BRICA-Dx™ — The Breast Immuno-Carcinogenic Assay

The Breast Immuno-Carcinogenic Assay (BRICA-Dx) uses a 64-gene signature to evaluate the transcriptional immunocarcinogenic phenotype in both early and advanced breast cancers, regardless of the molecular subtype or clinical background of the patient. With AI-powered algorithmic analysis of the expression and interactions among a wide range of HER2-regulated and immune checkpoint genes, BRICA-Dx clarifies the role of HER2 and immune checkpoint molecules in the molecular characteristics of breast cancer at any stage. As a result, BRICA-Dx™ serves as a complementary diagnostic tool for enhancing diagnosis, prognosis, and treatment strategies on a robust functional basis.

Six clinical applications

• Application 1: Differentiation between malignant and benign breast tissue, including assessment of benign-like, premalignant lesions and early breast carcinogenesis.
• Application 2: Detection of transcriptional immunocarcinogenic patterns and evaluation of recurrence risk in DCIS or early-stage invasive breast cancers and their tumor bed biopsies, to support negative margin control following breast-conserving surgery (lumpectomy) with intraoperative radiotherapy (IORT).
• Application 3: Quantification of HER2 functionality based on HER2-regulated gene expression in HER2 3+/Enriched breast cancer.
• Application 4: Assessment of HER2 functionality through HER2-regulated gene expression in triple-negative/HER2-low breast cancer.
• Application 5: Identification of immunosuppressive phenotypes in Luminal A/B breast cancers.
• Application 6: Characterization of HER2-regulated and non-HER2-regulated immune suppressive phenotypes in breast cancer.

Application 1: Distinguishing malignant versus benign breast tissue

BRICA-Dx enables the identification of early malignant transformation prior to overt morphological changes and beyond the capabilities of conventional pathology, which may be limited by biological ambiguity or technical constraints.

Recommended applications include

• B3 lesions, classified as having “uncertain malignant potential” due to their indeterminate morphology and histology showing features common to both benign proliferation and early malignancy.
• Clinical–pathological discordance, such as cases where imaging and clinical presentations (e.g., BI-RADS 4/5 lesions) are suggestive of malignancy but biopsy results indicate benign tissue.
• Rapidly growing lesions deemed benign in high-risk patients despite low-grade pathology.

Provided information

• BRICA-Dx detects activation of transcriptional immunocarcinogenic pathways, facilitating enhanced breast cancer screening and risk stratification.
• It quantifies “field carcinogenesis” activity by comparing gene expression patterns between benign and malignant breast tissue at specific sites within "normal-appearing" tissue, aiding in cancer risk assessment.
• The tool reclassifies B3 lesions as representing an initial phase of malignancy within a "sick lobe," underscoring a risk that the entire lobe is affected by field cancerization and suggesting that removal of the complete lesion and associated tissue (lobe) may be warranted, rather than focusing solely on the site identified via imaging.
• BRICA-Dx distinguishes functionally malignant lesions characterized by HER2-associated signaling and activated tumor-bed biology from benign/reactive proliferations, allowing identification of HER2-primed environments with oncogenic potential.
• Additionally, it identifies molecularly pre-invasive lesions and reports their HER2 Functional Activity Score, providing insight into HER2-centered signaling with potential clinical significance.

Application 2: Immunocarcinogenic patterns and recurrence risk in DCIS / early-stage invasive breast cancers (lumpectomy + IORT)

Employing a 62-gene signature, BRICA assesses the transcriptional immunocarcinogenic phenotype in ductal carcinoma in situ (DCIS) and early-stage invasive breast cancers, regardless of intrinsic molecular subtype, in patients undergoing breast-conserving surgery (lumpectomy) with intraoperative radiotherapy (IORT).

The main goal is to define the molecular phenotype by integrating tumor microenvironment immune checkpoint gene expression with HER2-regulated genes, allowing for occult HER2 functionality scoring.

Additionally, BRICA can also analyze samples from the surgical margin (tumor bed site) post-lumpectomy and IORT to generate a post-IORT Immunocarcinogenic Risk Score. This score provides insight into tumor bed changes after treatment and supports decision-making regarding the potential need for additional whole-breast irradiation.

Through comprehensive transcriptional profiling, BRICA identifies biological phenotypes that are not detectable by traditional ER/PR/HER2 classification systems and evaluates the impact of radiation on the immunocarcinogenic microenvironment and metastasis-associated signalling pathways.

Even in clinically defined “early-stage” cases, BRICA’s molecular profiling may reclassify patients as high-risk, informing recommendations for biologically rational escalation of therapy, such as neoadjuvant treatments, immune checkpoint inhibitor strategies, or microenvironment-targeted interventions.

Application 3: Scoring HER2 functionality through HER2-regulated gene expression

HER2 overexpression and gene amplification do not consistently correspond to active HER2 signaling in breast cancer, nor does a HER2-negative status necessarily indicate complete pathway inactivity. The discrepancy between HER2 expression and activity, along with the dynamic nature of HER2 status between primary and metastatic sites (30–50% discordance), substantially affects diagnostic reliability and treatment outcomes across all breast cancer subtypes.

BRICA identifies HER2-regulated genes in breast cancer, offering advantages over reliance on HER2 IHC/FISH assessments. The biological activity of the HER2 pathway, rather than simple protein or gene presence, is central to tumor aggressiveness and constitutes the principal therapeutic target.

