Uncovering the clinical relevance of unclassified variants in DNA repair genes: a focus on BRCA negative Tunisian cancer families.

Introduction: Recent advances in sequencing technologies have significantly increased our capability to acquire large amounts of genetic data. However, the clinical relevance of the generated data continues to be challenging particularly with the identification of Variants of Uncertain Significance (VUSs) whose pathogenicity remains unclear. In the current report, we aim to evaluate the clinical relevance and the pathogenicity of VUSs in DNA repair genes among Tunisian breast cancer families. Methods: A total of 67 unsolved breast cancer cases have been investigated. The pathogenicity of VUSs identified within 26 DNA repair genes was assessed using different in silico prediction tools including SIFT, PolyPhen2, Align-GVGD and VarSEAK. Effects on the 3D structure were evaluated using the stability predictor DynaMut and molecular dynamics simulation with NAMD. Family segregation analysis was also performed. Results: Among a total of 37 VUSs identified, 11 variants are likely deleterious affecting ATM, BLM, CHEK2, ERCC3, FANCC, FANCG, MSH2, PMS2 and RAD50 genes. The BLM variant, c.3254dupT, is novel and seems to be associated with increased risk of breast, endometrial and colon cancer. Moreover, c.6115G>A in ATM and c.592+3A>T in CHEK2 were of keen interest identified in families with multiple breast cancer cases and their familial cosegregation with disease has been also confirmed. In addition, functional in silico analyses revealed that the ATM variant may lead to protein immobilization and rigidification thus decreasing its activity. We have also shown that FANCC and FANCG variants may lead to protein destabilization and alteration of the structure compactness which may affect FANCC and FANCG protein activity. Conclusion: Our findings revealed that VUSs in DNA repair genes might be associated with increased cancer risk and highlight the need for variant reclassification for better disease management. This will help to improve the genetic diagnosis and therapeutic strategies of cancer patients not only in Tunisia but also in neighboring countries.

Gemcitabine-Related Atrial Fibrillation: A Case Report and Review of the Literature.

INTRODUCTION: Gemcitabine is a commonly used antimetabolite that has been effective in a broad spec- trum of tumors so far. The main grade three and four known toxicity of this drug is myelosuppres- sion. Cardiac adverse events have been rarely reported and gemcitabine-induced Atrial-Fibrillation (AF) has been described in only five previous cases so far. Here we report the 6th case of gemc- itabine-related AF. CASE PRESENTATION: A 68-year-old man diagnosed with metastatic nasopharyngeal cancer was referred to our oncology department. He started first-line chemotherapy with gemcitabine and cisplatin. He presented poorly tolerated atrial fibrillation related to gemcitabine infusion that lasted for six days. The treatment was then withdrawn, and the patient received the best supportive care. CONCLUSION: We conclude that medical oncologists and cardiologists should be aware of such toxicities of gemc- itabine, especially in the elderly who seem to be at a higher risk of such adverse events and which may dictate discontinuation of the drug.

Identification of BRCA2 Cis Double Heterozygous Breast Cancer Cases Using Whole Exome Sequencing: Phenotypic Expression and Impact on Personalized Oncology.

BRCA1 and BRCA2 are the most commonly mutated breast cancer susceptibility genes that convey a high risk of breast and ovarian cancer. Most BRCA1 or BRCA2 mutation carriers have inherited a single heterozygous mutation. In recent years, very rare cases with biallelic or trans double heterozygous mutations on BRCA1 and or BRCA2 have been identified and seem to be associated with distinctive phenotypes. Given that this genotype-phenotype correlation in cancer predisposing hereditary conditions is of relevance for oncological prevention and genetic testing, it is important to investigate these rare BRCA genotypes for better clinical management of BRCA mutation carriers. Here we present the first report on Cis double heterozygosity (Cis DH) on BRCA2 gene identified using Whole exome sequencing (WES) in a Tunisian family with two BRCA2 mutations namely: c.632-1G>A and c.1310_1313DelAAGA that are both reported as pathogenic in ClinVar database. Subsequent analysis in 300 high-risk Tunisian breast cancer families detected this Cis double heterozygous genotype in 8 additional individuals belonging to 5 families from the same geographic origin suggesting a founder effect. Moreover, the observed Cis DH seems to be associated with an early age of onset (mean age = 35.33 years) and severe phenotype of the disease with high breast cancer grade and multiple cancer cases in the family. The identification of unusual BRCA genotypes in this Tunisian cohort highlights the importance of performing genetic studies in under-investigated populations. This will also potentially help avoiding erroneous classifications of genetic variants in African population and therefore avoiding clinical misdiagnosis of BRCA related cancers. Our findings will also have an impact on the genetic testing and the clinical management of North African breast cancer patients as well as patients from different other ethnic groups in regard to several emerging target therapies such as PARP inhibitors.

