Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.Peer reviewe

Eating Habits of Postpartum Women in Nepal

The thesis aimed to explore the eating habits of postpartum women in Nepal. Previous evidences have revealed that maternal malnutrition is a huge challenge in Nepal. Despite of several intervention programs designated to improve the maternal nutritional status and reduce maternal anemia in Nepal, there has been poor improvements in both. Since nutritional status is directly related to food habits, a need to study about eating habits during postpartum phase was realized. Therefore the study was done to identify the food habits of postpartum women and how they define their own practices. The study was conducted in Changunarayan Village Development Committee of Nepal. Qualitative method was chosen for the study. 8 postpartum women were interviewed during individual semi structured interview sessions. The study was done in collaboration with a public health worker and female health care volunteers. The data was analyzed by using inductive content analysis method. The findings of the study suggest that the eating habits during this phase depended mostly upon the socio-cultural practice of food prescription and restriction and partly upon the mother's own perception on the diet she needs. The food believed to clean the postpartum wounds, heal the tissues, repair the digestive system, improve lactation, nourish the newborn, build up immune system of the child, healthy growth and development of newborn, and some special food for postpartum phase were allowed to eat. The food believed to harm the newborn and hinder the postpartum healing were restricted to eat. The mother's perception on the food she needs relied on her observation and suggestion from the health care workers. In conclusion, improving the maternal nutritional status in Nepal demands that the healthcare workers, government of Nepal and other intervening organizations need to understand the food culture as well as the beliefs associated with it and plan intervention programs accordingly in a reflective and analytical way. Postpartum women should be encouraged to eat food rich in micronutrients, follow diet plans and take supplements as needed. The thesis argues that further researches are required to identify the validity of the beliefs associated with food culture as identified by research

A comparative study of structural variants detected by 10X linked-read exome and whole exome sequencing in multiple myeloma

Structural variants comprise a large number of variations occurring in the human genome and are detected in many diseases including cancers. To a limited extent, whole exome sequencing (WES) is capable of detecting structural variations (SVs) using algorithms and tools utilizing local assembly, split-reads, discordant read-pairs and read depth methods. However, due to the significantly large size of SVs compared to the reads produced and the presence of repetitive regions in the genome, identification of SVs presents a major challenge. 10X Genomics has developed a technology that requires very low amounts of DNA and uses a linked-reads approach to produce long reads. Recently, linked-read technology has shown promising results in resolving complex SVs. In this thesis, we aimed to assess whether linked-read exome sequencing is able to infer more comprehensive information in SVs compared to WES in multiple myeloma (MM). The disease model was chosen based on the presence of high numbers of SVs in MM patient tumor cells. Here, we report that linked-read sequencing has led to the identification of a potential novel translocation t(1; 14) that significantly impacts the change in expression of genes and could potentially have impact on the prognosis and treatment of multiple myeloma patients. By Long Ranger analysis we detected t(1;14) in six out of eight samples. Further, to study whether the translocation differentially affects the expression levels of any genes, differential gene expression was performed between t(1;14) positive versus t(1;14) wild type samples. The analysis resulted in 107 differentially expressed genes where 4 upregulated and 103 downregulated genes were found in the translocation positive samples. Among the downregulated genes, we found S100A8 and S100A9 genes which are previously shown to be associated with chemoresistance to PAD (bortezomib, doxorubicin and dexamethasone) therapy. The related breakpoints of the event were identified by Manta tool (SV caller) using both linked-read and WES. Therefore, linked-read information does not appear necessary to detect this event. In this study, we found that linked-read sequencing has certain advantages over WES such as low input DNA, increased number and quality of calls and breakpoint information. However, linked-read sequencing technique is limited to the detection of certain SV types in addition to increased cost of sequencing. These two factors must be considered before choosing linked-read sequencing over WES. Somatic mutations and clinically relevant SV were detected equally efficiently by both techniques

Comparison of Structural and Short Variants Detected by Linked-Read and Whole-Exome Sequencing in Multiple Myeloma

