Associations of tissue tumor mutational burden and mutational status with clinical outcomes in KEYNOTE-042: pembrolizumab versus chemotherapy for advanced PD-L1-positive NSCLC.

BACKGROUND: We evaluated whether tissue tumor mutational burden (tTMB) and STK11, KEAP1, and KRAS mutations have clinical utility as biomarkers for pembrolizumab monotherapy versus platinum-based chemotherapy in patients with programmed death ligand 1 (PD-L1)-positive (tumor proportion score ≥1%) advanced/metastatic non-small-cell lung cancer (NSCLC) without EGFR/ALK alterations in the phase III KEYNOTE-042 trial. PATIENTS AND METHODS: This retrospective exploratory analysis assessed prevalence of tTMB and STK11, KEAP1, and KRAS mutations determined by whole-exome sequencing of tumor tissue and matched normal DNA and their associations with outcomes in KEYNOTE-042. Clinical utility of tTMB was assessed using a prespecified cut point of 175 mutations/exome. RESULTS: Of 793 patients, 345 (43.5%) had tTMB ≥175 mutations/exome and 448 (56.5%) had tTMB <175 mutations/exome. No association was observed between PD-L1 expression and tTMB. Continuous tTMB score was associated with improved overall survival (OS) and progression-free survival among patients receiving pembrolizumab (Wald test, one-sided P < 0.001) but not those receiving chemotherapy (Wald test, two-sided P > 0.05). tTMB ≥175 mutations/exome was associated with improved outcomes for pembrolizumab versus chemotherapy, whereas tTMB <175 mutations/exome was not {OS: hazard ratio, 0.62 [95% confidence interval (CI) 0.48-0.80] and 1.09 (95% CI 0.88-1.36); progression-free survival: 0.75 (0.59-0.95) and 1.27 (1.04-1.55), respectively}. Improved OS [hazard ratio (95% CI)] for pembrolizumab versus chemotherapy was observed regardless of STK11 [STK11 mutant (n = 33): 0.37 (0.16-0.86), STK11 wild-type (n = 396): 0.83 (0.65-1.05)]; KEAP1 [KEAP1 mutant (n = 64): 0.75 (0.42-1.35), KEAP1 wild-type (n = 365): 0.78 (0.61-0.99)], or KRAS [KRAS mutant (n = 69): 0.42 (0.22-0.81); KRAS wild-type (n = 232): 0.86 (0.63-1.18)] mutation status. CONCLUSION: tTMB with a cut point of ≥175 mutations/exome is a potential predictive biomarker for pembrolizumab monotherapy for advanced/metastatic PD-L1 tumor proportion score ≥1% NSCLC. Pembrolizumab is a standard first-line treatment in this setting regardless of STK11, KEAP1, or KRAS mutation status.

Time-resolved relaxation and fragmentation of polycyclic aromatic hydrocarbons investigated in the ultrafast XUV-IR regime.

Polycyclic aromatic hydrocarbons (PAHs) play an important role in interstellar chemistry and are subject to high energy photons that can induce excitation, ionization, and fragmentation. Previous studies have demonstrated electronic relaxation of parent PAH monocations over 10-100 femtoseconds as a result of beyond-Born-Oppenheimer coupling between the electronic and nuclear dynamics. Here, we investigate three PAH molecules: fluorene, phenanthrene, and pyrene, using ultrafast XUV and IR laser pulses. Simultaneous measurements of the ion yields, ion momenta, and electron momenta as a function of laser pulse delay allow a detailed insight into the various molecular processes. We report relaxation times for the electronically excited PAH*, PAH+* and PAH2+* states, and show the time-dependent conversion between fragmentation pathways. Additionally, using recoil-frame covariance analysis between ion images, we demonstrate that the dissociation of the PAH2+ ions favors reaction pathways involving two-body breakup and/or loss of neutral fragments totaling an even number of carbon atoms.

Molecular determinants of clinical outcomes with pembrolizumab versus paclitaxel in a randomized, open-label, phase III trial in patients with gastroesophageal adenocarcinoma.

