Prevalence of patientreported gastrointestinal symptoms and agreement with clinician toxicity assessments in radiation therapy for anal cancer.

Purpose Gastrointestinal (GI) symptoms pose a significant burden to patients receiving chemoradiation therapy (CRT) for anal cancer; however, the impact of symptoms from the patient perspective has not been quantified. This retrospective study examined and compared patient and clinician reports of acute GI toxicity during CRT. Materials and methods Patients treated with definitive RT using intensity-modulated radiation therapy for anal cancer between 9/09 and 11/12 were reviewed. Median RT dose was 56 Gy (range 45–56), and 76 patients (97%) received concurrent 5-fluorouracil-based chemotherapy. During RT, patients completed the 7-item Bowel Problem Scale (BPS) weekly. Clinicians assessed toxicity separately using CTCAE v. 3.0. Scores of BPS C 3 and CTCAE C 1 were considered to be clinically meaningful. Agreement of the two assessments was evaluated by Cohen’s kappa coefficient. Results Seventy-eight patients completed at least one BPS and had a corresponding clinician assessment. Patients reporting scores of C3 was highest at week 5 (n = 68) for diarrhea (44.1%), proctitis (57.4%), and mucus (48.4%), while urgency (47.6%), tenesmus (31.7%), and cramping (27%) were highest at week 4 (n = 63). Baseline bleeding scores (26.7%; score C3) improved during treatment (13.4% at week 5). ‘‘Poor’’ agreement was observed between patient- and clinician-reported proctitis (Cohen’s k = 0.11; n = 58); however, there was ‘‘good’’ agreement for diarrhea (Cohen’s k = 0.68; n = 58). Conclusions Acute GI toxicity during definitive CRT for anal cancer was most significant during weeks 4–5, while rectal bleeding improved during treatment. Discrepancies in patient- and clinician-reported symptoms demonstrate the potential for patient-reported outcomes to be useful tools for anal cancer clinical assessments

Targeting Homologous Recombination in Notch-Driven <i>C</i>. <i>elegans</i> Stem Cell and Human Tumors

<div><p>Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ <i>glp-1(ar202)</i>, a temperature-sensitive gain-of-function <i>C</i>. <i>elegans</i> NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, <i>glp-1(ar202)</i> is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.</p></div

Response of <i>C</i>. <i>elegans</i> germline tumors to ionizing radiation.

<p>(A) Time course of germ cell accumulation in wild-type and <i>glp-1(ar202)</i> hermaphrodites. Worms were stained with DAPI at the indicated times after egg laying and imaged (20x magnification). Data (mean±s.e.m) represent number of germ nuclei⁄gonad in a minimum of 10 gonad arms. (B) Representative images of germline tumors in adult <i>glp-1(ar202)</i> post radiation. Worms were irradiated at the L2-L3 stage (30h after egg laying) and DAPI stained at 40h post irradiation. (C) Stage sensitivity of germline tumors to ionizing radiation. <i>glp-1(ar202)</i> were irradiated at the L2-L3 or late L4 stage, and quantified as in (A). All experiments were performed at 25°C as described in Methods.</p

RNAi of DDR orthologs detects genes that enhance <i>ar202</i> tumor radiosensitivity.

<p>N* represents number of animals examined</p><p>RNAi of DDR orthologs detects genes that enhance <i>ar202</i> tumor radiosensitivity.</p

Knockdown of HDR genes results in abnormal morphology in <i>C</i>. <i>elegans</i> oocytes.

<p>15–25 animals were examined per group at each dose</p><p>Knockdown of HDR genes results in abnormal morphology in <i>C</i>. <i>elegans</i> oocytes.</p

Tumor cells in <i>glp-1(ar202)</i> arrest in G2 phase following radiation exposure, and are apoptosis resistant.

