Triple-negative breast cancer is defined by the absence of the three molecular handles that anchor most modern breast cancer therapy: the estrogen receptor, the progesterone receptor, and HER2. Without those targets, endocrine agents and HER2-directed drugs are ineffective, leaving cytotoxic chemotherapy as the systemic backbone of treatment. Although triple-negative tumors account for only about 10 to 15 percent of breast cancers, they carry a disproportionate share of the disease’s mortality because they tend to grow quickly, are more likely to metastasize, and historically have fewer therapeutic options. For patients whose tumors are caught early, the central treatment question is not whether chemotherapy helps but when it should be given.

That question is most acute for T1c tumors, which measure between one and two centimeters and have not spread to regional lymph nodes or distant sites. The T1cN0M0 designation describes a small, node-negative cancer, yet even at this stage triple-negative biology demands systemic therapy to reduce recurrence risk. The debate concerns sequencing. Neoadjuvant chemotherapy, delivered before surgery, offers the chance to shrink the tumor, potentially enabling less extensive operations, and provides real-time information about how the cancer responds to treatment. Adjuvant chemotherapy, given after surgery, achieves the same cytotoxic goal but leaves the surgeon operating on a tumor that has never been exposed to drugs. National guidelines leave room for either approach in this small-tumor setting, and practice varies considerably across institutions.

The reason sequencing remains contested is that neither strategy has demonstrated superior overall survival. Multiple prior analyses, including studies of the same National Cancer Database, have found that patients with clinically node-negative T1 triple-negative tumors fare equally well whether chemotherapy comes first or last. If survival outcomes are equivalent, the choice between the two approaches hinges on secondary benefits: the possibility of breast-conserving surgery, the prognostic information embedded in treatment response, and patient preference. The Johns Hopkins team, led by breast surgical oncology researcher Andrew Venardi, set out to sharpen that calculus by asking a more granular question: within this seemingly uniform early-stage population, which patients are actually likely to benefit from going first with chemotherapy?

The answer, they hypothesized, might lie in Ki-67, a nuclear protein expressed by cells that are actively dividing. Pathologists measure Ki-67 by immunohistochemistry, staining tumor samples and counting the percentage of positive cells, which yields a proliferation index reflecting how fast the cancer is growing. The logic connecting Ki-67 to neoadjuvant strategy is mechanistically straightforward: chemotherapy agents such as anthracyclines and taxanes preferentially kill rapidly proliferating cells, so tumors with a high fraction of dividing cells should, in principle, be more sensitive to upfront drug treatment. Previous studies have associated high Ki-67 expression with better responses to neoadjuvant chemotherapy in triple-negative disease, but the specific threshold that separates responders from non-responders in the earliest tumor category had not been clearly defined using large-scale national data.

To conduct the analysis, the researchers queried the National Cancer Database for female patients aged 18 and older diagnosed with T1N0M0 triple-negative breast cancer between 2018 and 2022 who underwent both chemotherapy and surgery. The National Cancer Database, jointly maintained by the American College of Surgeons and the American Cancer Society, captures roughly 70 percent of newly diagnosed cancer cases in the United States, providing the statistical power that single-institution series cannot match. Patients were divided into a neoadjuvant chemotherapy cohort and an adjuvant chemotherapy cohort. Because the two groups differ systematically in tumor size, grade, and other characteristics—surgeons and oncologists tend to select patients for upfront chemotherapy based on perceived risk—the team applied inverse probability of treatment weighting, a statistical technique that re-weights the cohorts to mimic the balance of a randomized trial.

The survival analysis produced a finding consistent with the existing literature: overall survival did not differ significantly between the neoadjuvant and adjuvant groups. Multivariate Cox regression, which adjusts for competing variables simultaneously, instead identified three factors associated with worse survival. Lymphovascular invasion, the presence of tumor cells within small blood or lymphatic vessels, signaled a higher risk of microscopic spread. The invasive lobular subtype, which arises in the milk-producing lobules rather than the ducts, and mixed lobular-ductal tumors were also linked to poorer outcomes. Lobular triple-negative cancers are rare and biologically distinct, and recent registry-based studies have shown they can carry worse survival despite appearing indolent, so their emergence as an adverse marker in this dataset aligns with a growing body of evidence.

The most consequential result came from the response analysis. Among patients who received neoadjuvant chemotherapy, those who achieved a pathologic complete response—meaning no residual invasive cancer was found in the breast or lymph nodes at the time of surgery—had markedly better survival than those who did not. Non-responders faced a hazard ratio of 3.59 for worse overall survival compared with patients who achieved a complete response. This echoes the findings of a large meta-analysis showing that pathologic complete response in triple-negative breast cancer is strongly associated with improved long-term event-free and overall survival. In other words, while the timing of chemotherapy may not change survival on average, achieving eradication of the tumor before surgery identifies a subgroup whose prognosis is transformed.