Germline copy number variations in BRCA1/2 negative families: Role in the molecular etiology of hereditary breast cancer in Tunisia.

Hereditary breast cancer accounts for 5-10% of all breast cancer cases. So far, known genetic risk factors account for only 50% of the breast cancer genetic component and almost a quarter of hereditary cases are carriers of pathogenic mutations in BRCA1/2 genes. Hence, the genetic basis for a significant fraction of familial cases remains unsolved. This missing heritability may be explained in part by Copy Number Variations (CNVs). We herein aimed to evaluate the contribution of CNVs to hereditary breast cancer in Tunisia. Whole exome sequencing was performed for 9 BRCA negative cases with a strong family history of breast cancer and 10 matched controls. CNVs were called using the ExomeDepth R-package and investigated by pathway analysis and web-based bioinformatic tools. Overall, 483 CNVs have been identified in breast cancer patients. Rare CNVs affecting cancer genes were detected, of special interest were those disrupting APC2, POU5F1, DOCK8, KANSL1, TMTC3 and the mismatch repair gene PMS2. In addition, common CNVs known to be associated with breast cancer risk have also been identified including CNVs on APOBECA/B, UGT2B17 and GSTT1 genes. Whereas those disrupting SULT1A1 and UGT2B15 seem to correlate with good clinical response to tamoxifen. Our study revealed new insights regarding CNVs and breast cancer risk in the Tunisian population. These findings suggest that rare and common CNVs may contribute to disease susceptibility. Those affecting mismatch repair genes are of interest and require additional attention since it may help to select candidates for immunotherapy leading to better outcomes.

Toxicity profile of taxanes in Tunisian cancer patients: A retrospective study of 90 cases.

INTRODUCTION: Taxanes are widely used in medical oncology. The aim of our study was to report and analyze the toxicity features of these drugs in Tunisian patients and to determine their impact on treatment response. METHODS: Our retrospective study concerned 90 patients treated by taxanes in a medical oncology unit, from January 2014 to January 2017. We collected their epidemiologic and anatomo-clinical data and we detailed toxicity features including types grades and impact on tumor response. RESULTS: Median age was 46 years. 80% of patients had breast cancer. Tumors were metastatic in 23.3% of cases. Nail toxicity was observed in 100% of patients. Grade I-II digestive toxicity was observed in 54.4% of cases. Hematological toxicity was noted in 42.2% of patients and it reached grade III-IV in five patients. Neurological toxicity occurred in 31% of patients and was grade III-IV in 6 cases. Alopecia was observed in 60% of patients. Fatigue was noted in 57.8% of patients. Myalgia was observed in 42.2% of patients. Toxicity did not affect the response to treatment. CONCLUSION: The taxanes' toxicity profile in Tunisian patients is characterized by more frequent digestive and nail toxicities and less frequent hematological toxicities, dose reduction and treatment delays than other populations.

The Predictive Value of 2D Myocardial Strain for Epirubicin-Induced Cardiotoxicity.

INTRODUCTION: Although epirubicin has significantly improved outcome in breast cancer (BC) patients, it is responsible for myocardial dysfunction that affects patients' quality of life. The use of 2D global longitudinal strain (GLS) has been reported to detect early myocardial dysfunction. The aim of this study was to evaluate how GLS changes can predict cardiotoxicity. METHODS: We conducted a prospective study from March 2018 to March 2020 on 66 patients with no cardiovascular risk factors, who presented with BC and received epirubicin. We measured left ventricular ejection fraction (LVEF) and GLS before chemotherapy, at three months (T3), and at 12 months (T12) from the last epirubicin infusion. Chemotherapy-Related-Cardiac-Dysfunction (CTRCD) was defined as a decrease of 10% in LVEF to a value below 53% according to ASE and EACI 2014 expert consensus. RESULTS: The mean age at diagnosis was 47 ± 9 years old. At baseline, median LVEF was 70% and median GLS was -21%. Shortly after chemotherapy completion, two patients presented with symptomatic heart failure while asymptomatic CTRCD was revealed in three other patients at T12. Three months after the last epirubicin infusion, median LVEF was 65%, median GLS was -19%, and median GLS variation was 5%. However, in patients who presented with subsequent CTRCD, median GLS at T3 was -16% and median GLS variation was 19% (p=0.002 and p < 0.001, respectively, when compared to patients who did not develop cardiotoxicity). Persistent GLS decrease at T3 was an independent predictor of CTRCD at T12. Age and left-sided thoracic irradiation did not increase the risk of cardiotoxicity in our study while the cumulative dose of epirubicin significantly affected cardiologic findings (p=0.001). CONCLUSION: This was the first North African study that assesses the value of measuring GLS to early detect cardiotoxicity. Patients whose GLS remained decreased after 3 months from anthracyclines-base chemotherapy had an increased risk for developing subsequent CTRCD. Further studies with larger sample size are warranted to identify the best cardioprotective molecules to be initiated in these patients before LVEF declines.