BACKGROUND: In the phase III KEYNOTE-061 trial (NCT02370498), pembrolizumab did not significantly improve overall survival versus paclitaxel as second-line therapy for gastric/gastroesophageal junction (GEJ) adenocarcinoma with programmed death-ligand 1 (PD-L1) combined positive score (CPS) ≥1 tumors. The association of tissue tumor mutational burden (tTMB) status and clinical outcomes was determined, including the relationship with CPS and microsatellite instability-high (MSI-H) status. PATIENTS AND METHODS: In patients with whole exome sequencing (WES) data [420/592 (71%); pembrolizumab, 218; paclitaxel, 202], the association of tTMB with objective response rate (ORR; logistic regression), progression-free survival (PFS; Cox proportional hazards regression), and overall survival (OS; Cox proportional hazards regression) were measured using one-sided (pembrolizumab) and two-sided [paclitaxel] P values. tTMB was also evaluated using FoundationOne®CDx [205/592 (35%)]. Prespecified equivalent cut-offs of 175 mut/exome for WES and 10 mut/Mb for FoundationOne®CDx were used. RESULTS: WES-tTMB was significantly associated with ORR, PFS, and OS in pembrolizumab-treated (all P < 0.001) but not paclitaxel-treated patients (all P > 0.6) in univariate analysis. The area under the receiver operating characteristics curve for WES-tTMB and response was 0.68 [95% confidence interval (CI) 0.56-0.81] for pembrolizumab and 0.51 (95% CI 0.39-0.63) for paclitaxel in univariate analysis. There was low correlation between WES-tTMB and CPS in both treatment groups (r ≤ 0.16). WES-tTMB remained significantly associated with all clinical endpoints with pembrolizumab after adjusting for CPS and with PFS and OS after excluding known MSI-H tumors (n = 26). FoundationOne®CDx-tTMB demonstrated a positive association with ORR, PFS, and OS in pembrolizumab-treated patients (all P ≤ 0.003) but not PFS or OS in paclitaxel-treated patients (P > 0.1). CONCLUSION: This exploratory analysis from KEYNOTE-061 is the first to demonstrate a strong association between tTMB and efficacy with pembrolizumab but not paclitaxel in patients with gastric/GEJ adenocarcinoma in a randomized setting. Data further suggest tTMB is a significant and independent predictor beyond PD-L1 status.

Anal cancer survival: a socioeconomic analysis.

INTRODUCTION: Squamous cell carcinoma is the most common variant of anal malignancy. Certain disease-related factors have been established in determining survival. These include tumour size, differentiation and nodal involvement. Other factors such as HIV status, human papillomavirus infection, smoking and socioeconomic disparity may have important roles, however few data are available on the UK population. We aim to correlate social deprivation and survival of anal cancer patients at a tertiary centre. MATERIALS AND METHODS: All consecutive cases diagnosed with anal squamous cell carcinoma and treated as per local protocol between July 2010 and April 2017 were included. The pathological and demographical details were collected from a prospectively maintained database. Socioeconomic deprivation was defined for each postcode using the Index of Multiple Deprivation decile compiled by local governments in England. Survival was estimated using Kaplan-Meier analysis and Cox regression was used to investigate the effect of different factors on overall survival. RESULTS: A total of 129 patients with anal squamous cell carcinoma over a median follow-up of 43 months were included. Overall survival for the entire patient cohort was 87.7% (95% confidence interval, CI, 82.0-93.7%), 75.5% (95% CI 67.5-84.5%) and 68.9% (95% CI 59.7-79.6%) at one year, three years and five years, respectively. On multivariate analysis, Index of Multiple Deprivation and income do not significantly influence overall survival (p = 0.79, hazard ratio, HR, 1.07; 95% CI 0.61-1.63), (p = 0.99, HR=1.00; 95% CI 0.61-1.63), respectively. Increased risk of death was observed for male sex (p = 0.02, HR=2.80; 95% CI 1.02-5.50) and larger tumour size (p = 0.01, HR=1.64; 95% CI 1.12-2.41). CONCLUSION: In contrast to US studies, there is little difference in survival between the least deprived and most deprived groups. We attribute this to equal access to intensity-modulated radiation therapy-based chemoradiotherapy. Thus, a highly effective treatment made available to all mitigates any survival difference between socioeconomic groups.

