Early-onset breast cancer (EOBC) is disproportionately common in Saudi Arabia, where women present nearly a decade earlier than in Western countries, suggesting unique inherited susceptibility. While BRCA1/2 explain part of the hereditary risk, the contribution of rare coding variants in Arab EOBC remains unclear. Whole-exome sequencing was performed on germline DNA from 102 unrelated Saudi EOBC patients and 1395 cancer-free controls recruited from the same national Saudi cohort. Rare variants were defined by stringent frequency and quality thresholds and classified as rare loss-of-function (RLOF) or rare predicted damaging variants (RPDVs). Gene-level case-control analyses were conducted using burden tests, with exome-wide significance set at p < 2.5 × 10-6. RLOF variants in BRCA1 (6.9% of EOBC vs. 0.14% of controls; OR = 51.3; p < 1.0 × 10-10) and RPDVs in TP53 (4.9% vs. 0.36%; OR = 14.3; p = 5.39 × 10-8) demonstrated strong associations. Sequence Kernel Association Test (SKAT) analysis identified NOTCH4 and OR12D3 and reinforced burden-based significance in GUCY2F, FRMPD3, and SHROOM2. No enriched signaling pathway emerged, indicating heterogeneous rare-variant mechanisms. This first germline exome-wide rare-variant association study in Saudi EOBC identifies substantial enrichment driven by BRCA1, TP53, and additional candidate genes, supporting population-specific genetic risk evaluation and the need for replication in larger Arab cohorts.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy with especially high incidence in Middle Eastern populations. While classical hereditary syndromes explain a minority of cases, the broader germline landscape of non-syndromic PTC remains unclear. Whole-exome sequencing was performed on 245 unselected Saudi PTC patients to identify germline pathogenic or likely pathogenic variants (PVs/LPVs) in cancer predisposition genes. Clinical and molecular characteristics, and family history were integrated to assess phenotypic correlations. Eleven patients (4.5%) harbored germline PVs/LPVs in cancer susceptibility genes including STK11, TP53, BRCA1, BRCA2, FANCA, SLX4, RAD50, MSH6, POLD1 and NF1. Four patients (36.4%) carried PVs/LPVs in canonical FA pathway genes; this increased to five patients (45.5%) when RAD50 was included. Two unrelated patients harbored the same STK11 variant (p.R304Q) without classical Peutz-Jeghers syndrome features. A TP53 hotspot mutation (p.R175H) was identified in a patient with a personal history of gastric cancer, a malignancy associated with Li-Fraumeni syndrome. Notably, the BRCA1 PV detected matches a known Saudi founder mutation in hereditary breast cancer, now observed in PTC. Most germline positive cases lacked syndromic manifestations, underscoring limitations of phenotype or family history-driven genetic testing strategies. These findings suggest that a small subset of non-syndromic PTC cases may carry germline PVs/LPVs in cancer predisposition genes, highlighting the need for broader genetic screening frameworks. Unbiased whole-exome analysis in unselected cohorts can uncover under-recognized genetic risk and guide screening strategies to address the unique hereditary landscape of thyroid cancer in underrepresented populations.

BACKGROUND:HER2-low breast cancer (BC) has recently emerged as a therapeutically targetable entity, yet its biological and clinical relevance remains debatable. Limited data are available about HER2-low from non-Western populations, particularly the Middle East, where distinct tumor biology may influence phenotype and treatment response. METHODS:We retrospectively analyzed 1097 Saudi breast cancer patients for HER2 status by immunohistochemistry (IHC), classifying tumors as HER2-zero (IHC 0) or HER2-low (IHC 1+/2+ and FISH-negative). Clinicopathological characteristics, biomarker profiles (ER, PR, Ki-67), molecular alterations (PIK3CA, TP53, BRCA) and survival outcomes (overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS), and distant disease-free survival (DDFS)) were compared. RESULTS:HER2-low tumors comprised 34.5% (n = 378) of the cohort and were significantly associated with ER (p < 0.0001) and PR (p = 0.0226) positivity, lower triple-negative phenotype (p < 0.0001), and reduced Ki-67 proliferation index (p = 0.0136) compared to HER2-zero tumors. Trends toward higher PIK3CA mutation (p = 0.0875) and lower BRCA mutation (p = 0.0892) rates were observed in HER2-low tumors, though not statistically significant. Despite these favorable biological features, survival analyses revealed no significant differences between HER2-low and HER2-zero subtypes with regards to OS, CSS, DFS, and DDFS. CONCLUSION:In this large, ethnically homogenous Saudi cohort, HER2-low breast cancer represents a distinct molecular and clinicopathological subtype with luminal like features, yet no prognostic advantage. These findings reinforce the therapeutic, rather than prognostic, significance of HER2-low status, highlighting its relevance in targeted antibody-drug conjugate-based therapies rather than influencing baseline risk stratification.

