Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid.

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria.

Extra-nasal NK/T-cell lymphoma: A rare case with a rarer presentation.

Extra-nodal (NK/T) cell lymphoma (NKTCL) of nasal type (ENKL) presents with extranodal involvement and is associated with EBV. They are localized primarily to nasal and upper airway region but can rarely involve skin, testis, intestine, muscle, or present as a disseminated disease. We present a case of a 65-year-old male presented with bilateral orbital swelling, right parotid swelling, fever, mediastinal nodes, pleural effusion and hepatomegaly. Cytology of pleural fluid showed monomorphic lymphoid cells with folded nuclei, vesicular chromatin, inconspicuous nucleoli and granular cytoplasm. Few histiocytes showing hemophagocytosis were also noted in a background of inflammatory cells. Flow cytometric immunophenotyping of pleural fluid showed abnormal lymphoid cells which were positive for CD45, CD2 and CD56. Histopathological examination of the right eye swelling showed many atypical lymphoid cells which were positive for CD56 and negative for B and T cell markers. Based on morphological and immunophenotypic features a diagnosis of NKTCL, extra-nasal type was suggested. Patient succumbed to the disease despite aggressive therapy.

Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome.

Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE.

Assessing the Impact of the COVID-19 Pandemic on Maternal and Child Health Services: A Comprehensive Analysis of Government Initiatives in Northern India.

BACKGROUND: The coronavirus disease (COVID-19) pandemic has significantly impacted healthcare services globally, with particular challenges observed in maternal and child health (MCH) care. This study aimed to assess the impact of the pandemic on MCH services in northern India, focusing on key government programs. METHODS: Data were collected from four major MCH programs in India: Janani Suraksha Yojana (JSY), Janani Shishu Suraksha Karyakram (JSSK), Pradhan Mantri Surakshit Matritva Abhiyan (PMSMA), and the Universal Immunization Program (UIP). The study compared MCH service utilization and outcomes during the pandemic period (March-September 2020) with the same period in 2019. RESULTS: Preliminary findings indicated a significant reduction in the utilization of MCH services during the pandemic, with a decrease observed in institutional deliveries, antenatal care visits, and immunization coverage. For instance, institutional deliveries declined by 30% compared with the previous year, with a similar decrease observed in antenatal care visits. Immunization coverage also decreased by approximately 25%, indicating a substantial decline in preventive care services. Challenges in accessing essential treatments for sick infants have also been reported, with a 40% decrease in the utilization of free treatment services under the JSSK program. CONCLUSION: The COVID-19 pandemic has substantially impacted MCH services in northern India, highlighting the vulnerability of these essential health programs during public health emergencies. Addressing the challenges identified in this study is crucial to ensuring the continuity and resilience of MCH services in similar settings.

Urinary extracellular vesicles-encapsulated miRNA signatures: A new paradigm for urinary bladder cancer diagnosis and classification.

Bladder cancer (BCa) stands as prevalent malignancy of the urinary system globally, especially among men. The clinical classification of BCa into non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) is crucial for prognosis and treatment decisions. However, challenges persist in current diagnostic methods like Urine cytopathology that shows poor sensitivity therefore compromising on accurately diagnosing and monitoring BCa. In recent years, research has emphasized the importance of identifying urine and blood-based specific biomarkers for BCa that can enable early and precise diagnosis, effective tumor classification, and monitoring. The convenient proximity of urine with the urinary bladder epithelium makes urine a good source of noninvasive biomarkers, in particular urinary EVs because of the packaged existence of tumor-associated molecules. Therefore, the review assesses the potential of urinary extracellular vesicles (uEVs) as noninvasive biomarkers for BCa. We have elaborately reviewed and discussed the research that delves into the role of urinary EVs in the context of BCa diagnosis and classification. Extensive research has been dedicated to investigating differential microRNA (miRNA) expressions, with the goal of establishing distinct, noninvasive biomarkers for BCa. The identification of such biomarkers has the potential to revolutionize early detection, risk stratification, therapeutic interventions, and ultimately, the long-term prognosis of BCa patients. Despite notable advancements, inconsistencies persist in the biomarkers identified, methodologies employed, and study populations. This review meticulously compiles reported miRNA biomarkers, critically assessing the variability and discrepancies observed in existing research. By synthesizing these findings, the article aims to direct future studies toward a more cohesive and dependable approach in BCa biomarker identification, fostering progress in patient care and management.

