Oral cyclosporine A treatment is feasible after myeloablative conditioning in allogeneic hematopoietic stem cell transplantation.

WHAT IS KNOWN AND OBJECTIVE: The target level and route of administration of cyclosporine A (CsA) differ between transplantation centres. It is unclear whether oral CsA is sufficient to maintain target level of CsA. CASE SUMMARY: We retrospectively analysed data from 48 adult patients, who underwent myeloablative hematopoietic stem cell transplantation. Twenty-one patients (44%) tolerated CsA orally throughout the transplantation period without increased incidence of acute graft versus host disease(aGVHD). Low concentration of CsA in week 2 was associated with increased incidence of aGVHD. WHAT IS NEW AND CONCLUSION: Oral administration of CsA is safe, less time-consuming and economically advantageous. Close monitoring of CsA concentration is important.

Identification of a novel UTY-encoded minor histocompatibility antigen.

Minor histocompatibility antigens (mHags) encoded by the Y-chromosome (H-Y-mHags) are known to play a pivotal role in allogeneic haematopoietic cell transplantation (HCT) involving female donors and male recipients. We present a new H-Y-mHag, YYNAFHWAI (UTY(139-147)), encoded by the UTY gene and presented by HLA-A*24:02. Briefly, short peptide stretches encompassing multiple putative H-Y-mHags were designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were used to screen for peptide-specific HLA-restricted T cell responses in peripheral blood mononuclear cells obtained post-HCT from male recipients of female donor grafts. In one of these recipients, a CD8+ T cell response was observed against a peptide stretch encoded by the UTY gene. Another bioinformatics tool, HLArestrictor, was used to identify the optimal peptide and HLA-restriction element. Using peptide/HLA tetramers, the specificity of the CD8+ T cell response was successfully validated as being HLA-A*24:02-restricted and directed against the male UTY(139-147) peptide. Functional analysis of these T cells demonstrated male UTY(139-147) peptide-specific cytokine secretion (IFNγ, TNFα and MIP-1ß) and cytotoxic degranulation (CD107a). In contrast, no responses were seen when the T cells were stimulated with patient tumour cells alone. CD8+ T cells specific for this new H-Y-mHag were found in three of five HLA-A*24:02-positive male recipients of female donor HCT grafts available for this study.

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants.

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkin's disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkin's lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy+/-fludarabine. The cumulative incidence of acute GVHD grade II-IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61-71%. Patients with MM, HD and MDS and a history of > or =5% blasts had a less favourable outcome with a PFS of 19-38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Evidence for involvement of clonally expanded CD8+ T cells in anticancer immune responses in CLL patients following nonmyeloablative conditioning and hematopoietic cell transplantation.

We have analyzed the clonotype composition of CD8+ T cells following nonmyeloablative (NMA) conditioning and hematopoietic cell transplantation (HCT), of patients with chronic lymphocytic leukemia (CLL). Consecutive analyses of blood samples taken up to 2 years following HCT, demonstrated that CD8+ T-cell clonality was highly dynamic in the early phases after HCT, but became more stable after 4-5 months. Moreover, donor lymphocyte infusion (DLI) given for disease progression in one of the patients led to establishment of recurrent as well as new T-cell clonotypes. This coincided with disease remission, strongly suggesting that these T cells were engaged with anti-CLL cytotoxicity. To examine the functional capacity of stable clonally expanded T cells after HCT, CD8+ T cells isolated post-transplant from the recipients were stimulated ex vivo with CLL cells and subsequently analyzed by FACS for surface expression of the marker for cytotoxic activity, CD107a. Stimulation with CLL cells indeed led to surface expression of CD107a, and clonotype analyses of sorted cells demonstrated that CD107a positive T cells were stably expanded following HCT. Our data suggest that clonally expanded CD8+ T-cell clones participate in the ongoing T-cell response against CLL cells following HCT with NMA conditioning.

A dominated and resistant subpopulation causes regrowth after response to 1,3-bis(2-chloroethyl)-1-nitrosourea treatment of a heterogeneous small cell lung cancer xenograft in nude mice.

