Challenges and Strategies for Improving Access to Cancer Drugs in Malaysia: Summary of Opinions Expressed at the 2nd MACR International Scientific Conference 2022.

Considerable progress has been made in cancer drug development in recent decades. However, for people in low- and middle-income countries, including Malaysia, many of these drugs are not readily available. During the 2nd Malaysian Association for Cancer Research (MACR) International Scientific Conference, a forum discussion was held to address these challenges and explore strategies to improve access to cancer medicines in the country. This paper presents the results of the said forum discussion. A few challenges to cancer drug access were highlighted, including lengthy approval and regulatory practices, cost of medicines, and manufacturing barriers. Besides, a few strategies for mitigating some of these challenges were proposed, such as mechanisms for cost reduction, uptake of biosimilars and generics, local manufacturing, public-private partnerships, strengthening the role of insurance companies, funding and regulation, and advocacy for fair pricing, by drawing examples from cancer medicines access initiatives in Malaysia and initiatives for different disease groups. Overall, this paper provides a comprehensive overview of the challenges and strategies for improving access to cancer medicines in Malaysia and provides valuable insights for policymakers, healthcare providers, the pharmaceutical industry, cancer patients, cancer support groups, and other stakeholders working on this important issue.

Long-Range Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.

Whole blood hydroxychloroquine: Does genetic polymorphism of cytochrome P450 enzymes have a role?

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a wide range of clinical manifestations and multifactorial etiologies ranging from environmental to genetic. SLE is associated with dysregulated immunological reactions, with increased immune complex formation leading to end-organ damages such as lupus nephritis, cutaneous lupus, and musculoskeletal disorders. Lupus treatment aims to reduce disease activity, prevent organ damage, and improve long-term patient survival and quality of life. Antimalarial, hydroxychloroquine (HCQ) is used as a first-line systemic treatment for lupus. It has shown profound efficacy in lupus and its associated conditions. However, wide variation in terms of clinical response to this drug has been observed among this group of patients. This variability has limited the potential of HCQ to achieve absolute clinical benefits. Several factors, including genetic polymorphisms of cytochrome P450 enzymes, have been stipulated as key entities leading to this inter-individual variation. Thus, there is a need for more studies to understand the role of genetic polymorphisms in CYP450 enzymes in the clinical response to HCQ. Focusing on the role of genetic polymorphism on whole blood HCQ in lupus disorder, this review aims to highlight up-to-date pathophysiology of SLE, the mechanism of action of HCQ, and finally the role of genetic polymorphism of CYP450 enzymes on whole blood HCQ level as well as clinical response in lupus.

The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells.

Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.

Tissue Rigidity Increased during Carcinogenesis of NTCU-Induced Lung Squamous Cell Carcinoma In Vivo.

Increased tissue rigidity is an emerging hallmark of cancer as it plays a critical role in promoting cancer growth. However, the field lacks a defined characterization of tissue rigidity in dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) in vivo. Pre-malignant and malignant lung SCC was developed in BALB/c mice using N-nitroso-tris-chloroethylurea (NTCU). Picro sirius red staining and atomic force microscopy were performed to measure collagen content and collagen (diameter and rigidity), respectively. Then, the expression of tenascin C (TNC) protein was determined using immunohistochemistry staining. Briefly, all tissue rigidity parameters were found to be increased in the Cancer group as compared with the Vehicle group. Importantly, collagen content (33.63 ± 2.39%) and TNC expression (7.97 ± 2.04%) were found to be significantly higher (p < 0.05) in the Malignant Cancer group, as compared with the collagen content (18.08 ± 1.75%) and TNC expression (0.45 ± 0.53%) in the Pre-malignant Cancer group, indicating increased tissue rigidity during carcinogenesis of lung SCC. Overall, tissue rigidity of lung SCC was suggested to be increased during carcinogenesis as indicated by the overexpression of collagen and TNC protein, which may warrant further research as novel therapeutic targets to treat lung SCC effectively.

