Stability Indicating Spectrophotometric Methods Determination of Nicardipine in the Presence of its Alkaline Induced Degradation Products

Objective: Derivative, ratio spectra derivative and ratio difference spectrophotometric methods were developed and validated for simultaneous determination of Nicardipine (NIC) in the presence of its alkaline induced degradation products. Methods: In these methods the overlapped spectra of NIC and its alkaline induced degradation products were well resolved by measuring the amplitudes of first derivative (D1) spectra and the second derivative (D2) at 382.3 and 239 nm, respectively. NIC was determined by ratio spectra derivative by measuring the amplitude at 244 nm. The ratio difference spectrophotometric method was developed in which the difference between amplitudes at 237.5 nm and 260 nm of the ratio spectra were recorded. The linearity range for the applied methods was 2-18 μg/ml.Results: All the developed methods were validated according to ICH Guidelines, NIC was determined with acceptable accuracy and precision.Conclusion: These methods were suitable as stability indicating methods for the determination of NIC in the presence of its alkaline induced degradation products either in bulk powder or in a pharmaceutical formulation. Statistical analysis of the results with those obtained by applying a reported method has been carried out revealing high accuracy and good precision.Keywords: Nicardipine, Spectrophotometry, Pharmaceutical preparations, Stability indicating, derivative, ratio derivative and ratio differenc

Tumor Mutation Burden Prediction Model in Egyptian Breast Cancer patients based on Next Generation Sequencing

Objectives: This study aimed to identify the tumor mutation burden (TMB) value in Egyptian breast cancer (BC) patients. Moreover, to find the best TMB prediction model based on the expression of estrogen (ER), progesterone (PR), human epidermal growth factor receptor 2 (HER-2), and proliferation index Ki-67. Methods: The Ion AmpliSeq Comprehensive Cancer Panel was used to determine TMB value of 58 Egyptian BC tumor tissues. Different machine learning models were used to select the optimal classification model for prediction of TMB level according to patient’s receptor status. Results: The measured TMB value was between 0 and 8.12/Mb. Positive expression of ER and PR was significantly associated with TMB ≤ 1.25 [(OR =0.35, 95% CI: 0.04–2.98), (OR = 0.17, 95% CI= 0.02-0.44)] respectively. Ki-67 expression positive was significantly associated with TMB >1.25 than those who were Ki-67 expression negative (OR = 9.33, 95% CI= 2.07-42.18). However, no significant differences were observed between HER2 positive and HER2 negative groups. The optimized logistic regression model was TMB = −27.5 −1.82 ER – 0.73 PR + 0.826 HER2 + 2.08 Ki-67. Conclusion: Our findings revealed that TMB value can be predicted based on the expression level of ER, PR, HER-2, and Ki-67

Somatic Mutations Alter Interleukin Signaling Pathways in Grade II Invasive Breast Cancer Patients: An Egyptian Experience

This study aimed to investigate the impact of somatic mutations on various interleukin signaling pathways associated with grade II invasive breast cancer (BC) in Egyptian patients to broaden our understanding of their role in promoting carcinogenesis. Fifty-five grade II invasive BC patients were included in this study. Data for somatic mutations in 45 BC patients were already available from a previous study. Data for somatic mutations of 10 new BC patients were included in the current study. Somatic mutations were identified using targeted next-generation sequencing (NGS) to study their involvement in interleukin signaling pathways. For pathway analysis, we used ingenuity variant analysis (IVA) to identify the most significantly altered pathways. We identified somatic mutations in components of the interleukin-2, interleukin-6, and inter-leukin-7 signaling pathways, including mutations in JAK1, JAK2, JAK3, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Interestingly, six mutations which were likely to be novel deleterious were identified: two in the SCH1 gene, two in the IL2 gene, and one in each of the IL7R and JUN genes. According to IVA analysis, interleukin 2, interleukin 6, and interleukin 7 signaling pathways were the most altered in 34.5%, 29%, and 23.6% of our BC group, respectively. Our multigene panel sequencing analysis reveals that our BC patients have altered interleukin signaling pathways. So, these results highlight the prominent role of interleukins in the carcinogenesis process and suggest its potential role as promising candidates for personalized therapy in Egyptian patients

Frequency of Pathogenic Germline Mutations in Early and Late Onset Familial Breast Cancer Patients Using Multi-Gene Panel Sequencing: An Egyptian Study