In order to address the question of the influence of a primarily chemoresistant tumor cell subpopulation on the progression of a heterogeneous tumor after cytotoxic therapy, in vitro established human small cell lung cancer cell lines of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive (592) and a resistant (NYH) tumor were used to produce mixed solid tumors in nude mice. Mixtures of 592/NYH (9:1 and 1:1) were inoculated s.c. After 3-4 weeks of tumor growth, the mice were stratified according to tumor size and randomized to treatment with BCNU 40 mg/kg i.p. (10% of lethal dose) or no treatment. Tumor growth curves were used to calculate the effect of the treatment, and changes in the relative proportions of 592 and NYH in the mixed tumors were monitored by flow cytometric DNA analysis by which the two cell lines were distinguishable due to differences in DNA content. A significant response was demonstrated in the 9:1 mixed tumors in which only 592 cells were detectable at the start of the treatment. The response was short and less pronounced compared with tumors containing only 592. In the regrowing tumors after treatment, only NYH was detected. In untreated 9:1 mixed control tumors, only 592 cells were detectable throughout the entire observation period. It is substantiated that the 592 cells were able to inhibit the growth of the NYH cells completely when grown together in 9:1 mixed tumors. This was not the case in the 1:1 mixed tumors. The 1:1 mixed tumors did not respond to BCNU, although 592 was eradicated. These results indicate that resistant and undetectable (dominated) subpopulations in heterogeneous tumors may be responsible for relapse and that the fractional size and the growth characteristics of the resistant subpopulation may determine the magnitude of the clinical response to cytotoxic treatment.

Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice.

The effects of estradiol and tamoxifen (TAM) on the estrogen-dependent human breast cancer cell line MCF-7 grown in vitro and in nude mice were compared. The effect on growth was determined by cell number in vitro and by tumor growth curves in nude mice. The effects on the cell cycle kinetics were determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase in the G1 phase from 60% to 75%. In nude mice, MCF-7 only formed tumors in estradiol-supplemented mice. No differences were observed in growth and cell kinetics between 0.1 and 1.0 mg of estradiol. Daily i.p. injections of TAM resulted in tumor growth inhibition with shrinkage of tumors. The flow cytometric DNA analysis and percentage of labeled mitosis investigations revealed no significant differences in the proliferation kinetics of TAM-treated and control tumors. Calculating the cell loss factor demonstrated an increase from 69% in control tumors to 107% in TAM-treated tumors. These experiments have shown that the cell kinetic effect of TAM is different when MCF-7 cells are grown in vitro versus in vivo. In contrast to the in vitro data, the in vivo data indicate that the growth-inhibitory effect of TAM is not mediated through a perturbation of the cell cycle.

Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis.

Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination and a newly developed procedure for storing aspirates at -80 degrees. Thirty-eight different metastases in 30 consecutive patients with small-cell anaplastic carcinoma of the lung were examined with a total of 273 fine-needle aspirations. The results on ploidy are reported in this paper. The degree of contamination of the aspirates with normal cells was determined by differential counts. The ratio of the peak channel numbers for the G1 phase of the tumor cells to that of the diploid standard (DNA index) was calculated and used for ploidy identification. Twenty-nine patients were evaluable with respect to DNA index determination. The coefficient of variation of the DNA index determinations was estimated as 0.039. In 23 (79%) patients, only one cell line could be detected. Evidence of the presence of 2 tumor cell clones with different ploidy was obtained in the remaining 6 (21%) patients. Of the 35 malignant clones thus demonstrated, 26 (74%) were significantly different from diploid (p less than or equal to 0.01). Four (11%) were hypodiploid, 3 (9%) were hypotriploid, and 19 (54%) were hypo- or near-tetraploid. Clonal heterogeneity in the tumors of 21% of the patients is a conservative estimate. Assessment of the detection limit set by the methodology used and the restricted number of samples studied in each patient indicate that the true occurrence of clonal heterogeneity in small-cell carcinoma of the lung may be much higher.

Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small cell lung cancer cells: relationship to DNA integrity and topoisomerase II.

