Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection.

In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos.

In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Total skin electron beam therapy for cutaneous T-cell lymphomas in the Netherlands: A retrospective analysis of treatment outcomes and selection for high or low dose schedule.

PURPOSE: Total skin electron beam therapy (TSEBT) is used mostly in the treatment of cutaneous T cell lymphoma. In this study we describe the results of TSEBT applied in the Netherlands using two different schedules, a conventional dose schedule of 35 Gy and a low-dose schedule of 12 Gy. We aimed to evaluate the treatment results in and compare treatment outcomes between the two treatment groups and to further define indications for both doses. METHODS: In the LUMC, Leiden, we performed a retrospective analysis of 51 patients treated with TSEBT between January 2008 and December 2018, with follow-up untill December 2019. Thirty one patients were treated with 35 Gy and twenty with 12 Gy. The dose was chosen based on the severity of skin involvement. Outcome measures were time to meaningful progression, survival, response rate and toxicity. RESULTS: Time to meaningful progression was 5.1 months with no significant differences between dose groups (P = 0.77). Overall survival was 27.4 months. Both time to progression and survival were significantly better for T2 vs T3 stage. Overall response rate was 80.4 %. Both dose groups showed improvement of symptoms. Treatment was generally well tolerated. CONCLUSIONS: Both high-dose and low-dose TSEBT offer similar results for TMP and OS. It remains unclear which patients benefit most from a high-dose schedule. We propose to use the low-dose schedule as a standard for TSEBT and use supplementary boosts or escalation to high-dose treatment for patients unresponsive to the low-dose schedule.

Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers.

BACKGROUND: Current risk models for renal cell carcinoma (RCC) based on clinicopathological factors are sub-optimal in accurately identifying high-risk patients. Here, we perform a head-to-head comparison of previously published DNA methylation markers and propose a potential prognostic model for clear cell RCC (ccRCC). PATIENTS AND METHODS: Promoter methylation of PCDH8, BNC1, SCUBE3, GREM1, LAD1, NEFH, RASSF1A, GATA5, SFRP1, CDO1, and NEURL was determined by nested methylation-specific PCR. To identify clinically relevant methylated regions, The Cancer Genome Atlas (TCGA) was used to guide primer design. Formalin-fixed paraffin-embedded (FFPE) tissue samples from 336 non-metastatic ccRCC patients from the prospective Netherlands Cohort Study (NLCS) were used to develop a Cox proportional hazards model using stepwise backward elimination and bootstrapping to correct for optimism. For validation purposes, FFPE ccRCC tissue of 64 patients from the University Hospitals Leuven and a series of 232 cases from The Cancer Genome Atlas (TCGA) were used. RESULTS: Methylation of GREM1, GATA5, LAD1, NEFH, NEURL, and SFRP1 was associated with poor ccRCC-specific survival, independent of age, sex, tumor size, TNM stage or tumor grade. Moreover, the association between GREM1, NEFH, and NEURL methylation and outcome was shown to be dependent on the genomic region. A prognostic biomarker model containing GREM1, GATA5, LAD1, NEFH and NEURL methylation in combination with clinicopathological characteristics, performed better compared to the model with clinicopathological characteristics only (clinical model), in both the NLCS and the validation population with a c-statistic of 0.71 versus 0.65 and a c-statistic of 0.95 versus 0.86 consecutively. However, the biomarker model had limited added prognostic value in the TCGA series with a c-statistic of 0.76 versus 0.75 for the clinical model. CONCLUSION: In this study we performed a head-to-head comparison of potential prognostic methylation markers for ccRCC using a novel approach to guide primers design which utilizes the optimal location for measuring DNA methylation. Using this approach, we identified five methylation markers that potentially show prognostic value in addition to currently known clinicopathological factors.

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected.

Effect of conditioning regimens on graft failure in myelofibrosis: a retrospective analysis.

In myelofibrosis, the introduction of reduced-intensity conditioning (RIC) preceding allogeneic stem cell transplantation (SCT) resulted in lower transplant-related mortality rates compared with myeloablative conditioning. However, lowering the intensity of conditioning may increase the risk of graft failure in myelofibrosis, although hitherto this has not been indisputably proven. We here report the outcome of 53 patients who underwent allogeneic SCT with different conditioning regimens (RIC and non-myeloablative (NMA)) in three transplantation centers in the Netherlands. The cumulative incidence of graft failure within 60 days after SCT was high (28%), and this was primarily associated with the intensity of the conditioning regimen. Cumulative neutrophil engraftment at 60 days was lower in patients who received NMA conditioning compared with those who received RIC (56% vs 84%, P=0.03). Furthermore, of six patients who received a second transplantation after graft failure, the three patients with RIC regimens subsequently engrafted, whereas the three patients who received a second NMA regimen did not. This study indicates that in myelofibrosis, NMA regimens result in high engraftment failure rates. We propose the use of more intensive conditioning regimens, incorporating busulfan or melphalan.