Early-onset Breast Cancer (EOBC), defined as breast cancer diagnosed at or before the age of 40 years, represents a small but clinically aggressive subset of breast cancer cases. While pathogenic variants in BRCA1/2 might explain some of this risk, many EOBC cases remain unexplained. This study aimed to investigate rare germline variants in DNA repair genes that may contribute to EOBC among BRCA1/2-negative patients of Arab ancestry. We performed germline whole exome sequencing (WES) on a cohort of 79 BRCA1/2 negative EOBC patients (median age 28 years, range 13-39). Rare variants (minor allele frequency < 1%) in 217 curated DNA repair genes were filtered and prioritized using CADD and REVEL scores. Protein structure modeling was conducted for selected novel variants to evaluate their potential impact on protein function. Seventy rare deleterious variants were identified across the analyzed DNA repair genes. Among these, five novel variants were identified in DNA2, CLK2, EME2, SWI5 and RAD51B, the latter harboring a frameshift variant indicative of loss of function. Structured modeling revealed that three novel missense variants - DNA2, CLK2 and EME2 - were predicted to introduce localized structural changes that may affect protein function. Several patients carried multiple deleterious variants, suggesting a possible polygenic contribution to EOBC predisposition. These findings expand the spectrum of rare DNA repair gene variants observed in BRCA1/2-negative early-onset breast cancer and provide candidate variants for further investigation, rather than establishing definitive causal associations.

BACKGROUND:Whether synchronous (SDM) and metachronous (MDM) metastases in differentiated thyroid cancer (DTC) differ in clinical behavior or molecular profiles remains unclear. Prior studies suggest poorer outcomes in metachronous cases but regional molecular profiles and clinical outcome are not well defined. We aimed to assess whether molecular alterations, immune landscape, metastatic sites and survival outcomes differ between SDM and MDM DTC in a large Middle Eastern cohort. MATERIALS AND METHOD:Among 1,822 DTC patients, 178 developed distant metastases-88 (4.8%) SDM and 90 (4.9%) MDM. Clinicopathological features, mutational status, PD-L1 expression and radioactive iodine (RAI) response were compared. Kaplan-Meier analysis assessed overall survival (OS) and thyroid cancer-specific survival (TCSS). RESULTS:Molecular alterations were largely similar between SDM and MDM groups. TERT mutation alone occurred in 16.9% of SDM vs. 19.7% of MDM. BRAF V600E mutation alone was significantly higher in MDM cases (22.2% vs 10.4%). TERT and BRAF V600E co-mutations were comparable (22.1% vs 30.9%) between the two groups. NRAS, HRAS and TP53 mutations showed no significant difference. RAI refractoriness was 57.9% (SDM) vs 67.8% (MDM) and PD-L1 high expression was 42.2% (SDM) vs 54.5% (MDM). Cancer-specific mortality was 8.6% in SDM and 13.8% in MDM groups. Metastatic patterns revealed the lung as the most common site (69.3% SDM vs. 84.4% MDM), followed by bone (37.5% vs. 26.7%), brain (5.7% vs. 3.3%) and liver (1.1% in synchronous only). Multiple-organ metastasis were observed in 11.4% of SDM and 13.3% MDM patients. However, Kaplan Meier curves showed no statistically significant differences in OS or TCSS by metastatic timing or metastatic site. CONCLUSION:In this Middle Eastern cohort, no consistent molecular or clinical disparities across most parameters. These findings suggest the timing of metastasis may not independently influence prognosis and highlight the relevance of individualized, biology-driven management strategies.