Looking beyond the ovary for oncofertility care in women: uterine injury as a potential target for fertility-preserving treatments.

Treatment for cancer has the potential to significantly diminish fertility and, further, to negatively impact the obstetrical outcomes of pregnancies that do occur. Cancer survivors have decreased rates of fertility and increased rates of pregnancy complications, such as preterm birth and low birth weight, after exposure to chemotherapy. To date, research on the impact of chemotherapy and radiotherapy on fertility and pregnancy outcomes has focused largely on the gonadotoxic effect of cancer treatments on ovaries, while the uterus and endometrium have not been extensively studied. It is intuitive, however, that decreased fertility and poorer obstetrical outcomes may be substantially mediated through injury to a highly mitotic tissue like the endometrium, which is also central to embryo implantation and utero-placental exchange. Pregnancy complications in cancer survivors might be due to compromised blood supply to the endometrium and myometrium affecting placentation or altered remodeling of the pregnant uterus secondary to radiation fibrosis. Alterations in endometrial receptivity at the molecular level could affect pregnancy implantation and early pregnancy loss, but later complications also can occur. This review focuses on understanding the unintended effects of chemotherapy and radiotherapy on uterine function in female cancer survivors and the impact on pregnancy, and summarizes mechanisms to protect and treat the uterus before and after cancer chemotherapy and radiotherapy.

Clinical outcomes and inflammatory response to single-incision laparoscopic (SIL) colorectal surgery: a single-blinded randomized controlled pilot study.

AIM: Single-incision laparoscopic (SIL) surgery is expanding, but its benefits, efficacy and safety compared with conventional laparoscopic (CL) surgery remain unclear. This pilot study examined clinical outcomes and biochemical markers of inflammation for colorectal resections by SIL and CL in a randomized controlled pilot trial. METHOD: Fifty patients undergoing elective colorectal resection were randomized to either SIL or CL. Primary outcomes were operating time and length of stay (LoS); secondary outcomes included combined length of scars, pain scores, complications, Quality of Life EQ5D-VAS and the inflammatory markers interleukin-6 (IL-6), IL-8 and C-reactive protein (CRP) at baseline, 2, 6, 24 and 72 h. RESULTS: There was no difference in age, gender, body mass index, indications and site of surgery, American Society of Anesthesiologists grade or incidence of previous surgery between the groups. Except for one conversion from SIL to open surgery, surgery was completed as intended. No difference between SIL and CL was found for operating time [median 130 (72-220) vs 130 (90-317) min, respectively, P = 0.528], LoS [median 4 (3-8) vs 4 (2-19)days, P = 0.888] and time to first flatus [2 (1-4) vs 2 (1-5) days, P = 0.374]. The combined length of scars was significantly shorter for SIL [4 (2-18) vs 7 (5-8) cm, P < 0.001]; in each group, four postoperative complications occurred (16%). Postoperative pain scores were similar [mean 7.67 (interquartile range 4) vs 7.25 (interquartile range 3.75), P = 0.835] to day 3. EQ5D-VAS was no different for both groups at discharge [72.5 (40-90) vs 70 (30-100), P = 0.673] but slightly higher for CL at 3 months [79 (45-100) vs 90 (50-100), P = 0.033].The IL-6, IL-8 and CRP levels between both groups showed similar peaks and no significant differences. CONCLUSION: SIL colorectal surgery by experienced laparoscopic surgeons appears to be safe and equivalent to CL, with no discernible difference in its effect on the physiological response to surgical trauma.

Effect of dietary supplementation of garlic, ginger and their combination on feed intake, growth performance and economics in commercial broilers.