Urinary extracellular vesicles miRNA-A new era of prostate cancer biomarkers.

Prostate cancer is the second most common male cancer worldwide showing the highest rates of incidence in Western Europe. Although the measurement of serum prostate-specific antigen levels is the current gold standard in PCa diagnosis, PSA-based screening is not considered a reliable diagnosis and prognosis tool due to its lower sensitivity and poor predictive score which lead to a 22%-43% overdiagnosis, unnecessary biopsies, and over-treatment. These major limitations along with the heterogeneous nature of the disease have made PCa a very unappreciative subject for diagnostics, resulting in poor patient management; thus, it urges to identify and validate new reliable PCa biomarkers that can provide accurate information in regard to disease diagnosis and prognosis. Researchers have explored the analysis of microRNAs (miRNAs), messenger RNAs (mRNAs), small proteins, genomic rearrangements, and gene expression in body fluids and non-solid tissues in search of lesser invasive yet efficient PCa biomarkers. Although the presence of miRNAs in body fluids like blood, urine, and saliva initially sparked great interest among the scientific community; their potential use as liquid biopsy biomarkers in PCa is still at a very nascent stage with respect to other well-established diagnostics and prognosis tools. Up to date, numerous studies have been conducted in search of PCa miRNA-based biomarkers in whole blood or blood serum; however, only a few studies have investigated their presence in urine samples of which less than two tens involve the detection of miRNAs in extracellular vesicles isolated from urine. In addition, there exists some discrepancy around the identification of miRNAs in PCa urine samples due to the diversity of the urine fractions that can be targeted for analysis such as urine circulating cells, cell-free fractions, and exosomes. In this review, we aim to discuss research output from the most recent studies involving the analysis of urinary EVs for the identification of miRNA-based PCa-specific biomarkers.

Bone marrow metastasis of testicular germ cell tumour: A rare case.

Germ cell tumour (GCT) is the most common testicular tumour that commonly presents as a painless mass. Bone marrow metastasis in cases of testicular GCT is rare; only few case reports are available till date in the literature. Here an adult male presented with an intra-abdominal mass in right iliac fossa with inguinal lymphadenopathy with a deranged kidney function test. Bone marrow (BM) aspirate smear revealed metastatic tumour cells, but BM-biopsy was unremarkable. High serum Beta - HCG (38286 mIU/L) pointed towards germ cell lesion. Lymph node biopsy along with immunomarkers confirmed metastatic foci from germ cell tumor and managed as per standard protocol. Rarely BM aspirate is seen positive for malignancy, while biopsy turns out to be negative. Secondly, BM metastasis of GCT should be considered while dealing with cases like this. Informed consent: This is certified that the informed consent has been obtained from the patient.

CD34 negative HLA-DR negative acute myeloid leukaemia: A higher association with NPM1 and FLT3-ITD mutations.