Background: Precision oncology has been increasingly used in clinical practice and rapidly evolving in the oncology field. Thus, this study was performed to assess the frequency of germline mutations in early and late onset familial breast cancer (BC) Egyptian patients using multi-gene panel sequencing to better understand the contribution of the inherited germline mutations in BC predisposition. Moreover, to determine the actionable deleterious mutations associated with familial BC that might be used as biomarker for early cancer detection. Methods: Whole blood samples were collected from 101 Egyptian patients selected for BC family history, in addition to 50 age-matched healthy controls. A QIAseq targeted DNA panel (human BC panel) was used to assess the frequency of germline mutations. Results: A total of 58 patients (57.4%) out of 101 were found to have 27 deleterious germline mutations in 11 cancer susceptibility genes. Of them, 32 (31.6%) patients carried more than one pathogenic mutation and each one carried at least one pathogenic mutation. The major genes harboring the pathogenic mutations were: ATM, BRCA2, BRCA1, VHL, MSH6, APC, CHEK2, MSH2, MEN1, PALB2, and MUTYH. Thirty-one patients (30.6%) had BRCA2 mutations and twenty (19.8%) had BRCA1 mutations. Our results showed that exon 10 and exon 11 harbored 3 and 5 mutations, respectively, in BRCA1 and BRCA2 genes. Our analysis also revealed that the VHL gene significantly co-occurred with each of the BRCA2 gene (p = 0.003, event ratio 11/21), the MSH2 gene (p = 0.01, 4/10), the CHEK2 gene (p = 0.02, 4/11), and the MSH6 gene (p = 0.04, 4/12). In addition, the APC gene significantly co-occurred with the MSH2 gene (p = 0.01, 3/7). Furthermore, there was a significant mutually exclusive event between the APC gene and the ATM gene (p = 0.04, 1/36). Interestingly, we identified population specific germline mutations in genes showing potentials for targeted therapy to meet the need for incorporating precision oncology into clinical practice. For example, the mutations identified in the ATM, APC, and MSH2 genes. Conclusions: Multi-gene panel sequencing was used to detect the deleterious mutations associated with familial BC, which in turns mitigate the essential need for implementing next generation sequencing technologies in precision oncology to identify cancer predisposing genes. Moreover, identifying DNA repair gene mutations, with focus on non-BRCA genes, might serve as candidates for targeted therapy and will be increasingly used in precision oncology

Novel homozygous CARD11 variants in two patients with combined immunodeficiency and atopic skin disease

Abstract Background Caspase recruitment domain family, member 11 (CARD11) is an important protein which plays a fundamental role in the activation of NF-κβ pathway in lymphocytes. CARD11 deficiency can be inherited in either autosomal dominant or autosomal recessive forms and present with different phenotypes including combined immunodeficiency, atopic dermatitis, and other variable manifestations. The present report describes clinical phenotypes and immunological defects of two unrelated patients with missense homozygous variants in CARD11 presenting with combined immunodeficiency (CID) and atopic skin disease resembling that reported in dominant negative CARD11 deficiency. The patients underwent next generation sequencing, immunophenotyping of T and B subsets by flow cytometry, T cell stimulation, and evaluation of CARD11 expression. Results Both patients had features suggesting CID including repeated pneumoniae with ICU admissions, chronic diarrhea, and itchy atopic skin disease. Patient-1 has homozygous missense variant in the C terminal domain (c.2839G > A, p.Glu947Lys), and patient-2 has homozygous variant in the inhibitory domain (c.1073C > G, p.Pro568Arg). Both have profound defects in Tregs with normal recent thymic emigrants, memory, and naïve CD4+ T cells. However, in response to stimulation, T cells failed to upregulate the expression of CD25. CARD11 expression by flow cytometry was decreased rather than abolished as previously described in patients with autosomal recessive CARD11 deficiency. B cells showed marked deficiency of switched memory and increase in transitional B cells. Conclusion Missense variants causing CARD11 deficiency may affect the protein function rather than the expression and can result in a phenotype combining the atopic skin disease and the features of CID

Multigene Panel Sequencing Reveals Cancer-Specific and Common Somatic Mutations in Colorectal Cancer Patients: An Egyptian Experience