AIM: The present study was carried out to evaluate the effect of supplementation of garlic, ginger and their combination in the diets of broiler chickens and assessment in terms of feed intake, growth performance and economics of feeding. MATERIALS AND METHODS: A total of 240 1-day-old Cobb-400 broiler chicks were randomly assigned to four dietary treatments each with three replicates of 20 chicks per replicate (n=60). Four experimental diets were formulated in such a way that control diet (T1) contained neither ginger nor garlic. While, birds in group T2 and T3 were fed with diets containing 1% garlic and ginger, respectively. Diet 4 (T4 group) contained a combination of 1% of garlic and ginger. The feeding experiment was carried out for 42 days, and different parameters evaluated includes feed intake, weight gain, feed conversion ratio, gut morphometry, and economics of feeding in terms of return over feed cost (ROFC) and European Performance Efficiency Index. RESULTS: Feed intake of experimental birds in ginger and mixture of garlic and ginger supplemented groups, i.e., T3 and T4 groups have significantly (p<0.05) higher feed intake as compared to control. While, feeding of garlic have non-significant effect on feed intake as compared to other groups. A body weight gain (g/bird) was found to be significantly (p<0.05) higher in garlic (T2 group) and ginger (T3 group) supplemented group as compare to control and garlic and ginger mixture supplemented group (T4 group). Feed conversion ratio was significantly (p<0.05) lower in ginger (T3 group) supplemented group as compare to other groups. Mean villi length, villi width and cryptal depth were significantly (p<0.05) higher in T3 group than rest of all three groups, indicating increased absorptive surface area. ROFC was significantly (p<0.05) lower in T3 and T4 groups as compare to control. However, it was not significantly different between control and T2 group. CONCLUSION: On the basis of the results of the study, it is concluded that supplementation of garlic improves the performance of broilers when added at the rate of 1% of broiler ration and can be a viable alternative to antibiotic growth promoter in the feeding of broiler chicken.

Bilateral ovarian maldescent: Unusual cause of infertility - A case report and literature review.

Infertility due to ovarian maldescent is extremely rare and diagnosis can be challenging in the absence of uterine anomalies. We present a case of infertility due to bilateral ovarian maldescent with normal uterine anatomy who conceived after in vitro fertilization and did not require removal of the ovaries. A 19-year-old woman presented with primary infertility. After three failed cycles of ovulation induction/intrauterine insemination and one failed cycle of fresh embryo transfer, the patient underwent laparoscopy, which showed bilateral ovaries above the pelvic brim. The patient conceived after second in vitro fertilization and delivered at 34 weeks. Accurate diagnosis of ovarian maldescent in a patient of unexplained infertility and normal uterus is crucial for successful follicular aspiration and infertility treatment. It is important to counsel patients about the benign nature of this rare entity and future fertility.

Effect of feeding varying levels of groundnut haulms on feed intake and growth performance in broiler chickens.

AIM: This study was carried out to evaluate groundnut haulms (GNH) as alternate feed source by its incorporation and assessment in terms of feed intake and growth performance in the diets of broilers. MATERIALS AND METHODS: A total of 240 1-day-old Cobb-400 broiler chicks were randomly assigned to four dietary treatments each with three replicates (n=60). Experimental Birds in group T1 were fed with conventional feed while birds in T2, T3, T4 were fed containing 2%, 4%, and 6% of GNH replacing maize and soyabean on iso-nitrogenous basis. RESULTS: Feed intake increases significantly (p>0.05) with increasing level of GNH in the diets of experimental birds. Highest feed intake was recorded in T4 (6% GNH), followed by T3 (4% GNH) than T2 (2% GNH) and T1 (control). Birds fed GNH gained significantly (p<0.05) higher body weight than birds fed the control diet. Birds in T4 [6% GNH] gained highest body weight, followed by T3 (4% GNH) than T2 (2% GNH) and T1 (control). However, feed conversion ratio (FCR) remained non-significant for all treatment groups. CONCLUSION: On the basis of the results of this study, it is concluded that supplementation of GNH can successfully replace costly ingredients like maize and soybean meal in the diets of broiler birds up to the level of 6 percent of concentrate mixture without any harmful effects on feed intake, growth and FCR.

Intra-arterial methylene blue injection into ex vivo colorectal cancer specimens improves lymph node staging accuracy: a randomized controlled trial.