The effect of combinations of the anthracyclines aclarubicin and daunorubicin was investigated in a clonogenic assay using the human small cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations of aclarubicin. Coordinately, aclarubicin inhibited the formation of daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein cross-links in OC-NYH cells when assayed by the alkaline elution technique. Aclarubicin had no influence on the accumulation of daunorubicin in these cells. In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by more than 300% when the cells were simultaneously incubated with the MDR modulator verapamil, aclarubicin, or the two agents combined. Yet the cytotoxicity of daunorubicin was potentiated significantly only by verapamil. The increased cytotoxicity of daunorubicin in the presence of verapamil was completely antagonized when aclarubicin was used together with the MDR modulator. Finally, the effect of daunorubicin on the DNA cleavage activity of purified topoisomerase II in the presence and absence of aclarubicin was examined. It was found that daunorubicin stimulated DNA cleavage by topoisomerase II at specific DNA sites. The addition of aclarubicin completely inhibited the daunorubicin-induced stimulation of DNA cleavage. Taken together, these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced cytotoxicity is due mainly to a drug interaction with the nuclear enzyme topoisomerase II. This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even though aclarubicin is able to increase the intracellular accumulation of daunorubicin in a MDR cell line.

Antagonistic effect of aclarubicin on the cytotoxicity of etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide in human small cell lung cancer cell lines and on topoisomerase II-mediated DNA cleavage.

The effect of combinations of the anthracycline aclarubicin and the topoisomerase II targeting drugs 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost completely antagonized by preincubating cells with nontoxic concentrations of aclarubicin. The inhibition of cytotoxicity was not seen when the cells were exposed to aclarubicin after exposure to VP-16. The inhibition was significant over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above which the toxicity of aclarubicin became apparent. A similar effect was seen on the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase II and naked DNA, aclarubicin did not stimulate the formation of cleavable complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1 microM inhibited the baseline formation of cleavable complexes elicited with the enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at aclarubicin concentrations above 1 microM an antagonistic effect was obtained. In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks were completely inhibitable by aclarubicin preincubation with no synergic dose levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated DNA cleavage. This inhibition could represent the mechanism of action of the drug and explain the lack of cross-resistance to the classical anthracyclines. The observed antagonism could have consequences for scheduling of aclarubicin with topoisomerase II-active anticancer drugs.

YKL-40 in allogeneic hematopoietic cell transplantation after AML and myelodysplastic syndrome.

YKL-40, also called chitinase-3-like-1 protein, is an inflammatory biomarker that has been associated with disease severity in inflammatory and malignant diseases, including AML, multiple myeloma and lymphomas. The objective of the current study was to assess the prognostic value of pretransplant recipient and donor plasma YKL-40 concentrations in patients with AML (n=624) or myelodysplastic syndrome (n=157) treated with allogeneic hematopoietic cell transplantation (HCT). In recipients, the plasma YKL-40 concentrations were increased when the HCT-comorbidity index was ⩾5 (P=0.028). There were no significant associations between plasma YKL-40 concentrations in recipients and any outcome measures. In donors with YKL-40 plasma concentrations above the age-adjusted 95th percentile, a trend toward increased grade II-IV acute GvHD in recipients was observed (adjusted hazard ratio 1.39 (95% confidence interval 1.00-1.94), P=0.050), with no significant associations with overall survival, treatment-related mortality or relapse. In conclusion, our study shows that YKL-40 does not aid risk stratification of patients undergoing allogeneic HCT, but suggests that YKL-40 may aid donor selection when multiple, otherwise equal, donors are available.

The superiority of combination chemotherapy including etoposide based on in vivo cell cycle analysis in the treatment of extensive small-cell lung cancer: a randomized trial of 288 consecutive patients.

