Breast cancer is the most prevalent malignancy worldwide and continues to be the primary cause of cancer-associated deaths in women. Recent statistics from GLOBOCAN 2022 indicate that breast cancer is the fourth deadliest cancer type overall, preceded only by lung, colorectal, and liver cancers. With approximately 2.3 million new diagnoses and over 666 000 fatalities reported in 2022, the disease represents a major global health challenge (Figure 1).1-3

Alarmingly, 2020 data revealed a disproportionate burden of breast cancer in low-resource settings, with developing regions accounting for over 50% of global cases and nearly 70% of mortality. This disparity highlights critical healthcare inequities in cancer management worldwide.2 In North Africa, the incidence of breast cancer has been rising, with rates increasing from 24.3 to 43.6 cases per 100 000 individuals between 2000 and 2015.4 In Morocco, according to the Casablanca Cancer Registry (2013–2017), breast cancer accounted for 22.5% of all cancer cases across both sexes. Among women specifically, breast cancer represented 38.1% of all cancer diagnoses. The age-adjusted breast cancer incidence among women reaches 45.6 cases annually per 100 000 women.5

According to the Global Cancer Observatory, the crude incidence rate of breast cancer in Morocco for individuals aged 0–74 years across both sexes was 65.4 per 100 000 in 2022.

Furthermore, Africa exhibits a higher incidence of hormone receptor-negative and triple-negative breast cancers (TNBC)6, with North Africa showing particularly high rates of these aggressive forms of breast cancer.7,8 This trend highlights the urgent need for comprehensive research on TNBC, which is particularly deadly. It accounts for approximately 10-17% of all breast cancer cases worldwide, with a higher prevalence in North Africa. In Morocco, TNBC accounts for around 16.5% to 17.5% of breast cancer cases.9,10

The aggressive nature of TNBC poses significant challenges in terms of prognosis and treatment, primarily due to the absence of estrogen receptors, progesterone receptors, and HER2 amplification, which limits treatment options and results in higher rates of recurrence and metastasis. In addition, TNBC tends to affect younger women and is associated with poorer survival outcomes, making it imperative to focus on targeted research in this area.11-13

The genomic landscape of TNBC is characterized by unique genetic alterations, which may serve as potential biomarkers and therapeutic targets. In this context, several relevant genes and variants associated with TNBC have been identified.14,15 Currently, data regarding these associations in Moroccan and North African populations remain sparse.10,16,17 This gap in knowledge emphasizes the importance of conducting studies that specifically address the genetic characteristics of TNBC within these populations.

The focus on whole-exome sequencing (WES) specifically for TNBC, as opposed to other forms of breast cancer, is paramount to unraveling its unique genetic underpinnings and finding more effective and tailored therapeutic strategies.18

Given this context, the primary objective of this case series is to investigate the genetic variations associated with TNBC in a Moroccan cohort through WES. By elucidating the genetic signatures of TNBC, this research aims to advance the understanding of this aggressive breast cancer subtype and contribute to the development of more effective, tailored therapeutic strategies for diverse populations.

This study included 10 unrelated female patients diagnosed with TNBC, all recruited from the National Institute of Oncology in Rabat, Morocco, between June 2022 and June 2023. Informed consent was obtained from all participants, and the study protocol was approved by the Ethics Committee for Biomedical Research at the Faculty of Medicine and Pharmacy of Rabat (Approval No. N° C53/20). The clinical characteristics of the patients, including tumor location, Scarff-Bloom-Richardson (SBR) grade, lymph node involvement that tested for metastatic cancer cells, histological type, and Ki-67 index, were collected from their medical records. All patients had a confirmed diagnosis of TNBC, characterized by negative hormone receptor (estrogen and progesterone) and HER2 status, determined through immunohistochemistry. Detailed demographic, epidemiological, and clinical features are provided in Table 1, which is intended as a descriptive summary.

Case 1: A 63-year-old postmenopausal female with a family history of cancer presented with a right breast nodule, initially classified as cT2N0M0. Histopathological examination revealed an invasive carcinoma of no special type (NST), classified as Scarff-Bloom-Richardson (SBR) grade III, with no lymph node involvement. Hormone receptor status (estrogen receptor [ER]: 0%, progesterone receptor [PR]: 0%) and HER2 were negative (Score 0, HercepTest).