Abstract Background: Risk stratified treatment strategies have become a focus in the treatment of Differentiated Thyroid Cancer (DTC). Tumor size at diagnosis has been widely used as a major mortality risk factor in risk stratification of DTC, but whether this is generally applicable, particularly in patient with different BRAF genetic backgrounds, is unclear. The current study was designed to analyze whether tumor size at diagnosis is a major prognostic factor in Middle Eastern DTC. Methods: We conducted a comparative study of the relationship between tumor size at diagnosis and event free survival with respect to BRAF status in 1709 consecutive patients treated surgically for Middle Eastern DTC. Patients were divided into four groups according to the size of tumor (≤4 cm vs. >4 cm) and BRAF mutation status: Group 1 (≤4 cm without BRAF mutation, n = 587), Group 2 (≤4 cm with BRAF mutation, n = 765), Group 3 (>4 cm without BRAF mutation, n = 198) and Group 4 (>4 cm with BRAF mutation, n = 159). Predictors of event-free survival (EFS) were compared using the Log-rank test and Cox proportional hazards models. Results: Following multivariate analysis, patients with tumors >4cm did worse than patients with tumors <4cm with respect to EFS (Group 3 vs Group1 HRadjusted=1.38, 95% CI=1.02-1.86, p = 0.0359; Group 4 vs Group 2 HRadjusted=1.61, 95% CI=1.20-2.16, p = 0.0017). However, patients did not differ according to EFS, regardless of the presence of BRAF mutation within each size category (Group 2 vs Group 1 HRadjusted=0.98 CI=.78-1.23, p=0.8415 ; Group 4 vs Group 3 HRadjusted=1.17, 95% CI=0.84-1.62, p=0.3523] Conclusion: Tumor size is an independent predictor of EFS in Middle Eastern DTC patients, regardless of BRAF mutational status. Citation Format: Rong Bu, Abdul K. Siraj, Sandeep K. Parvathareddy, Kaleem Iqbal, Saud Azam, Zeeshan Qadri, Maha Al-Rasheed, Wael Haqawi, Allianah D. Benito, Khawla S. Al-Kuraya. Effect of tumor size and BRAF mutational status in Middle Eastern differentiated thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6278.

Background: Colorectal cancer (CRC) displays a complex pattern of inheritance. It is postulated that much of the missing heritability of CRC is enriched in high-impact rare alleles, which might play a crucial role in the etiology and susceptibility of CRC. Methods: In this study, an exome-wide association analysis was performed in 146 patients with high-risk CRC in the Middle East and 1395 healthy controls. The aim was to identify rare germline variants in coding regions and their splicing sites associated with high-risk CRC in the Middle Eastern population. Results: Rare inactivating variants (RIVs) in APC had the strongest association with high-risk CRC (6/146 in cases vs. 1/1395 in controls, OR = 59.7, p = 5.13 × 10−12), whereas RIVs in RIMS1, an RAS superfamily member, were significantly associated with high-risk CRC (5/146 case vs. 2/1395 controls, OR = 24.7, p = 2.03 × 10−8). Rare damaging variants in 17 genes were associated with high-risk CRC at the exome-wide threshold (p < 2.5 × 10−6). Based on the sequence kernel association test, nonsynonymous variants in six genes (TNXB, TAP2, GPSM3, ADGRG4, TMEM229A, and ANKRD33B) had a significant association with high-risk CRC. RIVs in APC—the most common high-penetrance genetic factor—were associated with patients with high-risk CRC in the Middle East. Individuals who inherited APC RIVs had an approximate 60-fold increased risk of developing CRC and were likely to develop the disease earlier. Conclusions: We identified new potential CRC predisposition variants in other genes that could play a role in CRC inheritance. However, large collaborative studies are needed to confirm the association of these variants with high-risk CRC. These results provide information for counseling patients with high-risk CRC and their families in our population.

Correlation of Smad4 mutation with clinico-pathological parameters in colorectal carcinoma

Supplementary Materials and Methods

The PALB2 gene is a breast cancer (BC) and ovarian cancer (OC) predisposition gene involved in the homologous recombination repair pathway. However, the prevalence and clinicopathological association of PALB2 pathogenic/likely pathogenic (PV/LPV) variants in Middle East is still not fully explored. Total 918 BC/OC patients from Saudi Arabia were selected for PALB2 mutations screening using capture sequencing technology. Five heterozygous PVs or LPVs were identified in six cases, accounting for 0.65% (6/918) of entire cohort. Two cases (33.3%) harbored PVs and four cases (66.7%) carried LPVs. Four PVs/LPVs (80%) were frameshift along with one novel splicing LPV (c.2835-2_2835-1delinsTT). One recurrent LPV (c.3425delT: p.L1142fs) was identified in two cases. All six affected carriers have breast cancer diagnosis with median age of 39.5 years (range 34–49 years). Only two cases (33%) have documented family history of cancer. Breast cancer phenotype was invasive ductal unilateral cancer in all cases with 66.7% of hormone receptor positive and 16% of triple negative tumors. Germline PVs/LPVs in the PALB2 gene were observed in low frequency of 0.65% in Saudi BC and/or OC. Our study confirms one recurrent LPV and one novel LPV in Saudi breast cancer patients.