Rigid Tissue Increases Cytoplasmic pYAP Expression in Pre-Malignant Stage of Lung Squamous Cell Carcinoma (SCC) In Vivo.

Increased tissue rigidity is able to activate the Hippo signaling pathway, leading to YAP inactivation by phosphorylation and translocation into the cytoplasm. Accumulating evidence suggests that cytoplasmic pYAP serves as a tumor suppressor and could be a prognostic biomarker for several solid cancers. However, the relationship between tissue rigidity and cytoplasmic pYAP expression in the early stage of lung squamous cell carcinoma (SCC) remains elusive; this was determined in this study by using a mouse model. Female BALB/c mice were assigned into two groups (n = 6; the vehicle (VC) and the pre-malignant (PM) group, which received 70% acetone and 0.04 M N-nitroso-tris-chloroethylurea (NTCU) for 15 weeks, respectively. In this study, the formation of hyperplasia and metaplasia lesions was found in the PM group, indicating the pre-malignant stage of lung SCC. The pre-malignant tissue appeared to be more rigid as characterized by significantly higher (p < 0.05) epithelium thickness, proliferative activity, and collagen content than the VC group. The PM group also had a significantly higher (p < 0.05) cytoplasmic pYAP protein expression than the VC group. In conclusion, increased tissue rigidity may contribute to the upregulation of cytoplasmic pYAP expression, which may act as a tumor suppressor in the early stage of lung SCC.

A Cell-Based Systematic Review on the Role of Annexin A1 in Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is often associated with a poorer prognosis and does not respond to hormonal therapy. Increasing evidence highlights the exploitability of Annexin A1 (AnxA1), a calcium dependent protein, as a precision medicine for TNBC. To systematically summarize the role of AnxA1 and its associated mechanisms in TNBC, we performed data mining using three main databases: PubMed, Scopus, and Ovid/Medline. The papers retrieved were based on two different sets of key words such as "Annexin A1" or "Lipocortin 1" and "Breast cancer" or "TNBC". A total of 388 articles were identified, with 210 chosen for comprehensive screening and 13 papers that met inclusion criteria were included. Current evidence from cell culture studies showed that AnxA1 expression is correlated with NF-κB, which promotes migration by activating ERK phosphorylation. AnxaA1 also activates TGF-ß signaling which upregulates MMP-9 and miR196a expression to enhance epithelial-mesenchymal transition and migratory capacity of TNBC cells. AnxA1 can steer the macrophage polarization toward the M2 phenotype to create a pro-tumor immune environment. Existing research suggests a potential role of AnxA1 in the metastasis and immune landscape of TNBC tumors. Preclinical and clinical experiments are warranted to investigate the feasibility and effectiveness of targeting AnxA1 in TNBC.

Engaging community pharmacists to eliminate inadvertent doping in sports: A study of their knowledge on doping.

This study aimed to evaluate the community pharmacists' knowledge of tackling the issue of inadvertent doping in Malaysia. A cross-sectional survey was conducted among 384 community pharmacists working in Malaysia using a self-administered questionnaire. All the respondents were pharmacists fully registered with the Pharmacy Board of Malaysia and had been working in the community setting for at least one year. Of the 426 community pharmacists approached, 384 community pharmacists participated in this study, giving a response rate of 90.14%. The majority of the respondents were females (63.5%), graduated from local universities (74.9%), with median years of practising as a community pharmacist of six years (interquartile range, IQR = 9 years). The respondents were found to have moderate levels of doping-related knowledge (median score of 52 out of 100). Anabolic steroids (95.8%), stimulants (78.6%) and growth factors (65.6%) were recognised as prohibited substances by most of the respondents. Around 65.9% did not recognise that inadvertent doping is also considered a doping violation. Most of them (90%) also have poor levels of knowledge of doping scenarios in the country. Community pharmacists in Malaysia have limited knowledge in the field of doping. More programmes and activities related to doping and drugs in sports should be held to enhance the community pharmacists' knowledge on the issue of inadvertent doping.