Case 2: A 41-year-old premenopausal female, with no family history of cancer, was evaluated for a right breast nodule, initially classified as cT1N0M0. Biopsy confirmed invasive NST, classified as SBR grade II, with no lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest).

Case 3: A 49-year-old premenopausal female with a family history of breast cancer, with 2 of her sisters having been diagnosed with the disease (1 of whom had passed away), presented with a left breast nodule, initially classified as cT3N0M0. Histopathology confirmed NST, classified as SBR grade II, with no lymph node involvement. The patient’s hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest).

Case 4: A 68-year-old postmenopausal female with no family history of cancer was evaluated for a left breast nodule, initially classified as cT2N0M0. Biopsy confirmed the diagnosis of invasive secretory breast carcinoma, categorized as SBR grade II, with no lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 (Score 0, HercepTest Negative) were negative. The Ki-67 index was recorded at 10%.

Case 5: A 52-year-old postmenopausal female with a family history of cancer was referred for a right breast nodule evaluation, initially classified as cT2N1M0. Biopsy confirmed invasive florid-appearing metaplastic breast carcinoma, classified as SBR grade III, with no lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest). The Ki-67 index was notably high at 75%.

Case 6: A 30-year-old premenopausal female with a family history of cancer presented with a left breast nodule, initially classified as cT1N0M0. Biopsy confirmed the diagnosis of NST, categorized as SBR grade II, with no lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest). The Ki-67 index was recorded at 30%, indicating moderate tumor proliferation.

Case 7: A 40-year-old premenopausal female with no family history of cancer presented with a left breast nodule, initially classified as cT3N0M0. Histopathology confirmed NST, classified as SBR grade III, with no lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest). The Ki-67 index was notably high at 80%, indicating an aggressive proliferative rate.

Case 8: A 49-year-old postmenopausal female was referred for a right breast nodule evaluation, initially classified as cT2N0M0. She had no family history of cancer. Biopsy confirmed NST, categorized as SBR grade IV, with lymph node involvement and metastatic spread to the brain and bones. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest). The Ki-67 index was high at 60%.

Case 9: A 52-year-old postmenopausal female was referred for evaluation of a right breast nodule, initially classified as cT2N0M0. She reported a family history of cancer, with a maternal aunt having passed away from breast cancer. Biopsy confirmed the diagnosis of NST, classified as SBR grade II, with lymph node involvement. Hormone receptor status (ER: 0%, PR: 0%) and HER2 were negative (Score 0, HercepTest). The Ki-67 index was recorded at 40%, indicating moderate tumor proliferation.

Case 10: A 60-year-old postmenopausal female with no family history of cancer was referred for a left breast nodule, initially classified as cT2N0M0. Histopathology examination confirmed NST, categorized as SBR grade III, with lymph node involvement and metastatic spread to the brain. Hormone receptor status (ER: 0%, PR: 0%) and HER2 (Score 1+, HercepTest) were negative. The Ki-67 index was recorded at 44%, indicating moderate tumor proliferation.

The average age of TNBC patients in this study was 50.5 years, which is consistent with earlier studies in Morocco. Aznag et al. 19 reported an average age of around 48 years, while Khalis et al. 20 found the highest incidence rates in Moroccan women aged 45 to 59 years. This alignment not only reinforces the demographic trends observed in our cohort but also underscores the significant burden of TNBC among middle-aged women in Morocco. Such findings point to the importance of early screening and targeted interventions in this age group.

The present study on TNBC in Moroccan patients reveals significant insights into the genetic variants that may influence the pathogenesis of the disease. The application of WES in this study allowed for an in-depth exploration of the genetic underpinnings of TNBC in Moroccan patients. Through a rigorous filtering process, we identified 9 variants across 6 genes: IGSF3, CTBP2, LRRK2, NMNAT1, TPRG1, and ZNF334. These genetic variants are likely involved in the pathogenesis of TNBC, providing valuable insights into the molecular mechanisms driving this aggressive subtype of breast cancer.