INTRODUCTION: CD34 and HLA-DR negativity is often used as a characteristic immunophenotypic feature of acute promyelocytic leukaemia (APL) that differentiates APL from other subtypes of acute myeloid leukaemia (AML). However, other subtypes of AML, without expression of CD34 and HLA-DR antigens, have also been reported. METHODS: We analysed the HLA-DR negative de novo non-APL AML cases by dividing HLA-DR negative non-APL group into 2 sub-groups based on CD34 expression and compared the characteristics of CD34 negative HLA-DR negative with CD34 positive HLA-DR negative non-APL AML cases with respect to morphologic, immunophenotypic, molecular and clinical parameters. RESULTS: There were 70 cases (8.54%) which were CD34 negative HLA-DR negative and 52 cases (6.34%) were CD34 positive HLA-DR negative. The median age at diagnosis was higher in CD34 negative HLA-DR negative AML than in CD34 positive HLA-DR negative AML group (38 years vs. 12 years, p < 0.001). DIC rate was higher in CD34 negative HLA-DR negative group than the other group (p < 0.001). Median total leucocyte count was higher with higher blast count in peripheral blood and bone marrow in CD34 negative HLA-DR negative AML cases than the other group (p < 0.05). CD34 negative HLA-DR negative AML was more associated with normal karyotype (96.2% vs. 38.5%; p < 0.001), NPM1 mutation (67.8% vs. 8.3%; p < 0.001) and FLT-ITD mutation (37.3% vs. 13.9%; p < 0.05). In CD34 negative HLA-DR negative group, 16 cases had co-occurrence of NPM1 and FLT3-ITD mutations, whereas no case of CD34 positive HLA-DR negative group had such dual mutation positivity. There was poor median overall survival [3.8 months (95%CI: 2.3-7.8 months) vs. 20.4 months (95% CI: 12.8-25.7 months); p = 0.0148] in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases. CONCLUSION: We found that the CD34 negative HLADR negative non APL AML is highly associated with NPM1 and FLT3-ITD mutation, older age at diagnosis, DIC, higher total leucocyte count, higher blast counts and normal karyotype in comparison to CD34 positive HLA-DR negative AML group. Co-occurrence of NPM1 and FLT3-ITD mutation was also exclusively seen in CD34 negative HLA-DR negative group. There was poor overall survival in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases.

NF-κB signaling in prostate cancer: a promising therapeutic target?

Prostate carcinoma (PCa) displays a wide variety of genetic alterations, versatile expression profiles as well as cell surface markers. Despite this heterogeneity, a common treatment for advanced PCa is androgen deprivation therapy (ADT). ADT targets the androgen receptor-a member of the nuclear receptor superfamily-which is required for development and function of the prostate and critical for PCa growth and survival. After an initial regression of the tumor during ADT, a large fraction of tumors progress to so-called castration-resistant prostate carcinoma (CRPca) which is highly resistant toward chemotherapy. The ensuing high mortality rates illustrate the importance of novel therapeutic targets for CRPCa. The transcription factor NF-κB was recently proposed as such a potential target for therapeutic intervention in CRPCa. Although NF-κB is essential for the regulation of innate and adaptive immunity recent data suggest a role of NF-κB in cancer initiation and progression. However, the exact function of NF-κB signaling in PCa is still a matter of debate. Here, we review known roles of NF-κB signaling in PCa and emphasize the crosstalk of NF-κB and androgen receptor signaling. Finally, we discuss potential therapeutic relevance of blocking NF-κB in PCa.

Top 100 cited articles on Silver diamine fluoride-A bibliometric analysis.

Background: Identification of top cited articles aids in providing comprehensive and cumulative standardized quality work to research community. The present bibliometric study was aimed at identifying 100 top cited articles on Silver diamine fluoride, highlighting its landmark publications available in the current literature and aids in providing comprehensive and cumulative standardised quality work to research community. Methodology: Four major databases (Web of science, Pubmed, Scopus and Embase) were searched using search terms like 'silver fluoride' OR 'silver diamine fluoride' OR 'silverdiammine fluoride' OR 'diamine silver fluoride' OR 'diammine silver fluoride' OR silver ammonia fluoride AND dentistry. Selected publications were studied to tabulate information like journal, authors, year etc and contents of the publications were summarised. Variables such as journal name, first author, journal rank, citation count and average citations per year, authorship patterns, author's country of origin, most productive authors, journals and countries were identified. Additional outcome variables were SDF concentrations used, study designs adopted and SDF use like caries arrest/prevention/anti-hypersensitive agent and as an irrigant. Results: Among them maximum articles were clinical trials(n = 24) however systematic(n = 12), literature(n = 14) and narrative reviews(n = 12) had an equal share. The countries with the highest number of most-cited papers were China and USA. The most studied aspect was caries arrest. Journal of Dentistry and Journal of Dental Research had major publications among top cited papers. Conclusion: This particular review will prove to be a great source of information for research community worldwide and will aid in directing them towards relevant articles.