AIM: A randomized controlled trial was carried out to study the effect of a recently proposed technique of ex vivo intra-arterial methylene blue injection of the surgical specimen removed for colorectal cancer on lymph node harvest and staging. METHOD: Between May 2012 and February 2013, 100 consecutive colorectal cancer resection specimens in a single institution were randomly assigned to intervention (methylene blue injection) and control (standard manual palpation technique) groups before formalin fixation. The specimen was then examined by the histopathologist for lymph nodes. RESULTS: Both groups were similar for age, sex, site of tumour, operation and tumour stage. In the intervention group, a higher number of nodes was found [median 23 (5-92) vs. 15 (5-37), P < 0.001], with only one specimen not achieving the recommended minimum standard of 12 nodes [1/50 (2%) vs. 8/50 (16%), P = 0.014]. However, there was no upstaging effect in the intervention group [23/50 (46.0%) vs. 20/50 (40.0%); P = 0.686]. With a significantly lower number of nodes harvested in rectal cancer, the positive effect of the intervention was particularly observed in the patients who underwent preoperative neoadjuvant radiotherapy [median 30 nodes (12-57) vs. 11 (7-15); P = 0.011; proportion of cases with < 12 nodes 0/5 vs. 5/8 (62.5%), P = 0.024]. CONCLUSION: Ex vivo intra-arterial methylene blue injection increases lymph node yield and can help to reduce the number of cases with a lower-than-recommended number of nodes, particularly in patients with rectal cancer having neoadjuvant treatment. The technique is easy to perform, cheap and saves time.

Comparative assessment of pesticide residues in grain, soil, and water from IPM and non-IPM trials of basmati rice.

The integrated pest management (IPM) modules of pesticide schedule on Basmati rice were validated at field experiments conducted in Northern India for consecutive 3 years (2005-2008). The pesticide residues were found below the detectable limit (<0.01-0.001 mg/kg) in soil and irrigation water samples of Kaithal region. In Dehra Dun region of Uttrakhand, the residues of carbendazim in rice grains and soil were detected below <0.01 mg/kg level. In second year experiments (2006-2007), only four non-IPM soil samples indicated the presence of chlorpyrifos and endosulfan in the range of ND <0.001 to 0.07 mg/kg, out of 45 samples analyzed. Carbendazim applied as seed treatment at Dehradun and Kaithal field trials was found below detectable limit in both IPM and non-IPM rice grains (<0.01 mg/kg) and irrigation water (0.01 µl/ml). Chlorpyrifos was detected in five water samples from Kaithal and one from Pant Nagar in the range of 0.003-0.006 µl/L, α- and ß-isomer of endosulfan in the range of 0.005-0.03, and 0.005-0.02 µl/ml, respectively, in one sample from Pant Nagar and two from Kaithal, out of a total of 22 samples. In the region of Uttrakhand and Uttar Pradesh during 2007-2008, four non-IPM samples of soil indicated trace levels of endosulfan, out of 16 samples analyzed. The residues were detected below detection limit for carbendazim (<0.01 mg/kg) in soil samples of Dehradun IPM fields and for endosulfan and carbendazim (0.001-0.01 µl/L) in water samples each from IPM and non-IPM fields of Uttar Pradesh. The results of 3-year trials of IPM module indicated basmati rice as safe and economical with pesticide residue-free rice grains.

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PURPOSE: The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. METHODS: We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) RESULTS: CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CONCLUSION: CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

Comparative study of safety and outcomes of single-port access versus conventional laparoscopic colorectal surgery.

