Rationale: In informal conversation, a common concern expressed by nurses leaving our Blood & Marrow Transplant (BMT) unit included feeling overwhelmed by the amount of information required to practice safely. Given the nursing shortage and highly specialized nature of BMT nursing, it is vital that a BMT unit retain knowledgeable nursing staff.Purpose: This poster will describe how the BMT nursing staff at Froedtert Hospital assessed learning needs, presented education opportunities, and evaluated outcomes in order to increase knowledge and retention. Interventions: Early in 2006, our educator assessed education needs of our BMT nurses with a written survey. Staff indicated topics that interested them most: specific disease processes (leukemia, lymphoma, and multiple myeloma), the differences between autologous, allogeneic and non-myeloablative transplants, and oncologic emergencies. They also reported that live presentations were a preferred method of receiving the information. Our BMT Development Council, the arm of shared governance at Froedtert responsible for education, set up classes on the chosen topics. Experienced BMT nurses, and some less experienced nurses with support, chose a topic and presented a one hour-long class with Microsoft PowerPoint® slide shows and handouts. Our educator obtained approval for contact hours for attendees. Evaluation: Attendees at each class filled out an evaluation form. A five point Likert-type scale (5 = Strongly Agree, 4 = Agree, 3 = Slightly Agree, 2 = Disagree, 1 = Strongly Disagree) was used to rate the perceived increase in knowledge, the expertise of the presenter, and the appropriateness of the teaching strategies. Average ratings in these areas for all presentations were 4.6, 4.5 and 4.6 respectively. When the series of seven classes was complete, a follow-up survey on the overall effectiveness of the presentations was done. The same Likert-type scale was used to rate the effectiveness of the teaching strategies (4.3), perceived increase in knowledge (4.3), positive impact on patient care (4.2), support for peers presenting (4.3), and promoting retention (4.5). In the past year, we have had no resignations where lack of confidence in their level of knowledge was a reported factor in the decision to resign. Implications: When implemented with ongoing nursing input, peer presentations resulted in a strong feeling by nursing staff that their knowledge increased and retention was improved. Rationale: In informal conversation, a common concern expressed by nurses leaving our Blood & Marrow Transplant (BMT) unit included feeling overwhelmed by the amount of information required to practice safely. Given the nursing shortage and highly specialized nature of BMT nursing, it is vital that a BMT unit retain knowledgeable nursing staff. Purpose: This poster will describe how the BMT nursing staff at Froedtert Hospital assessed learning needs, presented education opportunities, and evaluated outcomes in order to increase knowledge and retention. Interventions: Early in 2006, our educator assessed education needs of our BMT nurses with a written survey. Staff indicated topics that interested them most: specific disease processes (leukemia, lymphoma, and multiple myeloma), the differences between autologous, allogeneic and non-myeloablative transplants, and oncologic emergencies. They also reported that live presentations were a preferred method of receiving the information. Our BMT Development Council, the arm of shared governance at Froedtert responsible for education, set up classes on the chosen topics. Experienced BMT nurses, and some less experienced nurses with support, chose a topic and presented a one hour-long class with Microsoft PowerPoint® slide shows and handouts. Our educator obtained approval for contact hours for attendees. Evaluation: Attendees at each class filled out an evaluation form. A five point Likert-type scale (5 = Strongly Agree, 4 = Agree, 3 = Slightly Agree, 2 = Disagree, 1 = Strongly Disagree) was used to rate the perceived increase in knowledge, the expertise of the presenter, and the appropriateness of the teaching strategies. Average ratings in these areas for all presentations were 4.6, 4.5 and 4.6 respectively. When the series of seven classes was complete, a follow-up survey on the overall effectiveness of the presentations was done. The same Likert-type scale was used to rate the effectiveness of the teaching strategies (4.3), perceived increase in knowledge (4.3), positive impact on patient care (4.2), support for peers presenting (4.3), and promoting retention (4.5). In the past year, we have had no resignations where lack of confidence in their level of knowledge was a reported factor in the decision to resign. Implications: When implemented with ongoing nursing input, peer presentations resulted in a strong feeling by nursing staff that their knowledge increased and retention was improved.

To evaluate the incidence, timing, nature and outcome of bulbo-membranous urethral strictures (BMUS) following high-dose-rate brachytherapy (HDRB) for prostate carcinoma, and to identify factors associated with their development. Data from 474 patients with clinically localised prostate cancer treated with HDRB between September 1997 and September 2005 were analysed. Ninety percent of patients received HDRB as a boost to external beam radiotherapy (HDRBB) and 10% received HDRB as monotherapy (HDRBM), using single implants. The first 108 patients receiving HDRBB and the 47 HDRBM patients were enrolled on prospective trials, and all patients without active follow-up at our institution had their treating physicians directly contacted. Concurrent hormonal therapy was used in 41%. Urethral strictures were diagnosed following investigation of urinary tract symptoms and were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v3.0. The median age was 66.1 years (60.3–70.1 years), with the HDRBB patients having more advanced tumours and longer follow-up than the monotherapy patients (p < 0.001 for both). The median external beam therapy dose was 46 Gy in 23 fractions, and the majority of HDRBB treatments were either 4 fractions of 4 (n = 54) or 5 Gy (n = 90), or 3 fractions of 6.5 Gy (n = 278). HDRBM was given using three fractions to a total dose of 30 Gy (n = 18), 31.5 Gy (n = 20), or 33 Gy (n = 9). At a median follow-up of 40.5 months (22.5–65.8 months), 35 patients (7.4%) had been diagnosed with BMUS, corresponding to an actuarial rate of grade 2 toxicity estimated to be 10.8% (95% CI 7.0–14.9%), with a median time to diagnosis of 22 months (range 10–68 months). Strictures in the HDRBM group have to date occurred at a median 7 months sooner than those in the HDRBB group. Strictures were initially managed with either dilatation (n = 15) or optical urethrotomy (n = 20). Following stricture diagnosis, at an additional median duration of 16 months (95% CI 2–47 months) second line therapy was required in 17 cases (49%) and third line therapy in 3 cases (9%). One patient progressed to a grade 3 stricture (urethroplasty). On proportional hazards analysis three variables were identified as having independent prognostic significance: prior transurethral resection of prostate ([TURP], Hazard Ratio [HR] 2.81, 95% CI 1.15–6.85, p = 0.023); hypertension (HR 2.83, 95% CI 1.37–5.85, p = 0.005); and dose per fraction used in HDR (HR for each 1 Gy increase per fraction 1.53, 95% CI 1.12–2.09, p = 0.008). BMUS is a post-radiotherapy toxicity that is relatively specific to hypofractionated treatment using HDRB. Its occurrence appears related to the dose per fraction of the brachytherapy and may be mediated by vascular injury, given the link to hypertension and previous TURP. Management is not standardised but is able to be effected endoscopically in the vast majority of cases. Research into potential predictive dosimetric indices in this condition is ongoing.