Identification of Novel BRCA1 and RAD50 Mutations Associated With Breast Cancer Predisposition in Tunisian Patients.

BACKGROUND: Deleterious mutations on BRCA1/2 genes are known to confer high risk of developing breast and ovarian cancers. The identification of these mutations not only helped in selecting high risk individuals that need appropriate prevention approaches but also led to the development of the PARP-inhibitors targeted therapy. This study aims to assess the prevalence of the most frequent BRCA1 mutation in Tunisia, c.211dupA, and provide evidence of its common origin as well as its clinicopathological characteristics. We also aimed to identify additional actionable variants using classical and next generation sequencing technologies (NGS) which would allow to implement cost-effective genetic testing in limited resource countries. PATIENTS AND METHODS: Using sanger sequencing, 112 breast cancer families were screened for c.211dupA. A set of patients that do not carry this mutation were investigated using NGS. Haplotype analysis was performed to assess the founder effect and to estimate the age of this mutation. Correlations between genetic and clinical data were also performed. RESULTS: The c.211dupA mutation was identified in 8 carriers and a novel private BRCA1 mutation, c.2418dupA, was identified in one carrier. Both mutations are likely specific to North-Eastern Tunisia. Haplotype analysis supported the founder effect of c.211dupA and showed its recent origin. Phenotype-genotype correlation showed that both BRCA1 mutations seem to be associated with a severe phenotype. Whole Exome Sequencing (WES) analysis of a BRCA negative family revealed a Variant of Unknown Significance, c.3647C > G on RAD50. Molecular modeling showed that this variant could be classified as deleterious as it is responsible for destabilizing the RAD50 protein structure. Variant prioritization and pathway analysis of the WES data showed additional interesting candidate genes including MITF and ANKS6. CONCLUSION: We recommend the prioritization of BRCA1-c.211dupA screening in high risk breast cancer families originating from the North-East of Tunisia. We also highlighted the importance of NGS in detecting novel mutations, such as RAD50-c.3647C > G. In addition, we strongly recommend using data from different ethnic groups to review the pathogenicity of this variant and reconsider its classification in ClinVar.

Clival chordoma in a young male patient: a case report.

Chordoma is a rare malignant tumor of the spine. We report the case of a 26-year-old man who presented with facial paralysis and upper limbs paresthesia. Cerebral CT-scan and cerebro-spinal MRI revealed a 58mm locally advanced middle clival mass with deviation of median cerebral structures. Endoscopic biopsy concluded to a chondroid chordoma. Skeletal survey and thoraco-abdomino-pelvic CT-scan were normal. Treatment consisted in complete surgical removal of the tumor followed by adjuvant radiotherapy. The patient is alive free of disease with a follow up of 12 months.

Urothelial bladder carcinoma in childhood: a case report.

We report an exceptional case of transitional cell carcinoma of the bladder in a 14-years old boy without personal nor family history who consulted for a total hematuria. Work-up showed a bladder lesion sized 5cm with histology of urothelial cancer. Treatment consisted of a transurethral surgery with carcinologic complete resection. Patient is alive, free of disease with a follow-up of 36 months.

Oxaliplatin-induced peripheral neuropathy risk factors and management in Tunisian population.