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis.

BACKGROUND: We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS: Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS: MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S): This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

Participation in colorectal cancer screening trials after first-time invitation: a systematic review.

BACKGROUND AND STUDY AIM: Colorectal cancer (CRC) screening is implemented by an increasing number of countries. Participation rates of screening programs influence the health benefit and cost-effectiveness of the applied method. The aim was to systematically review participation rate after first-time invitation for CRC screening with fecal occult blood test (FOBT), sigmoidoscopy, colonoscopy, and/or computed tomography (CT) colonography. METHODS: A systematic literature search was performed prior to October 1 2009. Prospective CRC screening studies of unselected populations reporting participation rates were included. RESULTS: After meta-analyses, overall participation rates were found to be 47 % for FOBT, 42 % for fecal immunologic tests (FITs), 35 % for sigmoidoscopy, 41 % for sigmoidoscopy combined with FIT/FOBT, 28 % for colonoscopy, and 22 % for CT colonography. Studies comparing screening methods showed higher participation rates for less invasive methods. Studies comparing invitation methods showed higher participation rates with general practitioner involvement, a more personalized recruitment approach, and reduction of barriers that discourage participation. CONCLUSIONS: Knowledge of identified factors affecting CRC screening participation can be used to improve screening programs.

Uterine fibrosarcoma in a Warmblood mare.

This paper describes a case of uterine fibrosarcoma in an 18-year-old Warmblood mare. The mare had exhibited bloody fluid accumulation inside the uterus and vaginal haemorrhagic discharge since the previous foaling. The mare was euthanized, and on pathological examination, in addition to the uterine neoplasia, multiple metastases were found in the lungs, liver and spleen. The histological and immunohistochemical examination determined that the tumour was a fibrosarcoma. To our knowledge, this is the first paper to describe a uterine fibrosarcoma in a mare.

The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test performance and transferability.

The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.

An abortion of monozygotic twins in a warmblood mare.

Naturally occurring monozygotic twins are extremely rare in the horse. This paper describes an abortion in a mare after 260 days of pregnancy with monozygotic twins, one a fresh foal and the other a mummified foal.

Magnesium intake and colorectal cancer risk in the Netherlands Cohort Study.

Energy-adjusted magnesium intake was nonsignificantly inversely related to risk of colorectal cancer (n=2328) in the Netherlands Cohort Study on Diet and Cancer that started in 1986 (n=58 279 men and 62 573 women). Statistically significant inverse trends in risk were observed in overweight subjects for colon and proximal colon cancer across increasing quintiles of magnesium uptake (P-trend, 0.05 and 0.02, respectively). Although an overall protective effect was not afforded, our results suggest an effect of magnesium in overweight subjects, possibly through decreasing insulin resistance.

Interaction between smoking, GSTM1 deletion and colorectal cancer: results from the GSEC study.

Cigarette smoking has inconsistently been associated with an increased risk of colorectal cancer. One of the enzymes responsible for the detoxification of the carcinogenic compounds present in tobacco smoke is glutathione S-transferase-mu (GST-mu). The gene that codes for this enzyme is GSTM1. In this study, we evaluated the associations and interaction between GSTM1 deletion, smoking behaviour and the development of colorectal cancer. We performed a pooled analysis within the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). We selected six studies on colorectal cancer, including 1130 cases and 2519 controls, and restricted our analyses to Caucasians because the number of patients from other races was too limited. In addition we performed a meta-analysis including the studies from the GSEC database and other studies identified on MEDLINE on the same subject. The prevalence of the GSTM1 null genotype was within the range reported in other studies: 51.8% of the cases had the GSTM1 null genotype versus 56.6% of the controls. No significant association between the GSTM1 null genotype and colorectal cancer was found (odds ratio 0.92, 95% confidence interval 0.73-1.14). Our results suggest a possible positive association between lack of the GST-mu enzyme and colorectal cancer for non-smoking women (odds ratio 1.47, 95% confidence interval 0.80-2.70). There was no interaction between the effects of smoking and GSTM1 genotype on colorectal cancer risk in men and women (chi2=0.007, p=0.97). Our findings do not support an association between the GSTM1 null genotype and colorectal cancer. In addition, we did not find any modification of the smoking-induced colorectal cancer risk by GSTM1 genotype

Flt-3 ligand (FL) drives differentiation of rat bone marrow-derived dendritic cells expressing OX62 and/or CD161 (NKR-P1).