Impact of B.1.617 and RBD SARS-CoV-2 variants on vaccine efficacy: An in-silico approach.

PURPOSE: The existing panels of COVID-19 vaccines are based on the spike protein of an earlier SARS-CoV-2 strain that emerged in Wuhan, China. However, the evolving nature of SARS-CoV-2 has resulted in the emergence of new variants, thereby posing a greater challenge in the management of the disease. India faced a deadlier second wave of infections very recently, and genomic surveillance revealed that the B.1.617 variant and its sublineages are responsible for the majority of the cases. Hence, it's crucial to determine if the current vaccines available can be effective against these variants. METHODS: To address this, we performed molecular dynamics (MD) simulation on B.1.617 along with K417G variants and other RBD variants. We studied structural alteration of the spike protein and factors affecting antibody neutralization and immune escape via In silico docking. RESULTS: We found that in seven of the 12 variants studied, there was a structural alteration in the RBD region, further affecting its stability and function. Docking analysis of RBD variants and wild-type strains revealed that these variants have a higher affinity for the ACE2 (angiotensin 2 altered enzymes) receptor. Molecular interaction with CR3022 antibody revealed that binding affinity was less in comparison to wild type, with B.1.617 showing the least binding affinity. CONCLUSIONS: The results of the extensive simulations provide novel mechanistic insights into the conformational dynamics and improve our understanding of the enhanced properties of these variants in terms of infectivity, transmissibility, neutralization potential, virulence, and host-viral replication fitness.

Effect of the sequence of pull of bone marrow aspirates on plasma cell quantification in plasma cell proliferative disorders.

INTRODUCTION: The evaluation of plasma cell (PC) compartment is influenced by the quality of bone marrow aspirate (BMA). Herein, we evaluated the impact of sequence of pull on quality of clinical assessment in plasma cell proliferative disorders (PCPDs). METHODS: Histomorphology along with smears from first pull and second pull BMA and flow cytometric immunophenotyping (FCMI) data from second pull aspirate were evaluated for cellularity and PC%. RESULTS: Of the 484 samples, BMA smears were adequate in 87.4% of first pull (median PC = 7%; IQR = 2-25%) and 51.2% of second pull samples (median PC = 2%; IQR = 0.5-12%; p < 0.001). Recovery of PC was least on FCMI (median PC = 0.59%; IQR = 0.14-3.07%), however, sample adequacy was met in 42.6% of samples with acquisition of ≥3 million events. Second pull smears under-reported PC% in 34% of newly diagnosed multiple myeloma (NDMM) (<10% PC) and 46% of MM on therapy (<5% PC), resulting in suboptimal assessment. Bone marrow biopsy (BMBx) was evaluated in a total of 309 cases (median PC = 10.0%; IQR 4.0-40.0%) with significantly higher numbers of BMPC% on BMBx compared with first pull smears (Mean ± 2SD: 25.9% ± 30.54 vs. 20.77% ± 20.20; p = 0.001). CONCLUSION: First pull BMA smears were of superior quality but inadequate in one-tenth of samples. Second pull smears underreported PC% and recovery of PC compartment was poorest on FCMI. Concurrent bone marrow biopsy and use of the first pull sample for FCMI along with acquisition of a higher number of cells on FCMI may enhance the quality of assessment in PCPDs.

Langherhans cell histiocytosis presenting as primary infertility in a young male.

Langerhans cell histiocytosis (LCH) is a rare clonal neoplastic disorder of Langerhans cells, with an incidence rate of 5 per million individuals. In adults LCH usually affects bone followed by lung, skin, pituitary gland, liver, spleen, and orbits. LCH presenting with endocrinopathy is rare and commonly involves posterior pituitary with central diabetes insipidus (DI). Here, we present a rare case of LCH involving posterior pituitary but presenting as infertility in a 25-year-old married man. Later the thyroid gland was also found to be involved in the form of multiple nodules. Fine needle aspiration cytology (FNAC) from right lobe of thyroid showed sheets of Langerhans cells along with entrapped residual thyroid follicular cells which were further confirmed by immunocytochemistry as well as cell block preparation followed by immunohistochemistry. A final diagnosis of LCH involving pituitary and thyroid was made and patient was then started on treatment according to LCH treatment protocol LCH III-6 consisting of prednisolone and vinblastine (6 weeks with daily 40 mg/m2 oral prednisolone, and 6 mg/m2 i.v. vinblastine every 7 days). Patient is responding well to the therapy and is on follow up.