Background: The PALB2 gene encoding protein is involved in the homologous recombination repair pathway and plays an important role in maintaining genomic integrity. PALB2 heterozygous pathological variants (PV) or likely pathogenic variants (LPV) are associated with increased risk of breast and ovarian cancer. However, the prevalence and clinicopathological association of PALB2 germline PV/LPV in Middle Eastern breast and ovarian cancers is not fully identified. Purpose: We retrospectively screened a cohort of 918 cancer patients (791 breast cancer and 127 ovarian cancer) from Saudi Arabia using targeted capture-based next generation sequencing (NGS) method for PALB2 germline variants regardless the family history of these patients or the presence of other hereditary cancer susceptibility genes mutations. Clinicopathological criteria were also fully analyzed. Results: Five heterozygous PVs or LPVs were identified in six cases, accounting for 0.65% (6/918) of entire cohort. Two cases (33.3%) harbored PVs and four cases (66.7%) carried LPVs. Four PVs/LPVs (80%) were frameshift along with one novel splicing LPV (c.2835-1 C>A). One recurrent LPV (c.3425delT) was identified in two cases. All six affected carriers have breast cancer diagnosis with median age of 39.5 years (range 34-49 years). Only two cases (33%) have documented family history of cancer. Breast cancer phenotype was invasive ductal unilateral cancer in all cases with 66.7% of hormone receptor positive and 16% of triple negative tumors. Conclusion: Germline PVs/LPVs in the PALB2 gene were observed in low frequency of 0.65% in Saudi breast and/or ovarian cancer. Our study confirms one recurrent LPV and one novel LPV in Saudi breast cancer patients. Citation Format: Rong Bu, Abdul K. Sira, Sandeep Parvathareddy, Kaleem Iqbal, Saud Azam, Zeeshan Qadri, Maha Al-Rasheed, Wael Haqawi, Mark Diaz, Ingrid G. Victoria, Khawla S. Al-Kuraya. PALB2 germline mutations in a large cohort Middle Eastern breast-ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6086.

Papillary thyroid carcinoma (PTC) is the commonest thyroid cancer. The majority of inherited causes of PTC remain elusive. However, understanding the genetic underpinnings and origins remains a challenging endeavor. An exome-wide association study was performed to identify rare germline variants in coding regions associated with PTC risk in the Middle Eastern population. By analyzing exome-sequencing data from 249 PTC patients (cases) and 1395 individuals without any known cancer (controls), GALNT9 emerged as being strongly associated with rare inactivating variants (RIVs) (4/249 cases vs. 1/1395 controls, OR = 22.75, p = 5.09 × 10−5). Furthermore, three genes, TRIM40, ARHGAP23, and SOX4, were enriched for rare damaging variants (RDVs) at the exome-wide threshold (p < 2.5 × 10−6). An additional seven genes (VARS1, ZBED9, PRRC2A, VWA7, TRIM31, TRIM40, and COL8A2) were associated with a Middle Eastern PTC risk based on the sequence kernel association test (SKAT). This study underscores the potential of GALNT9 and other implicated genes in PTC predisposition, illuminating the need for large collaborations and innovative approaches to understand the genetic heterogeneity of PTC predisposition.

