The Clinicopathological Characteristics of Young-Onset Versus Adult-Onset Colorectal Cancer: A Tertiary Hospital-Based Study.

Background: The prevalence of colorectal cancer (CRC) among young individuals is rising worldwide, especially in Malaysia. Investigations are currently employed to distinguish the features of young-onset CRC (YOCRC) from adult-onset CRC (AOCRC). This study aimed to compare the characteristics of patients with YOCRC and AOCRC diagnosed at Hospital Universiti Sains Malaysia (HUSM). Methods: This was a retrospective study of CRC cases from January 2013 to December 2021. The details of YOCRC (< 50 years old) and AOCRC (≥ 50 years old) patients were retrieved from the laboratory system and medical records. The Pearson's chi-square test, Fisher's exact test and multiple logistic regression were used to compare the AOCRC and YOCRC cases. Statistical significance was defined at a P-value of ≤ 0.05. Results: The AOCRC (254/319, 79.6%) was more prevalent than YOCRC (65/319, 20.4%), with a predominance of males (53.9%) and Malay sub-population (90.2%). AOCRC and YOCRC shared similarities in left-sided location, high occurrence of adenocarcinoma with moderately differentiated histology and advanced stage of diagnosis. More patients with YOCRC (23.1%) had a family history of cancer than patients with AOCRC. YOCRC also differed from AOCRC by having more specific histological subtypes, such as mucinous adenocarcinoma (15.4%) and signet ring carcinoma (6.2%). In addition, patients with YOCRC commonly presented with a low density of tumour-infiltrating lymphocytes (TILs) (60%). Multiple logistic regression showed a family history of CRC (adjusted odds ratio [AOR] = 3.75, P = 0.003) and histological type (AOR = 15.21, P < 0.001) are more likely to cause YOCRC than diabetes (AOR = 0.06, P < 0.001) and hypertension (AOR = 0.14, P < 0.001) comorbidities, which are associated with AOCRC. Conclusion: Our descriptive study presented the epidemiological and histopathological characteristics of AOCRC and YOCRC in HUSM, providing current information on distinguishing features between the groups.

Comparing the Biology of Young versus Old Age Estrogen-Receptor-Positive Breast Cancer through Gene and Protein Expression Analyses.

Background: Breast cancer developed at a young age (≤45 years) is hypothesized to have unique biology; however, findings in this field are controversial. Methods: We compared the whole transcriptomic profile of young vs. old-age breast cancer using DNA microarray. RNA was extracted from 13 fresh estrogen receptor (ER)-positive primary breast cancer tissues of untreated patients (7 = young age ≤45 years and 6 = old age ≥55 years). In silico validation for the differentially expressed genes (DEGs) by young-age patients was conducted using The Cancer Genome Atlas (TCGA) database. Next, we analyzed the protein expression encoded by two of the significantly down-regulated genes by young-age patients, Glycine N-acyltransferase-like 1 (GLYATL-1) and Ran-binding protein 3 like (RANBP3L), using immunohistochemical analysis in an independent cohort of 56 and 74 ER-positive pre-therapeutic primary breast cancer tissues, respectively. Results: 12 genes were significantly differentially expressed by young-age breast cancers (fold change >2 or <2- with FDR p-value < 0.05). TCGA data confirmed the differential expression of six genes. Protein expression analysis of GLYATL-1 and RANBP3L did not show heterogeneous expression between young and old-age breast cancer tissues. Loss of expression of GLYATL-1 was significantly (p-value 0.005) associated with positive lymph node status. Higher expression of RANBP3L was significantly associated with breast cancers with lower histopathological grades (p-value 0.038). Conclusions: At the transcriptomic level, breast cancer developed in young and old age patients seems homogenous. The variation in the transcriptomic profiles can be attributed to the other clinicopathological characteristics rather than the age of the patient.