A novel fabrication of electrospun polyacrylonitrile/NaYF4:Eu+3 light emitting nanofibers.

Polyacrylonitrile/NaYF4:Eu+3 nanophosphor composite nanofibers have been successfully prepared using the electrospinning technique. The electrospun nanofibers exhibited intense emission of gradient blue (X 2 = 0.254, Y 2 = 0.152 and X 3 = 0.233, Y 3 = 0.139) with different concentrations of nanophosphor under the excitation wavelength of 239 nm. The morphological and structural characterization of the nanofibers confirms the uniform dispersion of nanophosphor, while photoluminescence spectroscopy confirms tunability in luminescence properties.

Peripheral blood involvement in angioimmunoblastic T-cell lymphoma: a case report and review of the literature.

Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive variant of peripheral T-cell lymphoma, occurring in elderly patients without any gender predisposition. It accounts for 1-2% of all non-Hodgkin lymphoma. Although characterized by some peculiar histological features, diagnosis of AITL can sometimes be challenging and a definite diagnosis requires a complete immunophenotypic and molecular workup. Peripheral Blood (PB) involvement in AITL has not been studied in detail and there is a paucity of published data about leukemic presentation of AITL. We present a case of a 38-year-old female diagnosed as AITL with PB involvement. Flow cytometric (FCM) examination of PB showed 40% abnormal lymphoid cells which were CD45+, CD4+, CD2+, cCD3+, CD5+, CD10+, CD16+ and TCRγδ restricted. PB involvement by AITL appears to be more common and under-reported. Nevertheless, detection of these tumoral T lymphocytes needs to be assessed in large case studies for assessing the true incidence of PB involvement. FCM analysis is an effective and reliable approach in the identification of leukemic phase of AITL and can lead to timely and effective intervention.

Study of Predictors of Quality of Life and its Association with Anxiety and Depression in Chronic Obstructive Pulmonary Disease in Industrial Workers.

INTRODUCTION: Chronic diseases such as chronic obstructive pulmonary disease (COPD) have an adverse impact on the quality of life (QOL) of the patient. Anxiety and depression have an association with QOL in COPD. However, this area has not been studied in the Indian subcontinent, especially in reference to the industrial population, which is vulnerable to such chronic disease. The present study aims to assess the prevalence of anxiety and depression and its association with QOL in COPD patients. This study has also examined the other predictive factors associated with QOL in COPD. MATERIALS AND METHODS: This cross-sectional study involved 50 cases of COPD and an equal number of age- and sex-matched healthy controls. The severity of COPD was classified as per the global initiative for chronic obstructive lung disease recommendation. Participants were assessed for anxiety, depression, and QOL on generalized anxiety disorder 7, Patient Health Questionnaire nine-item and WHOQOL (World Health Organization Quality of Life Instrument)-BREF, respectively. RESULTS: The study sample was predominantly of men (n = 47) with a mean age of 57 years. The mean score of QOL in all domains was significantly lower in COPD cases than control. The prevalence of anxiety and depression was 38% and 44%, respectively, among COPD cases. Odds ratios predicted more risk of developing anxiety and depression in COPD as the OR for depression and anxiety, with 95% confidence intervals were 3.2 (1.2-8.3 and 4.8 (1.8-12.8), respectively. QOL had a strong association with anxiety, depression, chronicity, and the severity of the disease. CONCLUSION: Anxiety and depression are highly prevalent in COPD and appear to be strong predictors of poor QOL.

Trichobezoar without a clear manifestation of trichotillomania.

