Steen W. Hansen

Increased risk of myelodysplasia and leukaemia after etoposide, cisplatin, and bleomycin for germ-cell tumours

Among the cytostatic drugs only the alkylating agents have been firmly established as being leukaemogenic. This report describes 4 cases of acute myeloid leukaemia and 1 of myelodysplasia occurring in a cohort of 212 patients with germ-cell tumours treated with etoposide, cisplatin, and bleomycin. The mean cumulative risk of leukaemic complications was 4.7% (SE 2.3) 5.7 years after start of etoposide-containing chemotherapy, and, compared with the risk in the general population, the relative risk of overt leukaemia was 336 (95% CI 92-861). No leukaemias were detected in a previous cohort of 127 patients with germ-cell tumours treated with cisplatin, bleomycin, and vinblastine. The increased risk of leukaemia was most probably due to etoposide alone or in combination with cisplatin or bleomycin, since other published work has also not revealed an excess of leukaemias among patients with germ-cell tumours treated with only cisplatin, bleomycin, and vinblastine. The risk of leukaemia was dose related since all 5 patients with leukaemic complications were among the 82 who had received a cumulative dose of more than 2000 mg/m2 etoposide, whereas no leukaemias were observed among 130 patients who had received up to 2000 mg/m2 (p = 0.004). 3 of the leukaemic patients had balanced chromosome translocations affecting bands 11q23 and 21q22. These translocations, and perhaps also other balanced aberrations, seem to be characteristic of myelodysplasia and acute leukaemia occurring after therapy with cytostatic agents acting on DNA-topoisomerase II.

Effect of graded testicular doses of radiotherapy in patients treated for carcinoma-in-situ in the testis

PURPOSE To determine the effect of radiotherapy in doses 14 to 20 Gy on eradication of carcinoma-in-situ (CIS) testis and on the Leydig cell function. PATIENTS AND METHODS Forty-eight patients presented with unilateral testicular germ cell cancer and CIS of the contralateral testis. The CIS-bearing testis was treated with daily irradiation doses of 2 Gy, 5 days a week, to a cumulative dose of 20 Gy (21 patients), 18 Gy (three patients), 16 Gy (10 patients), and 14 Gy (14 patients). RESULTS All patients treated at dose levels 20 Gy to 16 Gy achieved histologically verified complete remission without signs of recurrence of CIS after an observation period of more than 5 years. One of 14 patients treated at dose level 14 Gy had a relapse of CIS 20 months after irradiation. Leydig cell function was examined before and regularly after radiotherapy in 44 of 48 patients. The levels of testosterone were lower after radiotherapy than before. Testosterone showed a stable decrease for more than 5 years after treatment (3.6% per year) without dose dependency. The levels of luteinizing hormone and follicle-stimulating hormone were increased after radiotherapy. The need of androgen substitution therapy was similar at all dose levels. CONCLUSION Testicular irradiation is a safe treatment at dose level 20 Gy (10 x 2 Gy). Decrease of dose to 14 Gy (7 x 2 Gy) might lead to risk of relapse of CIS. Impairment of hormone production without clinically significant dose dependency is seen in the dose range 14 to 20 Gy.

Long-term fertility and Leydig cell function in patients treated for germ cell cancer with cisplatin, vinblastine, and bleomycin versus surveillance.

Fertility and Leydig cell function were investigated in 31 patients previously treated for nonseminomatous testicular cancer. Twenty-two patients with metastatic cancer had received cisplatin-based chemotherapy, and the median follow-up was 64 months (range, 42 to 100 months). Nine patients without metastases were treated with orchiectomy alone, and follow-up in this group was a median of 61 months (range, 40 to 77 months). None of the patients have relapsed and retroperitoneal lymph node dissection was not performed in any patient. Both the concentration of spermatozoa and the volume of the remaining testis are significantly reduced in patients who had previously received chemotherapy when compared with patients treated with orchiectomy alone (P less than .05). There were no significant differences between groups when comparing morphology, motility, and penetration of the spermatozoa. Subclinical Leydig cell dysfunction with normal testosterone and elevated luteinizing hormone (LH) was observed in one patient (11%) treated with orchiectomy alone, while 59% of the patients who had received chemotherapy had elevated LH (P less than .05). We conclude that cisplatin-based chemotherapy leads to a persistent impairment of fertility and Leydig cell function in the majority of patients with testicular cancer.

Impaired Testicular Function in Patients With Carcinoma-In-Situ of the Testis

PURPOSE To elucidate the biologic association between germ cell neoplasia and testicular dysfunction, through investigation of Leydig cell function and semen quality in men with carcinoma-in-situ (CIS) of the testis. PATIENTS AND METHODS We examined two groups of men, unilaterally orchidectomized for testicular cancer. Biopsy of the contralateral testis had showed CIS in a group of 24 patients and no evidence of CIS in the other group of 30 patients. Semen quality and serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared in these two groups of men after orchidectomy but before further treatment for testicular cancer. RESULTS Significantly higher LH levels (median, 8.1 IU/L v 4.8 IU/L; P < .001) and generally lower testosterone levels (median, 12.5 nmol/L v 15.5 nmol/L; P = .13) were found in the CIS group. The proportion of patients with Leydig cell dysfunction was higher in the group of patients with CIS (11 of 24) than in the group of patients without (two of 30) (P = .01). Sperm concentration and total sperm count were significantly lower (P < .001) in patients with CIS (median, 0.03 x 10(6)/mL and 0.10 x 10(6), respectively) than in patients without (median, 9.1 x 10(6)/mL and 32 x 10(6), respectively), whereas the levels of FSH were significantly higher (P < .001) in the former group of men (median, 19.6 IU/L v 9.0 IU/L). CONCLUSION Not only spermatogenesis but also Leydig cell function is impaired in testes with CIS. This impairment could be due to common factors in the pathogenesis of germ cell neoplasm and testicular dysfunction. Alternatively, CIS cells may have a negative impact on Leydig cell function.