This aggressive subtype is characterized by a dysregulation of key signaling pathways, including Wnt and Notch, both of which are implicated in cancer progression. In particular, the Notch signaling pathway plays a central role in regulating tumor growth, metastasis, and chemoresistance in TNBC. 21, 22 Aberrant activation of this pathway is associated with poor prognosis and therapeutic resistance in TNBC. For example, mutations in NOTCH1 can lead to increased MYC expression, contributing to tumor initiation and metastasis. 23, 24 In TNBC, mutations can trigger abnormal activation of the Notch pathway, leading to tumor growth.21,25 Notch1 alterations in TNBC upregulate target genes like MYC, contributing to an oncogenic phenotype.26-28

Our findings suggest that the CTBP2-MYC axis may be a critical contributor to TNBC progression, with potential therapeutic implications for targeting this pathway. The CTBP2 gene, in particular, encodes a transcriptional co-repressor that regulates gene expression. The nonsynonymous p.D112A mutation in CTBP2 identified in the 10 patients could lead to the disruption of its normal regulatory functions, potentially contributing to tumorigenesis by affecting key signaling pathways like Wnt/β-catenin.29 The mutation is a key finding because CTBP2 functions as a transcriptional co-repressor that regulates gene expression. Disruption of its normal activity may lead to tumorigenesis, particularly in breast cancer.29-31 Additionally, CTBP2 modulates signaling pathways such as Wnt/β-catenin to promote cancer progression.32 Its inhibition has been shown to reduce cancer cell proliferation by decreasing c-Myc expression, a key oncogenic driver.29 In TNBC, where Notch1 mutations amplify MYC activity, a CTBP2 mutation could further dysregulate MYC signaling, contributing to cancer progression through Notch1-MYC crosstalk. This suggests that the CTBP2-MYC axis may serve as a promising therapeutic target in TNBC, highlighting the need for further investigation into its role in cancer biology.

Table 3 shows predicted and experimentally validated interactions between these proteins (STRING Database).

The ZNF334 gene, a member of the zinc-finger protein family, plays a critical role in regulating various biological processes, including differentiation and development.33 In TNBC, ZNF334 has been shown to suppress cancer progression by modulating the Wnt/β-catenin pathway, which is often aberrantly activated in many cancers.34 The aberrant activation of the Wnt/β-catenin signaling pathway is closely linked to the development and progression of various cancers.35,36 Numerous studies have highlighted its association with tumor proliferation, metastasis, stemness maintenance, and drug resistance in breast cancer.37,38

The involvement of the Wnt/β-catenin pathway in TNBC is particularly notable, as its activation leads to the expression of specific target genes, supporting the role of ZNF334 in modulating this pathway.39

The nonsynonymous variant identified in Exon 5 leads to a histidine-to-arginine substitution at amino acid position 339. This alteration may disrupt ZNF334's suppressive function, potentially impacting the Wnt/β-catenin pathway.

Given that ZNF334 is involved in regulating cancer progression and metastasis through the Wnt/β-catenin pathway, the presence of this variant in Cases 8 and 10, both with brain metastasis, raises the possibility that the disrupted function of ZNF334 could contribute to enhanced metastatic potential. It is possible that the alteration in ZNF334 function may allow for unchecked activation of the Wnt/β-catenin pathway, leading to increased tumor invasiveness and metastatic spread, particularly to distant organs like the brain.

While direct studies on ZNF334 variants in breast cancer are limited, its role as a transcriptional regulator implies potential effects on gene expression linked to cancer progression. The genetic interaction between ZNF334 and CTBP2, highlighted by GeneMANIA (Figure 3), suggests a regulatory network influencing MYC activity. As MYC drives cell proliferation and survival, mutations in ZNF334 or CTBP2 may enhance tumor progression and metastasis, pointing to these interactions as potential therapeutic targets in TNBC.

