Case #1
A male patient, 65 years old, presented with urinary bladder mass invading the left ureteric orifice with moderate backpressure on the left kidney, and the patient gave a history of two transurethral resections of bladder tumors (TURB) 1 year before their pathology was non-muscular invasive bladder cancer (NMIBC). The patient was hypertensive with no family history of bladder cancer, and serum creatinine was 5 mg/dl, total leukocyte count (TLC) 40 109/L, and platelets count 1.2.109/L. The patient underwent left nephrostomy for his renal insufficiency, bone marrow aspiration for leukocytosis, and thrombocytopenia Then, the patient was deteriorated as his bone marrow aspiration reported the presence of leukemia, and he died while being treated for leukemia.
Case #2
A male patient, 53 years old, smoker presented with hematuria and blood clots; radiological investigations revealed a 5-cm bladder mass at the base of the bladder; the patient gave a past history of cystolithotomy, cholecystectomy, and HCV treatment, no history of chronic medical diseases, and there was no family history of the same illness. TURBT revealed a high-grade muscle invasive urothelial carcinoma. After 1 month, radical cystectomy with ileal conduit diversion was done, and its histopathology revealed T2G3 with negative lymph node involvement. The patient was followed up, and at 8 months of follow-up, he developed a 4-cm left renal lower pole mass, for which partial nephrectomy was done, and its histopathology revealed papillary renal cell carcinoma (RCC).
Case #3
A male, 47 years old, heavy smoker presented to outpatient clinic with complaints of hematuria with blood clots and no chronic medical diseases (CMD); he had no family history of the same illness. The patient had a past history of internal fixation for left femur fracture. According to his imaging investigations, he was suffering from left bladder wall mass. TURBT was done, and its report revealed left lateral wall 4.5 cm mass high-grade transitional cell carcinoma (TCC) with muscle invasion. The patient was treated with trimodal therapy and followed up with CT with contrast. The patient responded to treatment, but his kidneys showed bilateral hypo-dense renal masses. Renal biopsy revealed Hodgkin lymphoma, and the patient was referred for chemotherapy.
Case #4
A male patient, 57 years old, presented to the outpatient clinic with complaints of hematuria with blood clots and history of diabetes mellitus on insulin treatment and irrelevant family history. His radiology revealed multiple bladder masses; diagnostic cystoscopy revealed multiple bladder masses, and TURBT was done. Pathology report revealed high-grade transitional cell carcinoma (TCC) with muscle invasion. The patient was treated with trimodal therapy.
Molecular analysis
DNA extraction
Genomic DNA was extracted from tissues and their matched blood samples using commercially available kits as follows: DNA extraction was done according to the manufacturer’s instructions of QIAamp DNA Mini blood kit (Cat No # 51104, Qiagen, Germany) based on spin column for DNA extraction method, while tissue DNA was extracted using Cat No # 51304, Qiagen, Germany from fresh tissue samples.
Both purity and the concentration for extracted DNA were detected by nano-drop spectrophotometer (Quawell, Q-500, Scribner, USA); then, extracted DNA was stored at − 80 °C till further assessments.
Library preparation and purification
The required regions for the examined genes BRCA1 and BRCA2 were amplified by Oncomine BRCA1 & BRCA2 research kit (Life Technologies). Amplification process was carried out by means of Ion AmpliSeq Library kit (Cat No# 4480441 Life technologies), according to the manufacturer’s protocol. After amplification, the primers were digested using FuPa Reagent; the samples were barcoded with Ion Xpress Barcode Adaptors. Barcoded libraries were then purified using Agencourt beads, and libraries were measured using the Ion Library TaqMan Quantitation kit (Cat No# 4468802 Life technologies). The quantified libraries were promoted to template preparation.
Template preparation
The obtained libraries from the previous step were pooled on molar equivalent rations to yield at least average 150x depth for coverage regarding every germline DNA sample and 750x for somatic samples. The assembled libraries were clonally amplified using Ion PGM Hi-Q view OT2 kit (Cat No# A29900 Life technologies) on the Ion OneTouch 2 instrument (Life technologies, USA) according to the manufacturer’s instructions. Then, the template ion sphere particles (ISP) were enriched using Ion PGM enrichment beads (Cat No# 4478525 Life technologies) by Ion OneTouch ES system (Life technologies, USA) according to the manufacturer’s instructions; the positive ISP Quality was assayed on Qubit 2.0 Fluorometer (Life technologies, USA) and then continued for accomplishing the sequencing process.
Sequencing using ion torrent PGM platform
Subsequently, calibrations and adjustments of the pH were done according to the manufacturer’s instructions using the Ion PGM Hi-Q View Sequencing kit (Cat No# A30044 Life technologies). Entirely barcoded enriched samples were sequenced on the Ion Torrent PGM Platform (Ion Torrent PGM, Life technologies, USA) using Ion 318 Chip Kit V2 BC (Cat No# 4488150 Life technologies).
Data analysis
Generated BAM files were uploaded to the cloud-based Ionreporter server version 5.10 on ThermoFisher website, and the Oncomine BRCA-specific plugin was used to analyze the paired normal and tumor sample for each patient using the default plugin parameters.