A GNAS Gene Mutation's Independent Expression in the Growth of Colorectal Cancer: A Systematic Review and Meta-Analysis.

Globally, colorectal carcinoma CRC is the third most common cancer and the third most common reason for cancer-associated mortality in both genders. The GNAS mutations are significantly linked with poor prognosis and failed treatment outcomes in CRC. A systematic review and meta-analysis of multiple studies executed following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) criteria and registered with PROSPERO (registration number: CRD42021256452). The initial search includes a total of 271 publications; however, only 30 studies that merit the eligibility criteria were eventually chosen. Data analysis via OpenMeta Analyst and comprehensive meta-analysis 3.0 (CMA 3.0) software were used to investigate the prevalence of GNAS gene mutation among CRC patients. The meta-analysis consisted of 10,689 participants with most being males 6068/10,689 (56.8%). Overall, prevalence of GNAS mutations was 4.8% (95% CI: 3.1−7.3) with I2 = 94.39% and (p < 0.001). In 11/30 studies, the frequency of GNAS gene mutations was majorly in codons R201C [40.7% (95% CI: 29.2−53.2%)] and in codon R201H [39.7% (95% CI = 27.1−53.8)]. Overall prevalence of GNAS mutations was highest among the male gender: 53.9% (95% CI: 48.2−59.5%: I2 = 94.00%, (p < 0.001), tumour location (colon): 50.5% (95% CI: 33.2−67.6%: I2 = 97.93%, (p < 0.001), tumour grade (Well): 57.5% (95% CI: 32.4−79.2%: I2 = 98.10%, (p < 0.001) and tumour late stage: 67.9% (95% CI: 49.7−84.3%: I2 = 98.%, (p < 0.001). When stratified according to study location, a higher prevalence was observed in Japan (26.8%) while Italy has the lowest (0.4%). Overall prevalence of GNAS gene mutations was 4.8% with codons R201C and R201H being the most mutated, and the results conformed with numerous published studies on GNAS mutation.

The Prediction of Survival Outcome and Prognosis Factor in Association with Comorbidity Status in Patients with Colorectal Cancer: A Research-Based Study.

Colorectal carcinoma (CRC) is rising exponentially in Asia, representing 11% of cancer worldwide. This study analysed the influence of CRC on patients' life expectancy (survival and prognosis factors) via clinicopathology data and comorbidity status of CRC patients. Methodology: A retrospective study performed in HUSM using clinical data from the Surgery unit from 2015 to 2020. The demographic and pertinent clinical data were retrieved for preliminary analyses (data cleansing and exploration). Demographics and pathological characteristics were illustrated using descriptive analysis; 5-year survival rates were calculated using Kaplan−Meier methods; potential prognostic variables were analysed using simple and multivariate logistic regression analysis conducted via the Cox proportional hazards model, while the Charlson Comorbidity Scale was used to categorize patients' disease status. Results: Of a total of 114 CRC patients, two-thirds (89.5%) were from Malay tribes, while Indian and Chinese had 5.3% each. The 50−69.9 years were the most affected group (45.6%). Overall, 40.4% were smokers (majorly male (95.7%)), 14.0% ex-smokers, and 45.6% non-smokers (p-value = 0.001). The Kaplan−Meier overall 5-year median survival time was 62.5%. From the outcomes, patients who were male and >70 years had metastasis present, who presented with per rectal bleeding and were classified as Duke C; and who has tumour in the rectum had the lowest survival rate. Regarding the prognosis factors investigated, "Gender" (adjusted hazard ratio (HR): 2.62; 95% CI: 1.56−7.81, p-value = 0.040), "Presence of metastases" (HR: 3.76; 95% CI: 1.89−7.32, p-value = 0.010), "Metastasis site: Liver" (HR: 5.04; 95% CI: 1.71−19.05, p-value = 0.039), "Lymphovascular permeation" (HR: 2.94; 95% CI: 1.99−5.92, p-value = 0.021), and "CEA-level" (HR: 2.43; 95% CI: 1.49−5.80, p-value = 0.001) remained significant in the final model for multiple Cox proportional hazard regression analyses. There was a significant mean association between tumour grades and the patient's comorbidity status. Conclusions: Histopathological factors (gender, metastases presence, site of metastases, CEA level, and lymphovascular permeation) showed the best prognosis-predicting factors in CRC.