The LRRK2 gene, located on chromosome 12, encodes a large multi-domain protein involved in cellular signaling and neuronal functions. Mutations in LRRK2, particularly the G2019S variant, are primarily linked to Parkinson’s disease (PD), where they enhance kinase activity, contributing to neurodegeneration.40,41 Notably, G2019S is the most common PD-associated variant, with age-related penetrance increasing from 17% at age 50 to 85% by age 70.42 However, penetrance is influenced by factors such as ethnicity, sex, and polygenic risk scores, which modulate the likelihood of PD onset. For instance, carriers with a high PD polygenic risk score have up to a 27-fold increased risk compared to non-carriers with a low score.43-45

In our cohort of TNBC patients, Cases 8 and 10 carried the G2019S mutation without exhibiting PD symptoms, consistent with incomplete penetrance. Interestingly, both cases had metastatic spread to the brain, a feature that raises intriguing questions about the role of LRRK2 in cancer metastasis. Although G2019S-related PD typically manifests later in life or with atypical features, such as a lack of prodromal symptoms45, the presence of brain metastases could suggest that the G2019S mutation may influence tumor progression or metastatic potential in addition to its neurodegenerative effects. This may reflect the dual role of LRRK2 in both neurodegeneration and oncogenesis, with its mutation potentially promoting tumor growth and facilitating metastasis.

Beyond neurodegeneration, recent studies have implicated LRRK2 mutations in cancer, including hormone-related cancers such as breast cancer.40 The G2019S variant has been shown to promote tumor growth by increasing cell proliferation, inflammation, and immune response alterations, as demonstrated in colitis-associated cancer models.40 Consistently, targeting LRRK2 with specific inhibitors like LRRK2-IN-1 has been found to reduce cancer cell viability in breast cancer cell lines by downregulating key signaling proteins such as STAT3 and AKT, which are critical for cancer progression.46 Our findings suggest a potential association between the LRRK2 G2019S mutation and TNBC in individuals without PD, raising questions about the dual role of LRRK2 in neurodegeneration and oncogenesis. This mutation may act through distinct pathways, influencing tumorigenesis independently of its neurodegenerative effects. Larger studies, particularly within the Moroccan population, are needed to confirm these associations and explore whether G2019S could serve as a biomarker for cancer risk. Such research may provide insights into LRRK2’s dual functionality and inform the development of targeted therapies for TNBC and other cancers.

The NMNAT1 gene, which encodes an enzyme involved in nicotinamide adenine dinucleotide (NAD+) biosynthesis, plays a crucial role in cellular energy metabolism, redox balance, and stress responses.47,48 The p.A13T variant in NMNAT1, identified in Cases 8 and 10, raises questions about its potential involvement in tumorigenesis. While this variant has been previously linked to Leber congenital amaurosis type 9 (LCA9), an early-onset autosomal recessive retinal degeneration disorder, it has not been directly associated with cancer.49,50 The heterozygous state of this variant in TNBC patients raises the question of whether NMNAT1 dysfunction could indirectly contribute to tumorigenesis. NAD+ metabolism is crucial for cellular survival, particularly in stress conditions such as those in tumors, where disruptions in NAD+ biosynthesis have been implicated in cancer cell proliferation and resistance to apoptosis.51,52 Given NMNAT1’s involvement in pathways like tryptophan catabolism and nicotinamide metabolism, its dysregulation could influence the tumor microenvironment by altering energy homeostasis, immune responses, or metabolic adaptation in cancer cells.53,54 Additionally, interactions between NMNAT1 and ZNF334 (Figure 3) suggest broader roles for NMNAT1 beyond retinal health. These interactions may point to potential mechanisms linking NMNAT1 mutations to cancer susceptibility, especially in conditions involving viral etiology or metabolic stress. Although NMNAT1 has not been widely reported in cancer, its central role in metabolic pathways relevant to cell survival suggests it may influence tumor progression. Further research, including functional studies, is warranted to explore whether NMNAT1 variants act as modifiers or interact with other genetic alterations in cancer. The identification of this variant in TNBC patients underscores the need for larger cohort studies to clarify its role in oncogenesis and its potential as a therapeutic target.

The IGSF3 gene, involved in cell adhesion and immune responses, has been implicated in various cancers, including hepatocellular carcinoma (HCC).55,56 Increased expression of IGSF3 is associated with poor prognosis and tumor progression, promoting cell migration, invasion, and growth through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. In HCC, silencing IGSF3 reduces these processes.56 Additionally, in vivo studies in mouse melanoma models further show that IGSF3 enhances metastasis by promoting cancer cell adhesion to the vascular endothelium, aiding in lung colonization.55 While its role in TNBC remains unexplored, these findings suggest IGSF3 may contribute to cancer progression through similar mechanisms.