This study aims at identifying common pathogenic somatic mutations at different stages of colorectal carcinogenesis in Egyptian patients. Our cohort included colonoscopic biopsies collected from 120 patients: 20 biopsies from patients with inflammatory bowel disease, 38 from colonic polyp patients, and 62 from patients with colorectal cancer. On top of this, the cohort included 20 biopsies from patients with non-specific mild to moderated colitis. Targeted DNA sequencing using a customized gene panel of 96 colorectal related genes running on the Ion Torrent NGS technology was used to process the samples. Our results revealed that 69% of all cases harbored at least one somatic mutation. Fifty-seven genes were found to carry 232 somatic non-synonymous variants. The most frequently pathogenic somatic mutations were localized in TP53, APC, KRAS, and PIK3CA. In total, 16 somatic mutations were detected in the CRC group and in either the IBD or CP group. In addition, our data showed that 51% of total somatic variants were CRC-specific variants. The average number of CRC-specific variants per sample is 2.4. The top genes carrying CRC-specific mutations are APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4. It seems obvious that TP53 and APC genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the APC and TP53 deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in APC and TP53 were CRC-specific variants, respectively. Moreover, we identified that, in 29 somatic mutations in 21 genes, their association with CRC patients was unprecedented. Ten detected variants were likely to be novel: six in PIK3CA and four variants in FBXW7. The detected P53, Wnt/βcatenin, Angiogenesis, EGFR, TGF-β and Interleukin signaling pathways were the most altered pathways in 22%, 16%, 12%, 10%, 9% and 9% of the CRC patients, respectively. These results would contribute to a better understanding of the colorectal cancer and in introducing personalized therapies for Egyptian CRC patients

Additional file 1 of Novel homozygous CARD11 variants in two patients with combined immunodeficiency and atopic skin disease

Additional file 1. List of the genes included in the panel

Circulating Serum miRNAs as Diagnostic Markers for Colorectal Cancer

<div><p>Aim</p><p>The study was designed to assess the possibility of using circulating miRNAs (serum miRNAs) as diagnostic biomarkers in colorectal cancer (CRC) and to identify their possibility as candidates for targeted therapy.</p><p>Methods</p><p>The study involved two sample sets: 1- a training set which included 90 patients with colorectal related disease (30 with CRC, 18 with inflammatory bowel disease (IBD), 18 with colonic polyps (CP) and 24 with different colonic symptoms but without any colonoscopic abnormality who were enrolled as control group) and 2- a validation set which included 100 CRC patients. Serum miRNAs were extracted from all subjects to assess the expression profiles for the following miRNAs (<i>miR-17</i>, <i>miR-18a</i>, <i>miR-19a</i>, <i>miR-19b</i>, <i>miR-20a</i>, <i>miR-21</i>, <i>miR-146a</i>, <i>miR-223</i>, <i>miR-24</i>, <i>miR-454</i>, <i>miR-183</i>, <i>miR-135a</i>, <i>miR- 135b and miR- 92a</i>) using the custom miScript miRNA PCR-based sybergreen array. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the studied miRNAs for colorectal cancer diagnosis.</p><p>Results</p><p>Data analysis of miRNA from the training set showed that; compared to control group, only <b><i>miR-19b</i></b> was significantly up-regulated in patients with IBD group (fold change = 5.24, p = 0.016), whereas in patients with colonic polyps, <b><i>miR-18a</i></b> was significantly up-regulated (fold change = 3.49, p-value = 0.018). On the other hand, <i>miR-17</i>, <i>miR-19a</i>, <i>miR-20a and</i> <b><i>miR-223</i></b> were significantly up-regulated (fold change = 2.35, 3.07, 2.38 and 10.35; respectively and p-value = 0.02, 0.015, 0.017 and 0.016; respectively in CRC patients. However, the validation set showed that only <b><i>miR-223</i></b> was significantly up-regulated in CRC patients (fold change = 4.06, p-value = 0.04).</p><p>Conclusion</p><p>Aberrant miRNA expressions are highly involved in the cascade of colorectal carcinogenesis. We have found that (<i>miR-17</i>, <i>miR-19a</i>, <i>miR-20a and miR-223</i>) could be used as diagnostic biomarkers for CRC. On the other hand, <i>miR-19b</i> and <i>miR-18a</i> could be used as diagnostic biomarkers for CP and IBD respectively.</p></div

The clinical data of the studied groups.

<p>The clinical data of the studied groups.</p

Differential expression of the studied miRNAs in CRC validation set versus control group.

<p>Differential expression of the studied miRNAs in CRC validation set versus control group.</p