Immunohistochemistry as a detection tool for ion channels involved in dental pain signaling.

Background: Despite advances in pain detection, diagnosis, and management, the prevalence of dental pain is still on the rise. Although dental pain is not directly related to fatal outcomes, the two most common types of dental pain-dental caries and dentin hypersensitivity-have a significant impact on an individual's quality of life. Understanding the mechanism of the pain pathway is one of the crucial steps in providing better treatment for these patients. Ion channels are critical biomolecules that have been the subject of dental study owing to their roles in the transmission and transduction of external stimuli, as well as in the control and perception of pain. Numerous immunohistochemical (IHC) staining approaches have also been used to identify the many ion channels implicated in peripheral pain signaling in dental pulp. Highlight: This review highlights the critical steps in IHC and its role in the detection of ion channels involved in the dental pain signaling pathway. Conclusion: The key ion channels identified using IHC and whose functions have been widely researched in dental tissues are addressed in this review article.

nNav1.5 expression is associated with glutamate level in breast cancer cells.

BACKGROUND: Glutamate and voltage-gated sodium channels, both have been the target of intense investigation for its involvement in carcinogenesis and progression of malignant disease. Breast cancer with increased level of glutamate often metastasize to other organs (especially bone), whilst re-expression of 'neonatal' Nav1.5, nNav1.5 in breast cancer is known to promote cell invasion in vitro, metastasis in vivo and positive lymph node metastasis in patients. METHODS: In this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was examined using pharmacological approach (VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA-nNav1.5). Effect of these agents were evaluated based on endogenous and exogenous glutamate concentration using glutamate fluorometric assay, mRNA expression of nNav1.5 using qPCR and finally, invasion using 3D culture assay. RESULTS: Endogenous and exogenous glutamate levels were significantly higher in aggressive human breast cancer cells, MDA-MB-231 cells compared to less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A. Treatment with TTX to MDA-MB-231 cells resulted in significant reduction of endogenous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells. CONCLUSIONS: An interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation.

Endocan-microvascular Density in Primary Ovarian Carcinoma.

BACKGROUND: Epithelial ovarian cancer is among the leading causes of death in women and is driven by angiogenesis. Microvascular density (MVD) can be used to evaluate angiogenesis in carcinomas and thus it can be used as a potential biomarker for ovarian cancer. This study is aimed to establish the association between endocan-MVD with clinicopathological factors in primary epithelial ovarian cancer. METHODS: The clinicopathological characteristics were acquired from the medical records filed between January 2008 and December 2018 of 89 epithelial ovarian cancer cases in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. Sectioned samples were analyzed for endocan through immunohistochemistry followed by the quantification of MVD. The association between clinicopathological characteristics and endocan-MVD was analyzed using the Pearson chi-square test and Fischer's exact test. RESULTS: All cases of epithelial ovarian carcinomas were positive for endocan. The mean ± standard deviation value of endocan-MVD level was 21.6±14.60 microvessels per 200x field. A total of 53 (59.6%) cases had low and 36 (40.04%) had high endocan-MVD values. High endocan-MVD level had a significant association with the older age group (p-value = 0.009), smaller tumor size (p-value<0.001), type II tumor (p-value<0.001), high-grade tumor (p-value<0.001), advanced FIGO stage (p-value=0.002), and presence of tumor recurrence (p-value=0.017). No significant association was found between endocan-MVD and the other clinicopathological characteristics such as race, pre-operative serum CA-125 level, presence of diabetes mellitus, endometriosis, lymph node involvement, distant metastasis, and family history of malignancy. CONCLUSION: Endocan-MVD showed a significant association with age, tumor size, tumor type, tumor grade, FIGO stage, and recurrence in primary epithelial ovarian cancer. Thus, endocan-MVD could be implemented as a reliable marker to predict prognosis in epithelial ovarian cancer in the future.