That is where the Ki-67 threshold enters. Using a receiver operating characteristic curve, the researchers plotted the relationship between Ki-67 expression and pathologic complete response and applied the maximum Youden index, a standard method for finding the cutoff point that best balances sensitivity and specificity. The threshold that emerged was 45.8 percent. Patients whose tumors expressed Ki-67 at or above that level achieved a pathologic complete response rate of 45.3 percent, while those below it responded at a rate of only 21.8 percent. Expressed differently, a high proliferation index more than doubled the odds that upfront chemotherapy would completely eliminate the tumor. Logistic regression confirmed Ki-67 expression as an independent predictor of complete response, suggesting the association held after accounting for other tumor characteristics.

The clinical implications are tangible for a patient population that often faces an anxious choice. A woman with a 1.5-centimeter, node-negative triple-negative tumor whose biopsy shows a Ki-67 index of 50 percent now has registry-scale evidence that chemotherapy before surgery is likely to produce a complete response, with the dual benefits of maximal prognostic information and a survival outcome that appears markedly better than that of non-responders. Conversely, a patient with a low proliferation index may reasonably opt for surgery first, since her probability of achieving a complete response is lower and adjuvant chemotherapy delivers equivalent survival. The authors emphasize that the study is retrospective and that Ki-67 measurement itself has well-known variability between laboratories and staining protocols, so the 45.8 percent cutoff should be validated prospectively before it reshapes guidelines. Still, as the field moves from one-size-fits-all recommendations toward personalized sequencing in stage I triple-negative breast cancer, a routine pathology marker already on every diagnostic report may prove to be the deciding variable.

Subject of Research: Ki-67 expression as a predictor of pathologic complete response to neoadjuvant chemotherapy in early-stage triple-negative breast cancer

Article Title: Ki-67 expression and rates of pathologic complete response in T1cN0M0 triple-negative breast cancer: a national cancer database analysis

Article References: Venardi, A., Shojaeian, F., Diaz, S., Schuster, C. R., Rath, P., Shaid, I., Singh, A., Santa-Maria, C., & Sogunro, O. (2026). Ki-67 expression and rates of pathologic complete response in T1cN0M0 triple-negative breast cancer: a national cancer database analysis. Breast Cancer Research and Treatment, 219(3), Article 21. https://doi.org/10.1007/s10549-026-08086-1

Image Credits: AI Generated

DOI: 10.1007/s10549-026-08086-1

Keywords: triple-negative breast cancer, Ki-67, neoadjuvant chemotherapy, pathologic complete response, National Cancer Database, T1cN0M0, proliferation index, biomarker, breast surgical oncology, lymphovascular invasion, invasive lobular carcinoma, personalized treatment

One of the most striking molecular signals described in the review is the loss of methylation in LINE-1, a repetitive DNA element that accounts for roughly 17 percent of the human genome. DNA methylation normally helps regulate genome stability and gene activity by attaching chemical methyl groups to cytosine bases, particularly at CpG sites. Cancer cells often show two apparently opposing epigenetic abnormalities: excessive methylation at promoters of tumor-suppressor genes, which can silence protective pathways, and widespread hypomethylation across the genome, which can destabilize chromosomes. Using bisulfite pyrosequencing, the Kumamoto researchers found that LINE-1 methylation was significantly lower in gastric tumor tissue than in matched normal gastric mucosa. Patients whose tumors had the lowest levels also experienced shorter overall survival. The finding suggests that LINE-1 hypomethylation may be more than a passive molecular signature. By increasing genomic instability, it could create a cellular environment in which additional mutations accumulate more readily, potentially linking environmental exposures, chronic inflammation and tumor progression.

The review also focuses on cancer stem-like cells, a small but influential population capable of self-renewal, tumor initiation, metastasis and resistance to chemotherapy. In gastric and other gastrointestinal cancers, the CD44 variant known as CD44v appears to help these cells survive oxidative stress. CD44v interacts with xCT, a transporter that supports production of reduced glutathione, one of the cell’s major antioxidant molecules. This system helps cancer cells neutralize reactive oxygen species, chemically aggressive molecules generated by metabolism, inflammation and some cancer treatments. When CD44 was removed, xCT disappeared from the cell surface, glutathione defenses weakened and oxidative stress activated the p38 mitogen-activated protein kinase pathway, followed by induction of the cell-cycle inhibitor p21. Tumor growth was suppressed in mouse models. The same redox-adaptation mechanism was connected to precancerous gastric metaplasia and to chronic Helicobacter pylori infection, which can suppress miR-328 and increase CD44 expression. In blood samples from patients with advanced gastric cancer, CD44-high circulating tumor cells also showed greater tumor-forming ability, suggesting that these cells may be both a source of metastasis and a tractable therapeutic target.

