Assessing the Therapeutic Impacts of HAMLET and FOLFOX on BRAF-Mutated Colorectal Cancer: A Study of Cancer Cell Survival and Mitochondrial Dynamics In Vitro and Ex Vivo.

Background and Objectives: Colorectal cancer (CRC) is a major global health challenge. The BRAF V600E mutation, found in 8-12% of CRC patients, exacerbates this by conferring poor prognosis and resistance to therapy. Our study focuses on the efficacy of the HAMLET complex, a molecular substance derived from human breast milk, on CRC cell lines and ex vivo biopsies harboring this mutation, given its previously observed selective toxicity to cancer cells. Materials and Methods: we explored the effects of combining HAMLET with the FOLFOX chemotherapy regimen on CRC cell lines and ex vivo models. Key assessments included cell viability, apoptosis/necrosis induction, and mitochondrial function, aiming to understand the mutation-specific resistance or other cellular response mechanisms. Results: HAMLET and FOLFOX alone decreased viability in CRC explants, irrespective of the BRAF mutation status. Notably, their combination yielded a marked decrease in viability, particularly in the BRAF wild-type samples, suggesting a synergistic effect. While HAMLET showed a modest inhibitory effect on mitochondrial respiration across both mutant and wild-type samples, the response varied depending on the mutation status. Significant differences emerged in the responses of the HT-29 and WiDr cell lines to HAMLET, with WiDr cells showing greater resistance, pointing to factors beyond genetic mutations influencing drug responses. A slight synergy between HAMLET and FOLFOX was observed in WiDr cells, independent of the BRAF mutation. The bioenergetic analysis highlighted differences in mitochondrial respiration between HT-29 and WiDr cells, suggesting that bioenergetic profiles could be key in determining cellular responses to HAMLET. Conclusions: We highlight the potential of HAMLET and FOLFOX as a combined therapeutic approach in BRAF wild-type CRC, significantly reducing cancer cell viability. The varied responses in CRC cell lines, especially regarding bioenergetic and mitochondrial factors, emphasize the need for a comprehensive approach considering both genetic and metabolic aspects in CRC treatment strategies.

Prognostic Value of Tumor Volume, Tumor Volume Reduction Rate and Magnetic Resonance Tumor Regression Grade in Rectal Cancer.

Background and Objectives: Rectal cancer poses significant treatment challenges, especially in advanced stages. Radiologic assessment, particularly with MRI, is critical for surgeons and oncologists to understand tumor dynamics and tailor treatment strategies to improve patient outcomes. The purpose of this study was to correlate MRI-based tumor volumetric and tumor regression grade analysis in patients with advanced rectal cancer, assessing the impact of preoperative chemotherapy (CT) alone or chemoradiotherapy (CRT) on surgical technique choices. Materials and Methods: Between 2015 and 2022, a prospective study was enrolled, including a cohort of 89 patients diagnosed with rectal cancer at stage II or III. The participants were divided into two distinct therapy groups, ensuring an equal distribution with a ratio of 1:1. The initial group was treated with the contemporary preoperative chemotherapy protocol FOLFOX4. In contrast, the alternative group received conventional preoperative chemoradiotherapy. Before surgery, each patient underwent a rectal MRI scan at 1.5 T, including T2-weighted and diffusion-weighted imaging (DWI) sequences. Results: The CT group showed a 36.52% tumor volume reduction rate (TVRR), and the CRT group showed 54.87%, with varying magnetic resonance and pathological tumor regression grades (mrTRG and pTRG). Analysis revealed a significant interaction between mrTRG and tumor volumetrics (volume and VRR) in both groups, especially CRT, underscoring the complexity of tumor response. Both treatment groups had similar initial tumor volumes, with CRT displaying a higher TVRR, particularly in higher pathological TRG (3/4) cases. This interaction and the strong correlation between mrTRG and pTRG suggest mrTRG's role as a non-invasive predictor for treatment response, highlighting the need for personalized treatment plans. Conclusions: Rectal tumor volume, volume reduction rate, and mrTRG are not just abstract measures; they are concrete indicators that have a direct and practical impact on surgical decision-making, planning, and prognosis, ultimately influencing the quality of care and life expectancy of patients with rectal cancer.