Mitochondrial DNA sequences and transcriptomic profiles for elucidating the genetic underpinnings of cisplatin responsiveness in oral squamous cell carcinoma.

OBJECTIVES: Functional genetic variation plays an important role in predicting patients' response to chemotherapeutic agents. A growing catalogue of mitochondrial DNA (mtDNA) alterations in various cancers point to their important roles in altering the drug responsiveness and survival of cancer cells. In this work, we report the mtDNA sequences, obtained using a nanopore sequencer that can directly sequence unamplified DNA, and the transcriptomes of oral squamous cell carcinoma (OSCC) cell lines with differing responses to cisplatin, to explore the interplay between mtDNA alterations, epigenetic regulation of gene expression, and cisplatin response in OSCC. DATA DESCRIPTION: Two human OSCC cell lines, namely H103 and SAS, and drug-resistant stem-like cells derived from SAS were used in this work. To validate our hypothesis that cisplatin sensitivity is linked to mtDNA changes, we sequenced their mtDNA using a nanopore sequencer, MinION. We also obtained the whole transcriptomic profiles of the cells from a microarray analysis. The mtDNA mutational and whole transcriptomic profiles that we provide can be used alongside other similar datasets to facilitate the identification of new markers of cisplatin sensitivity, and therefore the development of effective therapies for OSCC.

PCR enhancers: Types, mechanisms, and applications in long-range PCR.

Polymerase chain reaction is an important tool in molecular biology. Although the principles of the technique are relatively simple, amplifying complex or long DNA segments can be challenging. A variety of PCR additives are used to improve the performance and yield of difficult PCRs. Each PCR additive has unique properties and enhances PCR through a different mode of action. They are used to either improve PCR sensitivity, efficiency, and specificity, or mitigate the effects of PCR inhibitors. In this review, we categorise known PCR additives into four main groups. The first three groups comprise PCR additives with well-defined mechanisms, namely those that facilitate the amplification of GC-rich sequences, counteract the detrimental effects of PCR inhibitors, or alter PCR kinetics (nanomaterials). The fourth group is a loose mix of additives with unclear mechanisms of action. Then, we discuss how these additives may be used to tackle specific PCR-related challenges, particularly those associated with long-range PCR. We conclude the review with added insights into the use of PCR additives in enhancing the synthesis of complex and long DNA fragments.

NTCU induced pre-malignant and malignant stages of lung squamous cell carcinoma in mice model.

Mice have served as an excellent model to understand the etiology of lung cancer for years. However, data regarding dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) remain elusive. Therefore, we aim to develop pre-malignant (PM) and malignant (M) lung SCC in vivo using N-nitroso-tris-chloroethylurea (NTCU). BALB/C mice were allotted into two main groups; PM and M groups which received treatment for 15 and 30 weeks, respectively. Then, the mice in each main group were allotted into three groups; control, vehicle, and cancer (n = 6), which received normal saline, 70% acetone, and 0.04 M NTCU by skin painting, respectively. Histopathologically, we discovered a mix of hyperplasia, metaplasia, and dysplasia lesions in the PM group and intracellular bridge; an SCC feature in the M group. The M group was positive for cytokeratin 5/6 protein which confirmed the lung SCC subtype. We also found significantly higher (P < 0.05) epithelium thickness in the cancer groups as compared to the vehicle and control groups at both the PM and M. Overall, this study discovered that NTCU is capable of developing PM and M lung SCC in mice model at appropriate weeks and the vehicle group was suggested to be adequate as control group for future research.

CRISPR Gene-Editing Models Geared Toward Therapy for Hereditary and Developmental Neurological Disorders.