List of validated smad4 somatic mutation

Correlation of Smad4 alteration with clinico-pathological parameters in colorectal carcinoma

Breast cancer (BC) is the most prevalent malignancy among women worldwide with germline pathogenic variants/likely pathogenic variants (PVs/LPVs) in BRCA1/2 accounting for a large portion of hereditary cases. Recently, heterozygous PVs/LPVs in the ATM serine/threonine kinase or Ataxia-telangiectasia mutated gene (ATM) has been identified as a moderate susceptibility factor for BC in diverse ethnicities. However, the prevalence of ATM PVs/LPVs in BC susceptibility in Arab populations remains largely unexplored. This study investigated the prevalence of ATM PVs/LPVs among BC patients from Saudi Arabia, employing capture-sequencing technology for ATM PVs/LPVs screening in a cohort of 715 unselected BC patients without BRCA1/2 PVs/LPVs. In addition, founder mutation analysis was conducted using the PHASE program. In our entire cohort, four unique PVs/LPVs in the ATM gene were identified in six cases (0.8%). Notably, one recurrent LPV, c.6115G > A:p.Glu2039Lys was identified in three cases, for which haplotype analysis confirmed as a novel putative founder mutation traced back to 13 generations on average. This founder mutation accounted for half of all identified mutant cases and 0.4% of total screened cases. This study further reveals a significant correlation between the presence of ATM mutation and family history of BC (p = 0.0127). These findings underscore an approximate 0.8% prevalence of ATM germline PVs/LPVs in Arab BC patients without BRCA1/2 PVs/LPVs and suggest a founder effect of specific recurrent ATM mutation. These insights can help in the design of a genetic testing strategy tailored to the local population in Saudi Arabia, thereby, enabling more accurate clinical management and risk prediction.

Clinicopathological variables for the patient cohort (n=1050)

Effect of Smad4 manipulation on TGFβ induced CRC cell growth

Standard surgery followed by radioactive iodine (131I, RAI) therapy are not curative for 5–20% of papillary thyroid carcinoma (PTC) patients with RAI refractory disease. Early predictors indicating therapeutic response to RAI therapy in PTC are yet to be elucidated. Whole-exome sequencing was performed (at median depth 198x) on 66 RAI-refractory and 92 RAI-avid PTCs with patient-matched germline. RAI-refractory tumors were significantly associated with distinct aggressive clinicopathological features, including positive surgical margins (p = 0.016) and the presence of lymph node metastases at primary diagnosis (p = 0.012); higher nonsilent tumor mutation burden (p = 0.011); TERT promoter (TERTp) mutation (p < 0.0001); and the enrichment of the APOBEC-related single-base substitution (SBS) COSMIC mutational signatures 2 (p = 0.030) and 13 (p < 0.001). Notably, SBS13 (odds ratio [OR] 30.4, 95% confidence intervals [CI] 1.43–647.22) and TERTp mutation (OR 41.3, 95% CI 4.35–391.60) were revealed to be independent predictors of RAI refractoriness in PTC (p = 0.029 and 0.001, respectively). Although SBS13 and TERTp mutations alone highly predicted RAI refractoriness, when combined, they significantly increased the likelihood of predicting RAI refractoriness in PTC. This study highlights the APOBEC SBS13 mutational signature as a novel independent predictor of RAI refractoriness in a distinct subgroup of PTC.

Objective: Microsatellite instability (MSI) is a hallmark of Lynch Syndrome (LS). This study aims to determine the prevalence of LS and the optimal diagnostic method in women from Middle Eastern ethnicity with newly diagnosed epithelial ovarian cancer (EOC).

Mutation-induced activation of Wnt-β Catenin signaling pathway is frequent in CRC. The E3 ubiquitin ligase, RNF43, has been reported to negatively regulate the Wnt signaling pathway and RNF43 mutations are frequently seen in CRC. However, its role in Middle Eastern CRC remains unclear. Therefore, we employed Exome and Sanger sequencing technology to assess the frequency of RNF43 mutations and its association with other clinico-pathological features in Middle Eastern CRC. RNF43 mutations were found in 5.9% (13/220) of CRC cases and was inversely correlated to APC and TP53 mutations. A strong association of RNF43 mutations with right sided and sporadic microsatellite instable (MSI) CRC was observed. No association was identified between RNF43 mutation and other clinico-pathological features including BRAF mutation, age, tumor histological subtype, tumor grade or patients' prognosis. Multivariate logistic regression analysis revealed that MSI status and wild type APC were independent predictor of RNF43 mutation. We conclude that RNF43 mutations occur in Middle Eastern CRC at comparable frequencies with BRAF mutations and represent a distinct molecular subtype which further enhances our understanding of how different mutational subsets of Wnt tumor suppressor genes link to distinct tumor characteristics, which might be considered for treatment strategies for CRC patients.