MRI Evaluation of Rectal Cancer Lymph Node Staging Using Apparent Diffusion Coefficient.

Introduction Colorectal cancer is the third most diagnosed cancer globally. Lymph node metastases significantly affect prognosis, emphasizing the importance of early detection and management. Despite significant advances in conventional MRI's role in staging, improvements in advanced functional imaging such as diffusion-weighted imaging (DWI) in identifying lymph node metastases persist. Objectives The aim is to evaluate the effectiveness of apparent diffusion coefficient (ADC) MRI in evaluating lymph node staging in rectal cancer. Patients and methods In a prospective study, 89 patients with stage II-III rectal cancer were grouped into two treatments: pre-operative FOLFOX4 chemotherapy and standard pre-operative chemoradiotherapy. All underwent 1.5T MRI, with T2-weighted and DWI sequences. A radiologist defined regions of interest on the tumor, lymph nodes, and intact rectal wall to calculate ADC values. Results Rectal cancer ADC's receiver operating characteristic curve had an area under the curve (AUC) of 0.688 (P < 0.001), with optimal ADC cutoff at 0.99 x 10-3 mm2/s (sensitivity: 75%, specificity: 83%). For lymph nodes, AUC was 0.508 (P < 0.001), with a cutoff of 0.9 x 10-3 mm2/s (sensitivity: 78%, specificity: 67%). No correlation between tumor and lymph node ADC values was observed. In chemotherapy patients, "healthy" inguinal lymph nodes had higher ADC values than affected ones pre-treatment (P = 0.001), a disparity fading post-treatment (P = 0.313). For chemoradiotherapy patients, the ADC difference persisted pre and post-treatment (P = 0.001). Conclusion The study of ADC-MRI showed different ADC values between tumors and lymph nodes and highlighted ADC differences between treatment groups. Notably, no correlation was observed between tumor and lymph node ADC values. However, differences were apparent when comparing "healthy" inguinal nodes with lymph nodes affected by cancer.

HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations.

PURPOSE: Treatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro. METHODS: We treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k. RESULTS: The HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells. CONCLUSION: HAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells' respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.

DNA Methylation of HOXA11 Gene as Prognostic Molecular Marker in Human Gastric Adenocarcinoma.

Hypermethylation of tumor suppressor genes and hypomethylation of oncogenes might be identified as possible biomarkers in gastric cancer (GC). We aimed to assess the DNA methylation status of selected genes in GC tissue samples and evaluate these genes' prognostic importance on patient survival. Patients (99) diagnosed with GC and who underwent gastrectomy were included. We selected a group of genes (RAD51B, GFRA3, AKR7A3, HOXA11, TUSC3, FLI1, SEZ6L, GLDC, NDRG) which may be considered as potential tumor suppressor genes and oncogenes. Methylation of the HOXA11 gene promoter was significantly more frequent in GC tumor tissue (p = 0.006) than in healthy gastric mucosa. The probability of surviving longer (71.2 months (95% CI 57-85.3) vs. 44.3 months (95% CI 34.8-53.9)) was observed with unmethylated HOXA11 promoter in cancer tissues. Survival in patients with a methylation of HOXA11 promoter either in healthy gastric mucosa or gastric cancer tissue was twice as high as in patients with a methylation of HOXA11 promoter in both healthy gastric mucosa and cancer tissue (61.2 months (95% CI 50.9-71.4) vs. 28.5 months (95% CI 20.8-36.2)). Multivariate Cox analysis revealed the HOXA11 methylation as significantly associated with patients' survival (HR = 2.4, 95% CI 1.19-4.86). Our results suggest that the HOXA11 gene might be a potential prognostic molecular marker in patients with gastric adenocarcinoma.

Comparison of Changes in Disease-Free and Overall Survival of Resectable Rectal Adenocarcinoma between 2010 and 2015.