Hereditary or developmental neurological disorders (HNDs or DNDs) affect the quality of life and contribute to the high mortality rates among neonates. Most HNDs are incurable, and the search for new and effective treatments is hampered by challenges peculiar to the human brain, which is guarded by the near-impervious blood-brain barrier. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), a gene-editing tool repurposed from bacterial defense systems against viruses, has been touted by some as a panacea for genetic diseases. CRISPR has expedited the research into HNDs, enabling the generation of in vitro and in vivo models to simulate the changes in human physiology caused by genetic variation. In this review, we describe the basic principles and workings of CRISPR and the modifications that have been made to broaden its applications. Then, we review important CRISPR-based studies that have opened new doors to the treatment of HNDs such as fragile X syndrome and Down syndrome. We also discuss how CRISPR can be used to generate research models to examine the effects of genetic variation and caffeine therapy on the developing brain. Several drawbacks of CRISPR may preclude its use at the clinics, particularly the vulnerability of neuronal cells to the adverse effect of gene editing, and the inefficiency of CRISPR delivery into the brain. In concluding the review, we offer some suggestions for enhancing the gene-editing efficacy of CRISPR and how it may be morphed into safe and effective therapy for HNDs and other brain disorders.

Roles of Rho­associated kinase in lung cancer (Review).

Lung cancer is one of the most lethal forms of cancer known to man, affecting millions of individuals worldwide. Despite advancements being made in lung cancer treatments, the prognosis of patients with the disease remains poor, particularly among patients with late­stage lung cancer. The elucidation of the signaling pathways involved in lung cancer is a critical approach for the treatment of the disease. Over the past decades, accumulating evidence has revealed that Rho­associated kinase (ROCK) is overexpressed in lung cancer and is associated with tumor growth. The present review discusses recent findings of ROCK signaling in the pathogenesis of lung cancer that were conducted in pre­clinical studies. The significant role of ROCK in cancer cell apoptosis, proliferation, migration, invasion and angiogenesis is discussed. The present review also suggests the use of ROCK as a potential target for the development of lung cancer therapies, as ROCK inhibition can reduce multiple hallmarks of cancer, particularly by decreasing cancer cell migration, which is an initial step of metastasis.

G-quadruplex structures bind to EZ-Tn5 transposase.

Next generation DNA sequencing and analysis of amplicons spanning the pharmacogene CYP2D6 suggested that the Nextera transposase used for fragmenting and providing sequencing priming sites displayed a targeting bias. This manifested as dramatically lower sequencing coverage at sites in the amplicon that appeared likely to form G-quadruplex structures. Since secondary DNA structures such as G-quadruplexes are abundant in the human genome, and are known to interact with many other proteins, we further investigated these sites of low coverage. Our investigation revealed that G-quadruplex structures are formed in vitro within the CYP2D6 pharmacogene at these sites, and G-quadruplexes can interact with the hyperactive Tn5 transposase (EZ-Tn5) with high affinity. These findings indicate that secondary DNA structures such as G-quadruplexes may represent preferential transposon integration sites and provide additional evidence for the role of G-quadruplex structures in transposition or viral integration processes.

The Roles of Tissue Rigidity and Its Underlying Mechanisms in Promoting Tumor Growth.

Tissues become more rigid during tumorigenesis and have been identified as a driving factor for tumor growth. Here, we highlight the concept of tissue rigidity, contributing factors that increase tissue rigidity, and mechanisms that promote tumor growth initiated by increased tissue rigidity. Various factors lead to increased tissue rigidity, promoting tumor growth by activating focal adhesion kinase (FAK) and Rho-associated kinase (ROCK). Consequently, result in recruitment of cancer-associated fibroblasts (CAFs), epithelial-mesenchymal transition (EMT) and tumor protection from immunosurveillance. We also discussed the rationale for targeting tumor tissue rigidity and its potential for cancer treatment.

Mitochondrial DNA alterations may influence the cisplatin responsiveness of oral squamous cell carcinoma.

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.

Community Pharmacists' Knowledge, Attitude, and Practice in Providing Self-Care Recommendations for the Management of Premenstrual Syndrome.