The most important limits of oxaliplatin treatment is its peripheral neurotoxicity. The aim of our study was to evaluate the oxaliplatin-induced peripheral neuropathy, its impact on treatment and its management. One hundred chemo-naive patients treated with oxaliplatin-based regimen in the medical oncology department of the military hospital of Tunis between 2012 and 2017 were recruited retrospectively. Evaluation of neuropathy was done according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE V4). Fifty-six patients were aged more than 60 years. The sex-ratio was 1.56. Twenty-seven patients were overweight, 17 were obese and 56 had a BMI inferior to 25 kg/m2. Two patients were consuming alcohol. Twenty-three patients had diabetes. Sixty-four patients developed chronic peripheral neuropathy because of oxaliplatin (grade 1-2 in 58 cases and grade 3 in 6 cases). Sex, BMI, diabetes and alcohol consumption were not associated with the development of peripheral neuropathy. No association was found between grades of neuropathy and sex, alcohol consumption and diabetes. The median cumulative dose of oxaliplatin that induced neuropathy was 432.4 mg/m2. The most prescribed treatment was gabapentin (81%) and carbamazepine (16.8%). The treatment was not sufficient to stop neuropathy in 82.6% of cases. Dose reduction was done in 64.2% of cases, treatment delay in 10.7% of cases and treatment interruption in 10.7% of cases. We didn't find any association between known risk factors and peripheral neuropathy. The cumulative dose is interesting to define or to predict the timing of neurotoxicity.

Transformation of a prostatic adenocarcinoma into squamous cell carcinoma after luteinizing hormone-releasing hormone (LHRH) agonist and radiotherapy treatment.

Squamous cell carcinoma of the prostate is rare and represents 0.5% to 1% of prostatic carcinomas. Transformation of prostatic adenocarcinoma into squamous cell carcinoma after LH-RH agonist intake has been reported in only 8 cases in the literature. To our knowledge, our case is the second pure squamous cell carcinoma observed after hormonotherapy and radiotherapy. We reported a case of a patient with prostatic adenocarcinoma treated by radical prostatectomy followed by radiotherapy. Eleven years later, he had a vesical recurrence of prostatic adenocarcinoma. Our patient had an endoscopic resection followed by injections of Triptorelin. Six months later, he developed a local recurrence of a squamous cell carcinoma.

Ki-67: role in diagnosis, prognosis and follow-up after treatment of breast cancers.

AIM: To evaluate the literature data about diagnostic value, prognosis value and interest in follow-up of Ki-67 antibody after treatment for breast cancer. METHODS: We performed a literature search in pubmed using the keywords : Ki-67, anti-Ki-67, breast cancer, prognosis, proliferation, chemotherapy, hormone therapy. RESULTS: Ki-67 is routinely used as a static marker of proliferative activity and in follow-up-monitoring before and after treatment by chemotherapy and more recently hormonotherapy. Ki-67 was also used at a cut-off of 14% to differentiate between luminal A and B breast cancers. A high Ki-67 expression is probably related to a poorer prognosis but also a better response to neoadjuvant chemo and/or targeted therapy. More recently, genomic analysis is more reliable to classify the molecular breast cancer subtypes avoiding the possible cases of discordant Ki-67 rate. Ki-67 is also interesting in predicting histological response to neoadjuvant chemo and hormone therapy. CONCLUSION: Ki-67 evaluated by immunohistochemistry is important in routine in countries without bimolecular plateforms despite technical insufficiencies. When available, genomic grading is better to classify molecular subtypes and determine breast cancer prognosis in adjuvant and neoadjuvant setting.

Glioblastoma in Tunisia: A retrospective study about 41 cases.

BACKGROUND: Glioblastoma (GB) is the most common and lethal primary brain tumor in adults representing 25% of primary brain tumors in adults. The objective of our study was to report the epidemiologic, clinical and therapeutic features of GB in Tunisia. METHODS: Our retrospective study included 41 patients with histologically confirmed GB treated between 2006 and 2012 at the medical oncology departments of Abderrahmane Mami hospital in Ariana and the military hospital in Tunis. RESULTS: Median age was 54 years (13 to 72 years) and sex-ratio was 2.3. Karnofsky performance status (KPS) was <70% in 31.7% of cases, while Recursive partitioning analysis radiation therapy oncology group (RTOG-RPA) classification was III in 11 (26.8%), IV in 19 (46.3%), V in 10 (24.3%) and VI in 1 (2.4%) cases. Complete resection (CR) was achieved in 29 patients (70.7%), partial resection (PR) or tumor debulking in 5 patients (12.2%) and biopsy alone (BA) in 7 patients (17.1%). All patients received brain radiotherapy (RT) at a dose of 60 Gy combined with concurrent temozolomide (TMZ). Nineteen patients (46.3%) received adjuvant TMZ, 8 of them completed 6 cycles. Median overall survival (OS) was 12 months (2 to 56 months). Six, 12, 18 and 24-months OS rates were 84.6%, 57.6%, 35.4% and 20.7%, OS being correlated to age, KPS, RPA and quality of resection. CONCLUSION: Our retrospective study is the first African GB series. Despite it included predominantly poor prognosis patients with impaired neurocognitive function and adjuvant treatment discontinuation, our median OS was comparable to Stupp.