<p>(A) Worms were irradiated at L4 stage and after 12h stained with DAPI. Representative germline tumors are outlined. (B) Relative nuclear DNA content of distal germ nuclei in unirradiated (0Gy) or irradiated (480Gy) worms. A total of 207 nuclei from 5 unirradiated worms, and 147 nuclei from 5 irradiated worms were scored. (*<i>p</i><0.05; **<i>p</i><0.01 relative to non-irradiated control). (C) Wild-type and <i>glp-1(ar202)</i> unirradiated or irradiated germline are stained with anti-phospho-Tyr15-CDK-1 antiserum (red) and DAPI (blue) as in Methods. White bar indicates border of proliferative zone. Asterisk indicates position of distal end. Scale bar is 20 μm. (D) Comparison of radiation-induced germ cell apoptosis in wild type and <i>glp(ar202)</i>. Wild type and mutant worms were synchronized at 25°C and irradiated with 240Gy at the L4 stage. Germline apoptosis was scored in one gonad loop per worm. Incidence of germ cell death was quantified by dividing number of apoptotic germ cells by total germ cells. Data (mean±s.e.m) are from 10–12 worms⁄group. (E) Inactivation of apoptosis does not alter <i>ar202</i> response to radiation. <i>glp-1(ar202)</i> and <i>glp-1(ar202);ced-3(n717)</i> double mutant worms were irradiated at the L4 stage. Data (mean±s.e.m) are from 9–12 worms⁄group. Note the line of “<i>glp-1</i> 480Gy” is hidden behind the line of “<i>glp-1;ced-3</i> 480Gy”.</p

Radiation sensitivity of germline tumor after RNAi.

<p>(A) Knockdown of HDR ortholog genes radiosensitizes <i>ar202</i> tumors. L4 <i>ar202</i> worms were irradiated at 210Gy, an ineffective dose in this strain as shown in Fig 3B, and germline nuclei were quantitated at 72h post irradiation. Genes knocked down are classified according to their function in DNA damage repair pathways. Asterisks indicate significantly increased radiation sensitivity compared with empty vector control group, *<i>p<</i>0.01. (B) Inactivation of <i>rad-51</i> (left) or <i>mre-11</i> (right) enhances radiosensitivity of germ cells. L4 <i>ar202</i> worms were irradiated and germline nuclei counted 72h post radiation. Data (mean±s.e.m) are from 5–8 worms⁄group. Note the empty vector data set is the same in left and right panel. (C) Time course of germ cell accumulation after 210Gy combined with <i>rad-51</i> RNAi (<i>left</i>) or <i>mre-11</i> RNAi (<i>right</i>). Data (mean±s.e.m) are from 7–12 worms⁄group. Note the empty vector data set is the same in left and right panel. (D) <i>mre-11</i> inactivation extends survival of <i>glp-1(ar202)</i> after 180Gy treatment of L4 larvae. Survival assays were performed at 25°C. Data are from one representative of 3 experiments scoring ≥50 animals per group. (E) Knockdown of HDR genes radiosensitizes mitotic germ cells in distal gonad of wild-type worms. L4 stage-worms were irradiated with 60Gy and mitotic germ cells were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127862#pone.0127862.g001" target="_blank">Fig 1A</a>. Mitotic germ cells reside between the distal end of the gonad (indicated by bold asterisk in bottom panel) and the transition zone [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127862#pone.0127862.ref010" target="_blank">10</a>], which characteristically contains crescent-shaped nuclei (arrow). *<i>p<</i>0.05 and **<i>p</i><0.01 vs. empty vector control. (F) Knockdown of NHEJ genes results in vulval abnormalities post irradiation. Phenotypes were evaluated 120h post 120Gy using 75–85 worms⁄group. Somatic developmental phenotypes were quantified as wild-type vulva (WT), protruding vulva (Pvl), vulvaless (Vul), ruptured vulva (Rup) and uncoordinated (Unc). *<i>p<</i>0.05. (G) Knockdown of HDR genes in wild-type worms results in highly-abnormal oocyte chromosome morphology post irradiation. Chromosome morphology was quantified in the two oocytes (circled) closest to the spermatheca (arrow in right upper panel) at 18h post irradiation. Quantification of these data is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127862#pone.0127862.t002" target="_blank">Table 2</a>.</p