Background and objectives: Premenstrual syndrome (PMS) comprises a variety of physical and emotional symptoms that affect women of reproductive age. The distress caused by PMS often leads to self-medication, and many over-the-counter or non-prescription products are available for relieving PMS symptoms. The choice of a suitable product should be based on advice from a health professional, such as a community pharmacist. Hence, we assessed the knowledge, attitude, and practice of Malaysian community pharmacists in providing self-care recommendations for the management of PMS. Materials and Methods: A cross-sectional survey was carried out in Kuala Lumpur, Malaysia from September to November 2018 using a self-administered questionnaire. The respondents were community pharmacists working in Kuala Lumpur and were chosen from a list of Type A license holders in the city. Results: We achieved a response rate of 79% and included 181 questionnaires in the final analysis. Of the 181 respondents, most of them (76.8%; n = 139) had medium to good levels of knowledge of PMS. Likewise, most of the respondents (78.5%; n = 142) had positive attitudes toward their role in PMS management. Having taken courses on managing minor illnesses in women substantially enhanced their levels of knowledge of (p = 0.002), but not their attitude towards, PMS management. Among the PMS-relieving products, the most commonly recommended products were ibuprofen (79%; n = 143), mefenamic acid (74.5%; n = 135), and naproxen (66.9%; n = 121), which are well known for their anti-inflammatory effect. This suggests that the respondents based their product choice on sound evidence. Conclusions: Community pharmacists can play an important role in the management of PMS. In future work, a larger sample can be assembled to obtain more insight into the readiness of community pharmacists to help women in self-managing PMS and establish a specialized service to this end.

Metoclopramide-Induced Acute Dystonic Reactions May Be Associated With the CYP2D6 Poor Metabolizer Status and Pregnancy-Related Hormonal Changes.

We report two cases of metoclopramide-induced acute dystonia in pregnant women and consider the role of genetic variation in the pathogenesis of the adverse effect. By whole-gene sequencing, we found that both women were CYP2D6 poor metabolizers. We theorize that CYP2D6 governs the risk of metoclopramide-related acute dystonia through its role in the synthesis of serotonin, which inhibits the dopamine tone. The effect of CYP2D6 poor metabolism is exaggerated by rises in the estrogen levels during pregnancy, as the hormone augments dopamine sensitivity. Together, the two factors may create a hyper-dopaminergic state that is easily upset by metoclopramide, resulting in acute dystonia.

Revisiting the Role of Thiopurines in Inflammatory Bowel Disease Through Pharmacogenomics and Use of Novel Methods for Therapeutic Drug Monitoring.

Azathioprine and 6-mercaptopurine, often referred to as thiopurine compounds, are commonly used in the management of inflammatory bowel disease. However, patients receiving these drugs are prone to developing adverse drug reactions or therapeutic resistance. Achieving predefined levels of two major thiopurine metabolites, 6-thioguanine nucleotides and 6-methylmercaptopurine, is a long-standing clinical practice in ensuring therapeutic efficacy; however, their correlation with treatment response is sometimes unclear. Various genetic markers have also been used to aid the identification of patients who are thiopurine-sensitive or refractory. The recent discovery of novel Asian-specific DNA variants, namely those in the NUDT15 gene, and their link to thiopurine toxicity, have led clinicians and scientists to revisit the utility of Caucasian biomarkers for Asian individuals with inflammatory bowel disease. In this review, we explore the limitations associated with the current methods used for therapeutic monitoring of thiopurine metabolites and how the recent discovery of ethnicity-specific genetic markers can complement thiopurine metabolites measurement in formulating a strategy for more accurate prediction of thiopurine response. We also discuss the challenges in thiopurine therapy, alongside the current strategies used in patients with reduced thiopurine response. The review is concluded with suggestions for future work aiming at using a more comprehensive approach to optimize the efficacy of thiopurine compounds in inflammatory bowel disease.