In our TNBC cohort, we identified four IGSF3 variants: p.W575X, p.S579S, p.I619T, and p.Q212X. The p.W575X and p.Q212X truncations likely impair tumor-suppressive functions and enhance NF-κB-driven tumor growth and metastasis. While the synonymous p.S579S variant may affect mRNA stability or splicing, the p.I619T missense variant could disrupt critical protein interactions. The presence of these variants in all TNBC patients suggests a cumulative effect on tumor progression, with the pathogenic p.W575X mutation playing a key role. These findings underscore IGSF3's potential as a TNBC biomarker and therapeutic target, particularly through NF-κB signaling. Further research is needed to elucidate its role in TNBC pathogenesis and treatment, especially in this aggressive subtype with limited targeted therapy options.

The TPRG1 gene, located near TP63 on chromosome 3q28, is recognized as a tumor suppressor involved in cellular inflammatory responses. Dysregulation of TPRG1 is associated with early tumor recurrence.57 While the precise molecular role of TPRG1 is still under investigation, recent studies on its antisense RNA, TPRG1-AS1, suggest important contributions to cancer progression. For example, TPRG1-AS1 has been shown to promote apoptosis in liver cancer by regulating RBM24 through a competing endogenous RNA (ceRNA) mechanism.58 In this case series, a nonsynonymous variant (p.R64Q) was identified. This mutation could potentially disrupt TPRG1's function or regulation, and potentially disrupt similar pathways or interacting factors within breast cancer cells. Although direct links between TPRG1 and TNBC are limited, the presence of the p.R64Q variant suggests that TPRG1 may influence tumor progression, possibly through mechanisms similar to those of TPRG1-AS1 in other cancers. Large-scale studies could further explore whether mutations in TPRG1 affect its interaction with noncoding RNAs or tumor suppressive networks, as seen with TPRG1-AS1. In fact, despite its tumor-suppressive functions, the complexity of TPRG1 interactions in cancer suggests that its role may vary across different cancer types. This indicates a need for further research to fully understand its implications in oncogenesis.

Among the TNBC patients in our cohort, variants in NMNAT1, TPRG1, ZNF334, and LRRK2 were identified exclusively in Cases 8 and 10, who had the highest tumor grades (III and IV) and brain metastasis. The presence of these variants in these cases suggests a potential link to tumor aggressiveness and metastasis. However, it is important to note that the common variants in CTBP2 and IGSF3 were identified in all patients, regardless of tumor grade, highlighting their potential role in the overall pathogenesis of TNBC. Although direct evidence is limited, these findings highlight the need for functional studies to clarify their impact on disease pathogenesis and progression.

WES has significantly advanced our understanding of the genetic landscape associated with aggressive breast cancer subtypes, uncovering novel genetic factors that may influence cancer etiology across diverse populations. This study identified previously-unreported genetic variations in CTBP2, IGSF3, ZNF334, TPRG1, and NMNAT1, suggesting their potential roles as biomarkers and therapeutic targets. These findings emphasize the necessity of investigating genetic variations in underrepresented populations to refine breast cancer treatment strategies. Moreover, understanding these genetic alterations may help predict treatment responses, particularly to chemotherapeutic agents commonly used in TNBC, such as platinum-based compounds or poly (ADP-ribose) polymerase (PARP) inhibitors, which are more effective in tumors with specific genetic vulnerabilities. Furthermore, the involvement of these genetic alterations in key oncogenic pathways suggests potential therapeutic implications, including the use of Wnt-targeted regimens or demethylation therapies, which may enhance treatment efficacy for TNBC patients. Future research should focus on validating these findings in larger cohorts to confirm their clinical relevance and support the development of personalized therapeutic approaches.

This study adhered to the principles of the Declaration of Helsinki and was approved by the Ethical Committee for Biomedical Research at the Faculty of Medicine and Pharmacy of Rabat (12/23/21 /N° C53/20). Written informed consent for participation, including permission for data publication, was obtained from the patients at the time of recruitment.