Long-term effects on renal function and blood pressure of treatment with cisplatin, vinblastine, and bleomycin in patients with germ cell cancer.

Long-term effects of cisplatin on renal function were investigated in 34 patients with germ cell cancer observed for a median of 65 months (range, 43 to 97 months). All patients achieved a complete remission after treatment with cisplatin (median dose 583 mg/m2), vinblastine, and bleomycin. None of the patients relapsed during follow-up. During treatment the glomerular filtration rate (GFR) decreased by 18% (P less than .05). During follow-up kidney function recovered in ten patients and partly improved in eight patients. Changes in plasma creatinine did not consistently correspond to alterations in GFR. The mean increase in systolic blood pressure during follow-up did not differ from the increase seen in a group of age-matched healthy men. The mean increase in diastolic pressure, however, was significant (P less than .05), but was entirely due to hypertension observed in six patients. Renography of these patients was normal. We conclude that the decrease in GFR observed during treatment with cisplatin is partly reversible. Cisplatin-treated patients have an increased risk of developing hypertension years after treatment.

Raynaud's phenomenon in patients treated with cisplatin, vinblastine, and bleomycin for germ cell cancer: measurement of vasoconstrictor response to cold.

The prevalence of Raynauds phenomenon (RP) was studied in patients treated with cisplatin, vinblastine, and bleomycin. Thirty-two patients with germ cell cancer were followed for a median of 78 months (range, 49 to 106 months). All had obtained complete remission during treatment; none had relapsed at last follow-up examination. All blood samples, including serum magnesium, were normal. The arterial vasoconstrictor response to cold in the finger was measured by cuff- and strain-gauge techniques at 30, 15, and 10 degrees C. Blood pressure (BP) was measured auscultatorily, using a 12-cm broad cuff on the ipsilateral upper arm. Fourteen patients (44%) had developed anamnestic RP, and all showed an exaggerated response to cold; arterial closure was provoked in nine patients. Eighteen patients (56%) were without finger symptoms. These patients had an exaggerated response to cold in comparison with controls, and in two patients, RP was provoked. None of the patients had an increased systolic pressure gradient from arm to finger when compared with a control group. Thus, an exaggerated cold response was found to be a prolonged vasospastic side effect not only in patients with RP, but also in patients without finger symptoms.

Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5–51) and 9 days (range 5–51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4–99.4) and the median recovery was 52.3% (15.2–146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243–249.

Enhanced pulmonary toxicity in smokers with germ-cell cancer treated with cis-platinum, vinblastine and bleomycin: a long-term follow-up.

The long-term effects of bleomycin on pulmonary function were studied. Thirty-four patients with germ-cell cancer were followed for an average of 64 months (range 43-98 months). All had obtained complete remission during treatment and none had relapsed at the follow-up examination. Pulmonary function was tested by measurements of total lung capacity (TLC), vital capacity (VC), forced expiratory volume in one second (FEV1) and single breath diffusion capacity for carbon monoxide (TLCO). TLC and VC were significantly reduced by the treatment (P less than 0.05), but normalized during the follow-up. TLCO was initially reduced to a predicted median of 83%. In the smokers the initial TLCO was at a predicted median of 79%, while in non-smokers a median of 88% was predicted. During the first 2 months of treatment, TLCO increased both in smokers and non-smokers to predicted medians of 90% and 91%. Subsequently, however, a significant decrease to 72% was noted in the smokers, while the non-smokers had only an insignificant decrease to 84%. The decrease in TLCO was irreversible but not progressive. We conclude that bleomycin treatment is associated with a long-term sustained reduction in TLCO. The changes were most pronounced in the smokers.

Selective loss of progenitor subsets following clinical CD34+cell enrichment by magnetic field, magnetic beads or chromatography separation

In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88–98%), 86% (62–97%), and 49% (18–85%), and median yield of 65% (54–100%), 40% (21–74%), and 30% (8–55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I–II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2–4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin–avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10−5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.

Simultaneous estimation of the form factor and structure factor for globular particles in small-angle scattering

Small-angle scattering data from non-dilute solutions of particles are often analysed by indirect Fourier transformation using a specific model structure factor to obtain an estimate of the distance distribution function that is free from concentration effects. A new approach is suggested here, whereby the concentration effects are expressed solely through real space functions without the use of an explicit structure factor. This is done by dividing the total distance distribution function for the scattering into three different contributions, as suggested by Kruglov [(2005). J. Appl. Cryst. 38, 716–720]: (i) the single particle distribution which is due to intraparticle effects, (ii) the excluded volume distribution from excluded volume effects which is only dependent upon the geometry of the particles, and (iii) a structure distribution which is due to the remaining interaction between the particles. Only the single particle distribution and the structure distribution are allowed to vary freely (within the restrictions of a smoothness constraint). These two distributions may be separated mainly because they differ in their regions of support in real space. From the estimated distributions the structure factor can be calculated. For deviations of particles from spherical symmetry, the excluded volume distribution may be approximated by that of an ellipsoid of revolution. Excluded volume distributions have been calculated for ellipsoids of revolution of axial ratios between 0.1 and 10 and implemented in the program IFTc, which is described in the appendix. The validity of the approach is demonstrated for globular particles.