Two hundred and eighty-eight patients with extensive small-cell carcinoma of the lung (SCCL) were entered into a three-arm prospective randomized trial. The purpose was both to compare etoposide with methotrexate (MTX) in a combination chemotherapy regimen otherwise consisting of vincristine (VCR), lomustine (CCNU), and cyclophosphamide (CTX) and to evaluate a treatment design based on cell kinetic observations suggesting enhanced sensitivity to etoposide three to six days after administration of VCR, CCNU, and CTX. In all three treatment arms, VCR, CCNU, and CTX were administered on day 1 of a 28-day cycle. In arm A, MTX was administered on days 14 and 17, while in arm B, MTX was replaced by etoposide administered on days 14 through 17. In arm C, MTX was also replaced by etoposide, but administered on days 3 through 6. Overall survival was significantly longer for patients treated with "early" etoposide (arm C; median, 33 weeks) as compared with arm A (MTX; median, 23 weeks) (P less than .05), but not statistically different from "late" etoposide administration (arm B; median, 27 weeks). However, for patients with initial favorable performance status (0 + 1), a significantly longer survival was obtained for those treated with early etoposide (arm C. median, 51 weeks) as compared with patients in arm A (median, 32 weeks) and arm B (median, 36 weeks) (P less than .05). Two-year survival was obtained in six patients (7%) in arm C compared with three patients (3%) in arm B and none in arm A. The study confirmed that etoposide is an active drug in the treatment of SCCL and when combined with CTX, CCNU, and VCR, the cell kinetic approach of an early administration yields the best results.

In patients older than 55 years with AML in first CR, should we search for a matched unrelated donor when an old sibling donor is available?

Allogeneic hematopoietic transplantation is increasingly used in patients aged 55 years or more with AML. The question of whether outcomes can be improved with an allele-level 8/8 HLA-matched unrelated donor (MUD) rather than an older HLA-matched sibling (MSD, more than 55 years) is still unanswered. We thus analyzed outcomes in 714 patients aged 55 years and older with AML in first CR (CR1) who received PBSCs after a reduced-intensity conditioning hematopoietic cell transplant from a MUD (n=310) or a MSD (n=404) in a recent period (2005-2010). The 3-year cumulative incidences (CIs) of non-relapse mortality were 17% and 23% with MSD and MUD, respectively (P=0.17). The 3-year CIs of relapse were 37% and 30%, respectively (P=0.12), resulting in a 3-year CI of leukemia-free survival of 46% and 47%, respectively (P=0.51). The 3-year overall survival was 49% with both MSD and MUD. In conclusion, HLA-identical sibling donors aged 55 years or more should not be excluded because of age for patients aged 55 years and older with AML in CR1.

Clonal dominance between subpopulations of mixed small cell lung cancer xenografts implanted ectopically in nude mice.

Clonal evolution of neoplastic cells during solid tumour growth leads to the emergence of new tumour cell subpopulations with diverging phenotypic characteristics which may alter the behaviour of a malignant disease. Cellular interaction was studied in mixed xenografts in nude mice and during in vitro growth of two sets of small cell lung cancer (SCLC) subpopulations (54A, 54B and NYH, NYH2). The tumour cell lines differed in cellular DNA content enabling flow cytometric DNA analysis (FCM) to be used to monitor changes in the fractional composition of the mixed cell populations. The progeny clone 54B was found to dominate the parent 54A clone when grown as mixed subcutaneous xenografts in nude mice, whereas no dominance was exerted during in vitro growth. The in vivo dominance could not be explained by differences in growth kinetics between the two tumour cell lines, and the interaction was not dependent on 54B being in excess in mixed tumours. The dominance was dependent on close in vivo contact as no remote effect on the growth of 54A was found when the dominating 54B cells were growing in the opposite flank of tumour-bearing mice. Irradiation inactivated 54B cells were unable to exert the dominating effect, indicating that the interaction required viable and proliferating cells. Clonal dominance was not found in mixed NYH-NYH2 tumours indicating that the dominance mechanism(s) may not always be operational between subpopulations in heterogeneous tumours. Recognition of interaction between tumour cell populations may result in a better understanding of the behaviour of heterogeneous human malignancies.

Optimization and comparison of the MTT assay and the 3H-TdR assay for the detection of IL-2 in helper T cell precursor assays.