Simple Summary The wide variety of next-generation sequencing technologies requires thorough evaluation and understanding of their advantages and shortcomings of these different approaches prior to their implementation in a precision medicine setting. Here, we compared the performance of two DNA sequencing methods, whole-exome and linked-read exome sequencing, to detect large structural variants (SVs) and short variants in eight multiple myeloma (MM) patient cases. For three patient cases, matched tumor-normal samples were sequenced with both methods to compare somatic SVs and short variants. The methods' clinical relevance was also evaluated, and their sensitivity and specificity to detect MM-specific cytogenetic alterations and other short variants were measured. Thus, this study systematically demonstrates and evaluates the performance of whole-exome and linked-read exome sequencing technologies for detecting genetic alterations to aid in selecting the optimal method for clinical application. Linked-read sequencing was developed to aid the detection of large structural variants (SVs) from short-read sequencing efforts. We performed a systematic evaluation to determine if linked-read exome sequencing provides more comprehensive and clinically relevant information than whole-exome sequencing (WES) when applied to the same set of multiple myeloma patient samples. We report that linked-read sequencing detected a higher number of SVs (n = 18,455) than WES (n = 4065). However, linked-read predictions were dominated by inversions (92.4%), leading to poor detection of other types of SVs. In contrast, WES detected 56.3% deletions, 32.6% insertions, 6.7% translocations, 3.3% duplications and 1.2% inversions. Surprisingly, the quantitative performance assessment suggested a higher performance for WES (AUC = 0.791) compared to linked-read sequencing (AUC = 0.766) for detecting clinically validated cytogenetic alterations. We also found that linked-read sequencing detected more short variants (n = 704) compared to WES (n = 109). WES detected somatic mutations in all MM-related genes while linked-read sequencing failed to detect certain mutations. The comparison of somatic mutations detected using linked-read, WES and RNA-seq revealed that WES and RNA-seq detected more mutations than linked-read sequencing. These data indicate that WES outperforms and is more efficient than linked-read sequencing for detecting clinically relevant SVs and MM-specific short variants.Peer reviewe

Dermatoscopy of primary localised cutaneous amyloidosis ‐ A cross‐sectional study in a setting of South Asian public dermatology department

Abstract Background Amyloidosis, deposition of misfolded protein in body, is a fairly common condition. The deposition of misfolded proteins in skin which occurs in absence of systemic comorbidities, namely Primary Cutaneous Amyloidosis (PCA) is also a well‐known entity in skin of colour patients of Asian subcontinent. Primary Cutaneous Amyloidosis is usually diagnosed with good clinical acumen and typical clinical phenotype and involved site. Dermoscope has been used as an adjunct non‐invasive tool to confirm cases with diagnostic uncertainty and in those in whom biopsy is deferred. Typical dermoscopic features of PCA helps differentiate it from other pigmentary dermatoses and avoids unwanted invasive biopsies and investigations especially in resource poor settings with financial constraints. Objectives This study aims to identify and corroborate clinically, typical dermoscopic features in PCA in 42 patients which includes Macular Amyloidosis (MA) and Papular Amyloidosis (PA) predominantly in skin of colour patients from government based hospital of a south east Asian country. Materials and methods Patients with classic clinical features of PCA were selected. Primary Cutaneous Amyloidosis was subclassified into MA or PA and their corresponding clinically corroborative dermoscopic features were enlisted respectively. All patients (treatment naïve and previously treated), who consented to participate in the study were included. Patients were diagnosed based on the prototypical clinical features. Dermoscopy was done using DermLite III DL3N Polarised and Fluid Dermoscope w/PigmentBoost Brand (3Gen, DermLite LLC, San Juan Capistrano, CA, USA) and images were obtained to create digital dermoscopy system by attaching camera‐equipped mobile device via an optional connection kit (Redmi Note 11, MIUI version 13.0.5, CHINA) and the findings were enlisted concurrently. Results In this study of dermoscopic findings of PCA, 42 patients were evaluated for their clinical lesions along with its corroboration with the dermoscopic features. Macular Amyloidosis was seen in 30 patients and 12 patients had typical cutaneous phenotypic and dermoscopic feature of PA. The most common dermoscopic finding seen in patients with MA was shiny to dull white, circular or oval central hub surrounded with halo of light brown dots. Most common configuration of brownish pigmentation around central hub was fine streak type. Also eccrine clues were seen in some cases of MA, which was a unique finding. Similarly in the PA subtype, the central hub was replaced by scar like structureless translucent white area surrounded by brownish black dot like structures, especially in those with large and thick plaques. Conclusion Dermoscopic findings of PCA and their clinical corroboration is a much‐needed aspect in treating patients with pigmentary disorders and in those with skin of colour, especially in developing countries. Utilization of dermoscope in clinical settings of low income countries and in government based hospitals will decrease the add on economic burden of invasive diagnostic modalities like biopsy and other inadvertent tests done to rule out pigmentary conditions