Trichophagia is a compulsive condition characterized by eating hair, mostly seen in adolescent females. Persistence of ingestion of hair over many years results in the formation of trichobezoar. Surgical intervention is often needed in the management of large trichobezoars. We present a case of a 23 year old female with trichophagia which led to the formation of gastric trichobezoar. The patient reported with complaints of pain in abdomen for past 1-2 years, and loss of appetite and vomiting post meals for 6 months. On detailed examination, a diagnosis of trichobezoar was formed which was removed by laparotomy with gastrotomy under general anaesthesia. Psychiatric evaluation revealed a history of fluctuating mood symptoms during her childhood and depressive symptomatology presently without any associated urge of plucking hair and subsequent gratification. Hence, the management of the disease requires proper liaison between the surgeon, psychiatrist, and caregivers of the patient; and regular psychiatric follow-up and treatment of the underlying psychopathology to prevent the recurrent formation of bezoar.

Hotspots mutational analysis of Wilms tumor 1 gene in acute myeloid leukaemia; prevalence and clinical correlation in North Indian population.

BACKGROUND: The pathogenic role of Wilms tumor 1 gene (WT1) is well known in renal cancer. However, recently, its over expression is been documented in cases of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and myelodysplastic syndrome (MDS). WT1 mutations is found in about 6%-15% of cases of AML affecting mainly hotspot exon 7 and 9, and less frequently in other exon such as 1, 2, 3, and 8. Different studies have shown equivocal findings with few of them depicting poorer prognosis, while others suggesting lack of any significant clinical impact. OBJECTIVE: This study was planned to evaluate prevalence of WT1 gene mutation on exon 7 & 9 in de novo cases of AML and its correlation with their clinical features and disease course. METHODOLOGY: A total of newly diagnosed and treatment naive 100 cases of AML, having blast count of ≥20% in peripheral blood or bone marrow were enrolled. Genomic DNA of all participants was extracted from blood/bone marrow sample using Qiagen® DNA extraction kit. Haematological workup for counts and flow cytometry based immunophenotypes was done. Mutation on exon 7 & 9 were detected with the help of Sanger sequencing. RESULTS: WT1 mutations were detected in both types of cases having normal vs. abnormal cytogenetics. The overall prevalence of WT1 mutation of 2% was found. We have reported one novel mutation on exon 9 of WT1 gene. Twelve cases (12%) among all analyzed AMLs were found to have synonymous single nucleotide polymorphism (SNPs) on exon 7 which has been previously reported in SNP database (rs16754). CONCLUSION: In our study, presence of synonymous SNP was not associated with any change at protein level. We also evaluated mutational status with deaths during induction remission and concluded that presence of WT1 gene mutation was associated with death during induction therapy.

Next generation sequencing guided treatment modulation and prognosis in Acute myeloid leukemia: Case vignettes.

OBJECTIVE: The genomic mutational landscape of Acute Myeloid Leukemia has contributed to better treatment options, risk stratification and prognostication of this genetically heterogeneous disease. With several approved new drugs targeting specific mutations with better outcomes, we describe here two cases of AML in which, NPM1 was detected at diagnosis. The impact of age, type of treatment, stability of NPM1 mutation, and co-occurring mutations on survival are the essential parameters for investigation. METHOD: Both the cases of AML were females, >60 years of age with normal 46XX karyotype. Allele specific RT-PCR and fragment analysis was performed for the detection of NPM1-A mutation at diagnosis. Both the patients were unfit for intensive chemotherapy therefore reduced intensity induction chemotherapy regimen was initially administered. Next-generation sequencing was performed for comprehensive mutational profiling, which guided targeted treatment, prognostic stratification, and response assessment. RESULT: We report that the older AML patients with NPM1 mutation may not have a good outcome with intensive chemotherapy, especially patients with concurrent DNMT3A/IDH-1/2 mutations. In the second case with mutated NPM1, concurrent FLT3-ITD mutation served as a therapeutic target. The FLT3 inhibitor used in combination with standard therapy showed promising results in this case. CONCLUSION: Here, we emphasize on the utility of next generation sequencing in guiding the treatment initiation or modulation during the disease course and risk stratification in AML. In conclusion, conventional chemotherapy in AML gives very poor overall survival rates and targeted chemotherapy against specific mutations may drastically improve patient survival and treatment outcomes.