The helper T cell precursor (HTLp) assay is of value for predicting graft-versus-host disease after allogeneic bone marrow transplantation. The assay requires reliable detection of the amount of interleukin 2 (IL-2) produced by one cell. To optimize the IL-2 sensitivity of our HTLp assay we tested an IL-2 dependent cell line, CTLL-2, with two different measurement methods: a colorimetric assay with tetrazolium (MTT) and an isotope incorporation assay with 3H-thymidine (3H-TdR). The test conditions examined encompassed: time without IL-2, preincubation time in IL-2, CTLL-2 cell concentration and different human sera. Due to the different measurement procedures, the volumes of the IL-2 dilutions were 75 microl in assays with MTT and 150 microl in assays with 3H-TdR. We found that it was the amount of IL-2, not the concentration, that limited the growth of CTLL-2 cells. In the most optimal setting the MTT assay could detect 0.6 pg IL-2/well, corresponding to 8 pg/ml. For the 3H-TdR assay the sensitivity was 0.6 pg/well, corresponding to 4 pg/ml. Because of the possibility of IL-2 detection in the whole culture volume (150 microl), we found that the 3H-TdR assay was superior to the MTT assay with a 10-fold better sensitivity in different human sera.

Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation.

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequency determination by limiting dilution analysis (LDA) is of value for quantifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore important that the design of the experiment yields reliable frequencies with a minimum of effort and a realistic cell requirement. We have critically evaluated the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L.W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limiting dilution assays. Experimental design and statistical analysis. J. Immunol. Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y., 1996. Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comments)] and found them inadequate for this application. The estimation of the HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. The results were in favour of the single-hit model. Ten different LDA experimental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000-1:20,000 with a coefficient of variation of 10-20% and with a minimum of manual labour.

Autoreactivity, backstimulation and reproducibility in a helper T lymphocyte precursor assay.

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) have a predictive value for alloreactivity in allogeneic bone marrow transplantation. Methodological problems in LDA include autoreactivity in the responder or stimulator cell populations and interleukin 2 (IL-2) production by the stimulator cells as a response to the responder cells (backstimulation). The extent and impact of these aspects for IL-2 production and HTLp frequency determination were studied by autologous and allogeneic mixed lymphocyte reactions with healthy volunteers and HTLp determinations from bone marrow transplantation donor/recipient pairs. We found that autoreactivity occurred in the unirradiated cells with a reproducible inter-individual variation. The immunogenicity of the stimulator cells was preserved after gamma irradiation with 50 Gy and the risks of autoreactivity and backstimulation were limited. Higher doses of irradiation decreased the immunogenicity. Immune reactions to antigens present in the serum supplement of the culture medium were seen with foetal calf serum and to a lesser extent with pooled human sera. This could be avoided by the use of autologous serum. We were unable to ensure satisfactory culture conditions in serum-free medium. The reproducibility of the HTLp frequency determinations was tested for intra- and inter-assay variation. The coefficients of variation were estimated as 24% and 35%, respectively. This was acceptable considering the range of the HTLp frequencies (1:10(2) to 1:10(7)). The influence of the extent of autoreactivity of the bone marrow donors was investigated in 28 HLA-identical sibling transplantations. We found no correlation between the autoreactivity of the donors and the HTLp frequencies. The extent of autoreactivity of the donor did not correlate with the clinical outcome in terms of acute graft-versus-host disease, treatment-related mortality, risk of relapse and overall survival. In spite of methodological difficulties and interference from autoreactivity and backstimulation, reproducible quantification of clinically significant alloreactivity can be attained.

Evaluation of X-radiation and UV-B radiation for elimination of 3H-thymidine incorporation into responder cells in the IL-2 producing helper T lymphocyte precursor assay.

The helper T lymphocyte precursor (HTLp) assay is of value for predicting graft-vs.-host disease after allogeneic bone marrow transplantation. The assay is based on limiting dilution analysis and requires detection of the amount of interleukin 2 (IL-2) produced by one activated T cell. IL-2 is detected by 3H-thymidine (3H-TdR) incorporation into the IL-2 dependent cell line, CTLL-2. Detection of IL-2 in the whole culture volume of the wells in the microtiter plates increases sensitivity, but requires elimination of 3H-TdR incorporation into the responder cells in the HTLp assay. We have compared the ability of X-radiation and ultraviolet B (UV-B) radiation to eliminate 3H-TdR incorporation. X-irradiation of the cells reduced 3H-TdR incorporation by 90% with doses up to 400 Gy, but the incorporation was still 10 times higher than in wells with stimulator cells only. UV-B irradiation (with Philips TL 12 tubes) of the cells with > or = 2 J/cm2 decreased 3H-TdR incorporation to the level of wells with stimulator cells only. Neither X-irradiation nor UV-B irradiation of the cultures affected IL-2 produced in the assay or human recombinant IL-2 measured by 3H-TdR incorporation into the IL-2 dependent cell line, CTLL-2. HTLp frequencies determined after 25 Gy X-irradiation were higher (mean 1.5, range 1.02-2.2 times higher) than HTLp frequencies determined after UV-B irradiation with 2 J/cm2.