While FISH/IHC assays determine protein presence and quantify HER2 gene copy number, BRICA’s evaluation of HER2-regulated gene expression provides more definitive evidence of pathway signaling and its role in tumor progression, which is not always directly correlated.

Moreover, the HER2-HER3 heterodimer serves as the most robust activator of downstream survival mechanisms (PI3K/Akt). By assessing HER2-regulated genes, BRICA effectively quantifies the impact of this interaction, which holds greater clinical relevance for therapeutic success than mere enumeration of HER2 receptors.

In conclusion, the measurement of HER2-regulated genes by BRICA delivers a functional assessment of the tumor’s dependency on the HER2 pathway, presenting more meaningful information for treatment planning compared to solely measuring protein concentration.

Recommended applications

• All newly diagnosed cases of invasive breast cancer should undergo BRICA testing to evaluate HER2 functionality and inform treatment strategies.
• Detection of HER2-driven tumors that may be missed by conventional IHC testing.
• Assessment of primary resistance prior to neoadjuvant therapy (up to 35% of early-stage cases exhibit non-responsiveness to initial HER2-targeted treatment).
• Evaluation of acquired resistance post-neoadjuvant therapy (up to 88% of advanced-stage HER2-positive breast cancers progress despite an initial therapeutic response).
• Analysis of resistance to dual blockade following trastuzumab, pertuzumab, and chemotherapy regimens (up to 40% of cases fail to achieve a pathological complete response).

Application 4: Identifying the HER2-low phenotype in triple-negative and luminal breast cancers

Triple-negative (TPN) breast cancers represent a clinically diverse group, with approximately 55% exhibiting HER2-low expression—defined as IHC 1+ or IHC 2+/ISH-negative. The identification and segmentation of this subgroup are essential, as metastatic HER2-low cases may benefit from recently developed targeted antibody-drug conjugates.

BRICA utilizes a 13-gene signature algorithm to assess HER2-regulated tumor microenvironment (TME) genes, enabling the identification of hyperresponsive HER2-regulated TPN breast cancer phenotypes regardless of HER2 IHC score or ERBB2 mRNA status. Test results yield three predictive scores for TPN cancers: (1) defining the level of HER2 functionality; (2) identifying individuals most likely to respond to HER2-targeted therapies; and (3) predicting those who may experience extended relapse-free intervals.

Additionally, BRICA can detect high-risk subsets of Luminal A/B breast cancers with a HER2-low molecular phenotype who may be candidates for Trastuzumab Deruxtecan (T-DXd). While HER2-low tumors are often hormone receptor-positive and primarily luminal, distinguishing patients with more aggressive features is critical, as T-DXd has demonstrated improved progression-free survival and overall survival relative to conventional chemotherapy.

Application 5: Identifying the immunosuppressive phenotype in Luminal A/B breast cancers

Luminal-type cancers typically exhibit a lower tumor mutational burden and present a "cold" tumor microenvironment characterized by reduced immunogenicity. Although PD-L1 expression serves as an important marker, comprehensive profiling of additional immune checkpoints is essential, as Luminal B tumors—especially those resistant to aromatase inhibitors—demonstrate enhanced immune tolerance mechanisms that may not be adequately reflected by PD-L1 alone.

Assessing the complete immune checkpoint profile (beyond PD-L1) in HER2-negative/PD-L1-positive luminal breast cancers enables BRICA to identify "immune hot" tumors within this predominantly "cold" subtype. This approach supports the development of personalized strategies for integrating immunotherapy with endocrine treatments, particularly in the neoadjuvant setting for high-risk cases.

Historically, treatment options for triple-negative breast cancer (TNBC) have been limited; however, the combination of PD-1/PD-L1 inhibitors with chemotherapy has improved pathological complete response rates and progression-free survival in both neoadjuvant and first-line metastatic settings. Utilizing a HER2-regulated TME gene signature, BRICA also provides comprehensive immune checkpoint profiling in PD-L1-positive TNBC cases, regardless of HER2 status. These findings help define HER2-dependent and independent immune suppressive phenotypes, facilitating tailored combinations of immune checkpoint inhibitors and chemotherapy regimens with higher immunogenic potential.

Application 6: Identifying HER2-regulated and non-HER2-regulated immune-suppressive phenotypes

HER2 signaling can directly induce immune suppression in breast cancer through multiple mechanisms, such as immune checkpoint molecule and immunosuppressive cytokine overexpression, recruitment of immunosuppressive cells, and downregulation of MHC-I molecules. Consequently, although HER2-positive tumors frequently exhibit substantial immune cell infiltration, the signaling pathways actively transform the environment into an immunosuppressive state. Therefore, HER2-targeted therapies like trastuzumab not only inhibit tumor growth but also contribute to mitigating this immune suppression.

Conversely, HER2-independent immune suppression is a well-established phenomenon observed in both triple-negative and luminal breast cancers. The mechanisms involved include expression of immune checkpoint molecules, low immunogenicity, metabolic reprogramming, MHC-I downregulation, and increased infiltration of regulatory T cells and macrophages, which collectively create a microenvironment that inhibits CD8+ T cell function and promotes TGF-β production to facilitate epithelial-mesenchymal transition (EMT).

By integrating 31 HER2-regulated genes with 29 immune checkpoint genes, BRICA identifies HER2-dependent and HER2-independent immunosuppression across luminal, triple-negative, and HER2-positive breast cancers. This approach informs targeted immunotherapy for cases exhibiting HER2-independent immunosuppression and supports the use of HER2-targeted therapies to reverse immune suppression in breast cancers with elevated HER2 Functional Activity Score.