A Retrospective Hospital-Based Study of HMGCR Expression in HER2 IHC 2+ and 3+ Breast Cancer.

OBJECTIVE: The role of HMG-CoA reductase (HMGCR) in relation to prognostic and treatment predictive information of HER2 positive breast cancer has been newly explored. In this study, we aimed to determine the expression of HMGCR in HER2 immunohistochemistry (IHC) scores of 2+ and 3+ breast cancer and to correlate with the patients' outcomes. METHODOLOGY: Using a cross-sectional design, invasive breast carcinoma of no special type (NST) and HER2 IHC scores of 2+ and 3+ cases were selected over a 50-month period in Hospital Sultanah Bahiyah (HSB), Alor Setar. IHC staining for HMGCR was performed on paraffin-embedded tissues at the Pathology Laboratory, Hospital Universiti Sains Malaysia (HUSM), Kubang Kerian using the standard staining procedure. The results were correlated with the patient's demographic and clinicopathological data. RESULTS: A total of 59 cases of HER2 IHC 2+ and 3+ invasive breast carcinoma were identified. The cases were predominant in young Malay women with tumours smaller than 50mm, higher grade and positive for lymphovascular invasion, axillary lymph nodes involvement and ER/PR expressions. HMGCR was positively expressed in HER2 IHC 2+ and 3+ breast cancer cases, which the staining intensities varied from weak, moderate to strong. Majority of the cases were scored 1+ for HMGCR expression. A low-positive HMGCR was more likely to be associated with less favourable outcomes of patients with HER2 IHC 2+ and 3+. However, the associations were statistically not significant. CONCLUSION: A study in a larger cohort of tumour samples is needed to further validate HMGCR expression as a potential prognostic biomarker for HER2 positive breast cancer. It is also suggested that all the HER2 IHC 2+ and 3+ cases need to be gene amplified using FISH analysis.  
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The Unique Biology behind the Early Onset of Breast Cancer.

Breast cancer commonly affects women of older age; however, in developing countries, up to 20% of breast cancer cases present in young women (younger than 40 years as defined by oncology literature). Breast cancer in young women is often defined to be aggressive in nature, usually of high histological grade at the time of diagnosis and negative for endocrine receptors with poor overall survival rate. Several researchers have attributed this aggressive nature to a hidden unique biology. However, findings in this aspect remain controversial. Thus, in this article, we aimed to review published work addressing somatic mutations, chromosome copy number variants, single nucleotide polymorphisms, differential gene expression, microRNAs and gene methylation profile of early-onset breast cancer, as well as its altered pathways resulting from those aberrations. Distinct biology behind early-onset of breast cancer was clear among estrogen receptor-positive and sporadic cases. However, further research is needed to determine and validate specific novel markers, which may help in customizing therapy for this group of patients.

Exploring Two Protocols of FISH Using Cytocell SYT-SSX Probe on Formalin Fixed Paraffin Embedded Tissue Sections.