Interactions between three subpopulations of Ehrlich ascites tumor and a P388 murine leukemia in mixed solid tumors in immune competent mice.

Cellular interactions between three subpopulations of Ehrlich ascites tumor and between these and the P388 murine leukemia were studied during growth of solid tumors obtained by mixtures of the cells in immune competent N/D mice. An immunogenic Ehrlich cell line (E1.15) induced an immunologically based growth inhibition of the two other Ehrlich cell lines (E1.80 and E1.95) which themselves were non-immunogenic. E1.15 was, however, unable to induce an immunological response against the P388 cell line. It is therefore suggested that when in close contact, immunologically induced cellular responses imposed by an immunogenic cell line on other cell lines require genetic and thereby close immunogenic resemblance between the cell lines. Another type of interaction was found between the E1.95 cell line and the P388 line which showed nearly identical growth characteristics as determined by tumor weight day 14, tumor growth curves, cell cycle times (per cent labelled mitoses) and cell cycle distributions (flow cytometric DNA analysis). After 2 weeks of growth of mixed P388/E1.95 tumors, flow cytometric DNA analysis on fine-needle tumor aspirates showed nearly total dominance of P388. This type of interaction required close cellular contact of viable cells, and no cellular immune response was elicited by the host animals. A third finding was that a faster growing Ehrlich cell line E1.95 dominated the tumors when inoculated in mixture with a slower growing subpopulation E1.80. This could be explained on the basis of the cell kinetic differences between these two cell lines.

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

The predictive value of helper T lymphocyte precursor frequencies for graft-versus-host disease and graft-versus-leukaemia effects in allogeneic bone marrow transplantation.

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis were studied in the graft-versus-host direction to assess the predictive value for outcome in allogeneic BMT. The HTLp frequencies correlated with the degree of HLA disparity. HTLp frequencies from 28 HLA-identical sibling BMT pairs had a median of 1:557 362 (range 1:9511 to <1:2 500 000). The HTLp frequencies from 20 HLA-matched unrelated and partially HLA-matched related BMT pairs had a median of 1:88 110 (range 1:4139-1:736 123). The HLA-identical sibling BMT pairs were split evenly into high and low HTLp frequency groups above and below 1:500 000. There was a trend towards a higher risk for acute GVHD > or =grade II (P = 0.075) in the high frequency group. There was no difference in TRM. The high HTLp frequency group had a significantly higher risk for chronic GVHD (P = 0.04), a significantly lower risk for relapse (P = 0.01), as well as a significantly better overall survival (P = 0.045) and leukaemia-free survival (P = 0.008). The HLA-matched unrelated and partially HLA-matched related BMT pairs were split evenly into high and low HTLp frequency groups above and below 1:90 000. There was a significantly higher risk for acute GVHD > or = grade II (P = 0.007) in the high HTLp frequency group. There was a trend towards a higher TRM in the high HTLp frequency group (P = 0.05). There were no differences in chronic GVHD, risk of relapse, overall survival and leukaemia-free survival. Analyzing all 48 patients the risk of acute GVHD > or = grade II and TRM was significantly higher (P = 0.012 and 0.021, respectively) with HTLp frequencies >1:100 000 and there was a trend towards a higher risk of relapse (P = 0.058) with low HTLp frequencies <1:400 000. Patients in the intermediate HTLp frequency group 1:100 000-1:400 000 had a trend towards improved survival (P = 0.059). The HTLp frequency seems to detect clinically significant differences in alloreactivity, that can be useful in donor selection and graft engineering.