BACKGROUND: Management of rectal cancer (RC) has undergone many changes in recent decades. A multidisciplinary approach to this complex disease is essential, ensuring high-quality diagnostic, treatment, and outcomes. We aimed to compare treatment results of RC in a single-centre setting between 2010 and 2015. METHODS: A retrospective comparative study included patients with newly diagnosed and operated resectable RC. Patients' diagnostic and treatment data were analysed. Postoperative morbidity was measured according to the Clavien-Dindo classification. Survival data were received from the Lithuanian Cancer Registry. Continuous variables were expressed as mean and SD. Student t test and one-way ANOVA were used for parametric data and the Mann-Whitney test for non-parametric. A multivariate logistic regression analysis was used to identify independent factors for increased survival. Association between categorical variables was verified using Pearson χ2. RESULTS: The study included 179 patients: 80 from 2010 and 99 from 2015. Mean sample age was 67.1 ± 10.7 years. There was no significant difference regarding age, gender, median ASA (3 in both groups), but mean hospital stay was 2 days shorter (8 vs. 10 days) in 2015 (p = 0.002). There were only 8 patients (4%) admitted to the hospital on an emergency basis. Pelvis MRI and abdominal CT were performed more often in 2015: from 37.5 to 77.8% (p < 0.001) and from 52.5 to 97% in 2015, respectively. Circumferential margin evaluation increased from 13.8 to 36.4% (p = 0.001). Neoadjuvant therapy increased from 20% in 2010 to 44.9% in 2015 (p = 0.01). The overall postoperative Clavien-Dindo complication rate was higher in 2015 (13.8 vs. 20.2%, p = 0.596), but in-hospital mortality was lower (1 vs. 0 patients). Comparison of radiological TNM and pathological TNM with one-way ANOVA showed a significant difference staging between 2010 (p = 0.002) and 2015 (p = 0.001). The 2-year overall survival (OS) increased from 76.3 to 86.9% (p = 0.046) and the median disease-free survival from 27 (range 0-35) months to 28 (range 0-35) months (72.5-83.5%, p = 0.077). Multivariate logistic regression analysis determined that availability and performance of MRI were associated with an increased OS (OR = 1.529, 95% CI 0.916-2.554, p = 0.020). CONCLUSIONS: The expanded quantity of preoperative imaging, an improved radiological staging, and compulsory multidisciplinary team board discussions have led to selective neoadjuvant treatment decision followed by surgery which can positively affect the 2-year OS rate.

Long-term follow-up results of umbilical hernia repair.

INTRODUCTION: Multiple suture techniques and various mesh repairs are used in open or laparoscopic umbilical hernia (UH) surgery. AIM: To compare long-term follow-up results of UH repair in different hernia surgery groups and to identify risk factors for UH recurrence. MATERIAL AND METHODS: A retrospective analysis of 216 patients who underwent elective surgery for UH during a 10-year period was performed. The patients were divided into three groups according to surgery technique (suture, mesh and laparoscopic repair). Early and long-term follow-up results including hospital stay, postoperative general and wound complications, recurrence rate and postoperative patient complaints were reviewed. Risk factors for recurrence were also analyzed. RESULTS: One hundred and forty-six patients were operated on using suture repair, 52 using open mesh and 18 using laparoscopic repair technique. 77.8% of patients underwent long-term follow-up. The postoperative wound complication rate and long-term postoperative complaints were significantly higher in the open mesh repair group. The overall hernia recurrence rate was 13.1%. Only 2 (1.7%) patients with small hernias (< 2 cm) had a recurrence in the suture repair group. Logistic regression analysis showed that body mass index (BMI) > 30 kg/m2, diabetes and wound infection were independent risk factors for umbilical hernia recurrence. CONCLUSIONS: The overall umbilical hernia recurrence rate was 13.1%. Body mass index > 30 kg/m2, diabetes and wound infection were independent risk factors for UH recurrence. According to our study results, laparoscopic medium and large umbilical hernia repair has slight advantages over open mesh repair concerning early postoperative complications, long-term postoperative pain and recurrence.