BACKGROUND: Chromosomal translocation t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and have been identified as an alternative diagnostic strategy in differentiating synovial sarcoma from other histologic mimics. This study was carried out to test the efficacy of two FISH protocols using the SYT-SSX break apart probe from Cytocell. METHODOLOGY: Representative paraffin blocks of synovial sarcoma were utilized in this study. FISH study was performed on formalin-fixed paraffin embedded tissue sections using the SYT-SSX break apart probe from Cytocell, to detect two form of SYT-SSX transcript, SYT-SSX1 and SYT-SSX2. FISH protocol, including the hybridization was done following two different protocols, Cytocell FISH protocol and Optimized Dako FISH protocol. RESULTS: Tissue samples subjected to FISH using Cytocell FISH protocol showed the absence of signal corresponding to the probe used. Utilizing Optimized Dako FISH protocol, the two signals (red and green) corresponding to the break-apart probes was detected. These findings suggested that Optimised Dako FISH protocol is more suited for use with the tested probe on paraffin embedded tissues in comparison to Cytocell FISH protocol. CONCLUSION: Optimised Dako FISH protocol was noted to be more suited for detecting SYT-SSX FISH signals on paraffin embedded tissues in comparison to Cytocell FISH protocol.

Cellular and Molecular Changes in MNU-Induced Breast Tumours Injected with PF4 or bFGF

Background: Angiogenic activity has been considered to reflect important molecular events during breast tumour development. The present study concerned cellular and molecular changes of MNU-induced breast tumours subjected to promotion and suppression of angiogenesis. Methods: Female Sprague Dawley rats at the age of 21 days received MNU at the dose 70 mg/kg of body weight by intraperitoneal injection. Three months post-carcinogen initiation, mammary tumours were palpated and their growth was monitored. When the tumour diameter reached 1.0 ± 0.05 cm, rats were given bFGF or PF4 intratumourally at a dose of 10 µg/tumour. Entire palpable tumour were subsequently excised and subjected to histology examination, IHC staining, and RT-PCR. Results: No critical morphological changes were observed between pro-angiogenic factor, bFGF, and control groups. However, increase of tumour size with more necrotic and diffuse areas was notable in tumours after anti-angiogenic PF4 intervention. ER and PR mRNA expression was significantly up- and down-regulated in bFGF and PF4 groups, respectively. The trends were significantly associated with peri- and intratumoural MVD counts. However, irrespective of whether we promoted or inhibited angiogenesis, the expression of EGFR and ERBB2 continued to be significantly increased but this was not significantly associated with the MVD score. No significant differences in E-cadherin and LR gene expression were noted between intervention and control groups. Conclusion: ER and PR receptor expression shows consistent responses when tumour angiogenesis is manipulated either positively or negatively. Our study adds to current understanding that not only do we need to target hormonal receptors, as presently practiced, but we also need to target endothelial receptors to successfully treat breast cancer.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.

The association between phenotype and size of breast tumors induced by 1-methyl-1-nitrosourea (MNU) injection in rats.

BACKGROUND: Administration of 1-methyl-1-nitrosourea (MNU) is considered a simple and rapid method for inducing breast tumors in rats. While most studies focus on the time frame of tumor development, there are little data on the development of breast tumor in relation to tumor size. Thus the current study was carried out to analyze the phenotype of MNU-induced tumors in relation to tumor size. MATERIAL/METHODS: Twenty-one 21-day-old female Sprague Dawley rats were administered intraperitoneally with MNU at a dose of 70 mg/kg body weight. The entire palpable tumor was excised when the tumor size reached the diameters of 4.0+/-0.5 mm, 8.0+/-0.5 mm, 12.0+/-0.5 mm, and 16.0+/-0.5 mm and then subjected to histopathological assessment. RESULTS: Epithelial inclusion cysts and mammary adenomas made up most of the benign tumors, with four cases occurring together with malignant lesions. Ductal carcinoma in situ was seen in tumor sizes of 4.0+/-0.5 mm or less. Among the malignant tumors, the cribriform type was seen predominantly at tumor sizes of less than 12.0+/-0.5 mm, while those with sizes of 12.0+/-0.5 mm or greater were of papillary type. Infiltrating ductal carcinoma-no special type (IDC-NST) commonly seen in humans was also observed at tumor sizes greater than 12.0+/-0.5 mm. CONCLUSIONS: The tumors were found to be mainly of malignant type and the histological features of the induced tumors underwent changes as the tumor grew in size.