Bone marrow-derived dendritic cells (DC) of the rat have not been as well characterized as those from the mouse. Here, large quantities of bone marrow-derived rat DC were generated when Flt-3 ligand (FL) was used as an adjunct to granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). These cells displayed a typical DC phenotype, expressing MHC class II, CD54, CD80, CD86, and CD11b/c. These DC also uniformly expressed low levels of CD161 and expressed OX62 in a bimodal distribution. Few cells were recovered from cultures grown without FL, and they failed to express OX62 or CD161. The DC generated with FL were more potent antigen-presenting cells in mixed lymphocyte cultures than cells grown without FL, and among FL-derived cells, the OX62+ cells were slightly more stimulatory than OX62- cells. Thus, FL is a useful cytokine for obtaining large quantities of functional rat DC subsets in vitro.

Tibolone: a compound with tissue specific inhibitory effects on sulfatase.

The aim was to test whether sulfatase activity is differently regulated by tibolone in human bone, endometrium and breast cells since selective inhibition of sulfatases in various tissues may contribute to the tissue-specificity of tibolone. Tibolone, its 3 alpha- and 3 beta-hydroxy metabolites and their 3-sulfated forms, and its Delta(4)-isomer strongly (70-90%) inhibited the sulfatase activity in human breast cell lines (two T-47D clones) and intermediately (8-43%) in human endometrial cells (HEC-1A). In contrast, they did not inhibit sulfatase in two human osteoblast-like cell lines (MG 63, HOS TE-85). The specific sulfatase inhibitor, EMATE, showed inhibition in all cell lines. Just as estrone sulfate, 3 alpha-sulfated tibolone was also converted by sulfatase to the unconjugated 3 alpha-hydroxy-tibolone intracellularly in all cell lines. The tissue specific inhibition pattern of sulfatase activity by tibolone and its metabolites suggest that tibolone could be protective against development of mammary carcinomas, whereas it retains favorable estrogenic effects on bone.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Role of NK cells in adoptive immunotherapy of metastatic colorectal cancer in a syngeneic rat model.

This article reviews our immunotherapy research with natural killer (NK) cells in a syngeneic rat colorectal cancer liver and lung metastasis model. Using adoptive transfer of interleukin (IL)-2-activated NK cells, NK cells were shown to selectively infiltrate the tumors. More NK cells were found in tumors when the NK cells were directly injected into tumor-draining blood vessels than when the cells were injected in systemic blood vessels. Under optimal conditions, a limited, though significant, effect of adoptively transferred NK cells on tumor growth was shown. We observed that both endogenous and adoptively transferred NK cells were predominantly present in the stroma surrounding the tumor cell nodules. It is possible that they did not penetrate the nodules containing the tumor cells because of the presence of a basal membrane-like structure around these nodules. Adoptively transferred NK cells may initiate elimination of tumor cells by activating other effector cells, whereas some may eliminate tumor cells by direct cell-cell contact. A diverse array of molecules was shown to be involved in this process. CD45 on NK cells was found to be important in initiating the lysis-inhibitory signal upon binding of 'self' major histocompatibility complex (MHC) class I on potential target cells. Our results indicate that NK-cell cancer therapy is still promising and needs improvement.

NK cells modulate MHC class I expression on tumor cells and their susceptibility to lysis.

Cytotoxicity and production of cytokines are two important functions of NK cells. These two different NK functions were studied in a syngeneic rat model in relation to MHC class I expression. We focussed on the mechanism by which NK cells modulate MHC class I expression on target cells and how this interferes with NK cell-mediated lysis. Using transfection experiments an inhibitory role on NK cell cytotoxicity for expression of target cells of RT1.A, rat MHC class I, was found. Co-culturing syngeneic tumor cells and NK cells resulted in enhanced MHC class I expression on the surviving tumor cell fraction, which was less susceptible to NK lysis. Increased tumor cell MHC class I was due to production of a soluble factor by NK cells, most likely interferon gamma. The regulatory function of NK cells shows here, that the enhancing of MHC class I expression on tumor cells in vitro and in vivo, results in downregulation of their target cell killing, but at the same time may facilitate the cytotoxic T cell function.

The clinical pharmacology of alkylating agents in high-dose chemotherapy.

Alkylating agents are widely used in high-dose chemotherapy regimens in combination with hematological support. Knowledge about the pharmacokinetics and pharmacodynamics of these agents administered in high doses is critical for the safe and efficient use of these regimens. The aim of this review is to summarize the clinical pharmacology of the alkylating agents (including the platinum compounds) in high-dose chemotherapy. Differences between conventional and high doses will be discussed.

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography.

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N'-diethylene-N"-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen-phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25-2500 ng/ml for monochloroTEPA and 25-5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at -80 degrees C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.