BACKGROUND: Single-port access (SPA) offers cosmetic advantages in addition to the well-recognised benefits of conventional multi-port laparoscopic (CL) surgery, and can be carried out using standard straight instruments. We report the outcomes of our early experience with SPA colorectal resections in comparison with CL surgery. METHODS: We compared the following data, patient characteristics, operating time, morbidity, operative mortality, length of hospital stay and tumour variables, of patients who underwent SPA right, left, sigmoid and total colon resections, as well as high anterior resections and panproctocolectomies, with that of patients who underwent equivalent conventional laparoscopic (CL) operations. The 40 SPA and 78 CL patients studied underwent surgery between February 2008 and September 2011. RESULTS: There was no difference between the SPA and CL operations, as regards the patient's sex (55.0 vs. 62.8% males, p = 0.411), comorbidity (ASA I 10.0 vs. 12.8%; ASA II 57.5 vs. 59.0%; ASA III 32.5 vs. 25.6%; ASA IV 0 vs. 2.6%, p = 0.722) and body mass index (26.2 vs. 28.0 kg/m(2), p = 0.073). However, SPA patients were younger (mean age 54.1 vs. 64.8 years, p = 0.001), and malignancy was a less common indication for surgery (25.0 vs. 71.8%, p < 0.001). There were no conversions to open surgery, and one death occurred in the CL group (1.3%). Mean operating time (162 vs. 170 min, p = 0.547), median post-operative hospital stay (4 vs. 4 days, p = 0.255) and morbidity (7.5 vs. 12.8%, p = 0.538) were comparable. CONCLUSIONS: SPA laparoscopic surgery appears safe in the hands of experienced laparoscopic surgeons, with no increase in operating time, length of stay, morbidity and mortality. Selection of patients with indications for surgery for benign disease may be of importance to ensure an oncologically safe initial uptake of SPA colorectal practice.

Single-port access laparoscopic reversal of Hartmann operation.

BACKGROUND: Single-port access offers cosmetic advantages in addition to the well-recognized benefits of standard multiport laparoscopic surgery, and can be performed with the use of standard straight instruments. We describe a technique of single-port access reversal of Hartmann colostomy by use of the colostomy site for access. METHODS: After routine skin preparation and laparoscopic setup, the colostomy is mobilized from its mucocutaneous border, and the anvil of a circular stapler is secured to the distal lumen. By the use of a GelPoint system with 3 or 4 trocars, the intra-abdominal adhesions are divided and the splenic flexure is mobilized to achieve sufficient access to the abdominal and pelvic cavities and proximal colonic mobility. The rectal stump is mobilized to the mid rectum, starting from the posterior mesorectal fascia around to the anterior rectal wall. A tension-free colorectal anastomosis is secured with a standard circular stapling device inserted transanally, and leak tested. The colostomy wound is closed in standard fashion. RESULTS: Five patients underwent single-port access reversal of Hartmann resection (4 diverticular perforations and 1 pT3N0 colon cancer), with a mean operating time of 155 (range, 137-187) minutes and a median length of stay of 3 (range, 2-11) days. There were no conversions, major surgical morbidity, or deaths. CONCLUSION: Single-port access reversal of Hartmann colostomy through the stoma site is safe, and it offers additional cosmetic advantages with no apparent additional morbidity in comparison with standard multiport surgery.

Role of Helicobacter pylori in causation of diabetic gastropathies and non-gastrointestinal complications in type 2 diabetes.

A cross-sectional case-control study was conducted in 80 diabetic patients, to evaluate the incidence of gastropathy by endoscopy in type 2 diabetes mellitus. An association between Helicobacter pylori infection and non-gastrointestinal complication of diabetes mellitus was also looked into. Gastric biopsies were subjected to rapid urease test for demonstration of Helicobacter pylori. The fasting blood glucose levels among Helicobacter pylori positive diabetes were 175 +/- 36.5 mg %, and in Helicobacter pylori negative diabetics were 138 +/- 39.4 mg %. The prevalence of endoscopically detectable gastro-intestinal complications were higher in Helicobacter pylori infected diabetics (odd's ratio 4:2; p < 0.05). The total prevalence of Helicobacter pylori positive in diabetics by rapid urease test was statistically significant (p < 0.05). Coronary heart disease was more prevalent in diabetics with Helicobacter pylori infection than those without Helicobacter pylori (57%). The prevalence of H. pylori positivity in other complications such as peripheral vascular diseases, cerebrovascular diseases was not significant. The association between nephropathy, retinopathy and neuropathy with Helicobacter pylori, was also observed and the strong association was seen in diabetic retinopathy (p < 0.001), diabetic neuropathy (p < 0.01) and nephropathy (p < 0.001).