Forty-nine women with breast cancer were enrolled in a prospective, longitudinal study of the genetic damage caused by treatment. Assays of mutant frequency at the hprt locus in peripheral blood lymphocytes were performed at approximately 6-month intervals for 2 years. Treatment consisted of surgery alone or additional tamoxifen, radiotherapy, or chemotherapy in various combinations. At 6 months, there was an elevation of mean mutant frequency compared to initial values (P = 0.004) which persisted for as many as 2 years. A significant elevation at 6 months occurred only in the group of women who received combination chemotherapy (P = 0.005). Within this group, 5 of 15 patients had striking elevations of mutant frequency following chemotherapy (greater than 3 SD). Three of these 5 women had serum folate levels in the deficient range, while only one of 9 patients with lesser responses to chemotherapy were folate deficient. The change in mutant frequency after chemotherapy was inversely related to serum folate levels (P = 0.05) and to the number of years of smoking cigarettes (P = 0.01). We conclude that of the various modalities used to treat breast cancer, only chemotherapy was accompanied by a high risk of somatic mutation. A subset of patients manifested substantial increases in mutant frequency, often in association with low serum folate levels.

The frequency of 6-thioguanine resistant (TG(r)) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 × 10−6 (SD = 0.41 × 10−6; median value = 0.58 × 10−6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of γ-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR β and γ cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (1018) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5′ and 3′ regions of the hprt gene (418 and 418, respectively). These results demonstrate that γ-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TGr) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22-33 years) employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.

Somatic cell mutation which occurs in vivo in humans can be determined by measurement of the frequency of the 6-thioguanine-resistant (TGr) T lymphocytes in samples of peripheral blood. This frequency can be determined by either a short-term autoradiographic methodology or a longer cell-cloning methodology. The advantage of the former is the relative simplicity of the assay, while the latter allows recovery of mutant clones for further characterization. This report presents results of a longitudinal study of cancer chemotherapy nurses and other health care personnel by use of the autoradiography assay. The use of this assay in human mutagenicity monitoring and the analysis of the TGr cell frequencies are discussed in terms of age effects and validation of ‘elevated’ frequencies by use of the clonal assay.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.

Cell cloning by limiting dilution in 96-well microtiter plates has been employed to isolate colonies of human T-lymphocytes resistant to the purine analogue, 6-thioguanine (TG). These colonies show stability of the TG-resistant (TG1) phenotype, lack hypoxanthine guanine phosphoribosyl transferase (HPRT) activity and thus appear to be the result of in vivo somatic cell mutation events. In order to employ this T-lymphocyte cloning assay for quantitative determination of the in vivo TGr mutant frequency in humans, we have defined the optimal conditions for T-cell colony formation with both nonselected and TG-selected cells. The parameters investigated include medium, serum, amount of the mitogen phytohaemagglutinin, amount of T-cell growth factor (TCGF) and the number of irradiated feeder or accessory cells. Under the optimal conditions, the fraction of positive wells is proportional to the number of cells plated per well with both nonselection and TG selection conditions. T-cell cloning efficiencies therefore are independent of inoculum size. There was some evidence for a decline in TGr mutant cell cloning at densities greater than 2 x 10(4) cells per round-bottom well, possibly due to metabolic cooperation between wild-type and mutant cells. The conditions defined in this study appear to provide a quantitative measurement of the in vivo TGr mutant frequency in human T-lymphocytes.

Fifty wild-type and 164 in vivo-derived hprt mutant T-cell clones obtained from eight non-mutagen-exposed adult males with mutant frequency values in the normal range (usually less than 10 X 10(-6) were studied by Southern blot analyses to determine frequency and extent of gross structural alterations in the hprt gene. Sixteen (9.8%) of the mutant clones showed hprt changes. No site or type of lesion predominated. Relative frequencies of gross structural alterations in the recovered hprt mutants did not differ among the eight individuals, within limits detectable by the study. DNA from 201 of these 214 clones was also studied with a T-cell receptor (TCR) beta gene probe as a marker for independence of in vivo-derived clones. Some clones were also studied with a TCR gamma gene probe. Ninety-four percent of wild-type and 89% of the hprt mutants were found to originate from independent in vivo precursors. Therefore, most of the recovered hprt mutants in the study were presumably derived from separate in vivo mutations. For non-mutagenized adults with normal mutant frequencies, in vivo mutant frequencies are thus reasonable approximations of in vivo mutation frequencies, although elsewhere we show that this is not necessarily true for individuals with grossly elevated mutant frequencies.