistance to CY is mediated by elevated ALDH activity, may elucidate mechanisms for effective elimination of drug-resistant leukemic cells ex vivo and in vivo.

Invasive fungal disease in pediatric acute leukemia patients with fever and neutropenia during induction chemotherapy: a multivariate analysis of risk factors.

We evaluated the courses of 115 consecutive cases of pediatric acute leukemia treated with induction chemotherapy. Seventy-two patients developed fever associated with neutropenia; 15 developed systemic fungal infections. We reviewed multiple demographic and treatment characteristics of these patients in an attempt to identify potential risk factors for the development of invasive fungal disease (IFD). Risk factors identified in a univariate analysis included duration of neutropenia after first fever (P less than .0001), diagnosis of acute nonlymphocytic leukemia (ANLL) (P = .003), onset of fever and neutropenia within 5 days of starting induction chemotherapy (P = .009), and multiple (greater than one) surveillance culture sites positive for fungal organisms (P = .02). In a multiple logistic regression analysis, duration of neutropenia (P less than .001) remained a significant risk factor. The study group of patients had a significantly higher risk of fungal infections than a matched group of leukemia patients developing fever with neutropenia due to postremission consolidation chemotherapy (P = .003). In the first 48 patients, 14 (29%) developed IFD. In the subsequent patients (n = 24), intravenous miconazole (5 mg/kg every 8 hours) was begun at the time of the first fever. One of the 24 patients (4%) given miconazole developed IFD. The use of miconazole was a negative risk factor for the development of IFD in univariate (P = .01) and multivariate (P = .05) analysis. We conclude that pediatric leukemia patients who develop fever associated with neutropenia during induction chemotherapy are at high risk for developing IFD. The role of intravenous miconazole at the time of the first fever in this group deserves further study.

Positive stem cell selection--basic science.

Immunologic strategies for removal of malignant cells from autologous marrow grafts by "negative selection" (i.e., "purging") requiring multiple specific monoclonal antibodies for each tumor type. "Positive selection" of marrow stem cells for grafting is a possible alternative strategy, using a monoclonal antibody which selectively recognizes lymphohematopoietic stem cells. The human hematopoietic progenitor cell antigen, CD34, is an integral cell membrane glycoprotein of approximately 115 kD, which has been molecularly cloned and sequenced. Although its function has not been determined, the glycoprotein has been characterized biochemically, including preliminary epitope mapping. Collective results from several laboratories indicate that CD34 monoclonal antibodies (My10, BI-3C5, 12.8, etc.) have the appropriate specificity to warrant testing their utility in positive selection for autologous bone marrow transplantation. First, precursors for all human hematopoietic lineages assayed (including most CFU-GM, BFU-E, CFU-MEG, CFU-EO, CFU-MIX or CFU-GEMM, pre-CFU, CFUBLAST, and terminal transferase+ B [and probably T] lymphoid precursors) are CD34+. Second, only 1.5% (mean) of low density human marrow mononuclear cells express CD34; mature human blood and marrow cells are CD34-. Endothelial cells are the only fixed tissue cells which express CD34. Third, the expression of CD34 in malignancies appears to parallel normal cellular expression: of hematopoietic malignancies, some acute leukemias and chronic myelogenous leukemia blasts are CD34+, but chronic lymphois leukemias, lymphomas, myelomas and non-hematopoietic malignancies are uniformly CD34-. Fourth, it appears feasible to isolate CD34+ cells from clinical marrow harvest samples in large scale, using either columns or immunomagnetic microspheres. Fifth, recent studies in very small numbers of non-human primates and human patients suggest that isolated CD34+ cells include the true hematopoietic stem cell, since transplantation of CD34+ cells, into myeloblated recipients results in at least short-term hematopoietic engraftment. It is anticipated that transplantation of CD34+ marrow cells may have broad applicability in clinical bone marrow transplantation.

A subline of the Brown Norway myeloid leukemia in the Lewis x Brown Norway rat: in vivo growth characteristics and development of an in vitro clonogenic assay.

A subline of Brown Norway (BN) acute myelocytic leukemia (AML) which can be propagated in suspension culture (designated IPC-81) is described. Injection into Lewis x BN F1 hybrid (LBN) rats resulted in a log-linear correlation between tumor cell dose and time till death from the onset of leukemia even after multiple (greater than 16) passages in vitro. An in vitro clonogenic assay for IPC-81 colony formation (CFU-leuk) was developed with excellent cloning efficiency (55-82%). Colonies grew without the addition of specific growth factors; syngeneic spleen-conditioned medium inhibited CFU-leuk by 40%, but co-culture with untreated normal LBN rat bone marrow cells had no effect on CFU-leuk. CFU-leuk could be detected in the bone marrow 7 to 10 days before morphologic detection of leukemia in injected animals. This cell line should prove useful in the preclinical evaluation of new strategies for treating AML and evaluating new bone marrow purging methods.