A rare inherited syndrome called gastric adenocarcinoma and proximal polyposis of the stomach, or GAPPS, offered another window into how gastric tumors emerge. The syndrome is generally caused by a point mutation in promoter 1B of the APC gene and is inherited in an autosomal dominant pattern. Sequencing of normal mucosa, polyps and carcinomas from seven affected patients revealed a progression in which somatic APC mutations appeared in polyps, while additional KRAS mutations emerged in carcinomas. APC and KRAS mutations repeatedly occurred together in cancer samples and in separate tumor subclones, suggesting that their cooperation may be close to essential for the final malignant transition in GAPPS. This pattern differs from sporadic gastric cancer, in which APC-KRAS co-mutation is uncommon and TP53 mutations are more typical. The researchers propose that KRAS mutations could eventually help identify polyps at high risk of malignant transformation and guide the timing of preventive surgery. Circulating tumor DNA and other liquid-biopsy approaches might one day allow clinicians to monitor this evolution without repeatedly removing tissue.

The tumor microenvironment provides another explanation for why gastric cancer can invade, evade immunity and withstand drugs. Cancer-associated fibroblasts, or CAFs, are connective-tissue cells reprogrammed by tumors into active partners. They remodel the extracellular matrix, release growth factors and inflammatory molecules, influence blood-vessel formation and suppress immune attack. In diffuse-type gastric cancer, inflammatory signals from tumor cells induce the fibroblast protein RHBDF2, which enhances transforming growth factor beta signaling and increases CAF motility. These mobile fibroblasts then help tumor cells penetrate extracellular matrix and lymphatic vessels. CAFs can also release interleukin-8, activating NF-κB signaling in cancer cells and increasing PD-L1, a surface protein that weakens T-cell attack. Tiny membrane-bound particles called extracellular vesicles provide another route of communication: CAF vesicles carrying Annexin A6 stabilize β1 integrin on tumor cells, activating the FAK-YAP pathway and promoting drug resistance. In fibrotic tumors, PDGF signaling drives CAFs to produce chemokines that recruit suppressive myeloid cells, helping explain why anti-PD-1 immunotherapy may fail. Blocking PDGF receptors alongside immune checkpoint therapy improved tumor control in experimental models.

Microbes add a further layer of complexity. Helicobacter pylori remains the most important inflammatory trigger in the stomach, driving a sequence from gastritis and gland loss to intestinal metaplasia, dysplasia and carcinoma. Its virulence proteins, including CagA and VacA, disrupt cell signaling, stimulate proliferation and contribute to tissue injury. Epstein-Barr virus-associated gastric cancer, which accounts for about 10 percent of cases, forms a distinct molecular subtype marked by widespread CpG-island hypermethylation. The researchers have also investigated Fusobacterium nucleatum, a bacterium commonly found in the mouth and associated with periodontal disease. In a study of 325 resected esophageal cancers, F. nucleatum DNA was detected in 23 percent of tumors and was more abundant in malignant tissue than in normal mucosa. Bacterial positivity correlated with advanced disease and cancer-specific survival, with a hazard ratio of 1.78. The organism was linked to cytokine signaling, including the CCL20 chemokine pathway, and appeared to activate NF-κB and NOD1-RIPK2 signaling. In cell and animal models, F. nucleatum increased tumor growth and altered autophagy, a cellular recycling process, allowing esophageal cancer cells to resist 5-fluorouracil, cisplatin and docetaxel. Suppressing the autophagy gene ATG7 reversed this resistance.

The ability of gastric cancer cells to disseminate also depends on epithelial-mesenchymal transition, or EMT. During EMT, cells lose epithelial features such as E-cadherin-mediated adhesion and apical-basal polarity, while acquiring mesenchymal traits that increase movement, invasion, resistance to cell death and stem-like behavior. This transformation helps cells detach from a primary tumor, enter the circulation, survive transit and establish new colonies, although many must later undergo the reverse process, mesenchymal-epithelial transition, to grow efficiently at distant sites. In gastric cancer, miR-200b can suppress EMT by targeting the transcription factor ZEB2. Peritoneal dissemination is especially difficult to treat because detached tumor cells enter the abdominal cavity, survive without normal attachment to extracellular matrix, adhere to the mesothelial lining and form metastatic nodules. PLOD2 remodels collagen, while the receptor tyrosine kinase DDR2 may promote metastatic spread. In malignant ascites, inflammatory signals push CAFs into a senescent state and induce a senescence-associated secretory phenotype rich in interleukin-6. Epigenetic changes involving EZH2 sustain this inflammatory program, which activates JAK-STAT3 signaling and accelerates peritoneal tumor formation. Single-cell analyses further identified mesothelial cells undergoing mesothelial-mesenchymal transition as sources of chemokines and tenascin-C, helping recruit immunosuppressive cells and establish metastatic colonies.

The clinical findings in the review show how molecular biology can be paired with basic measures already available in hospitals. In 416 patients undergoing potentially curative gastric cancer surgery, a high preoperative Controlling Nutritional Status, or CONUT, score was strongly associated with poor outcome. The score combines serum albumin, lymphocyte count and cholesterol, reflecting nutritional reserve and systemic inflammation. Patients with a score of four or higher had more advanced disease and a markedly worse overall survival; after adjustment for other variables, the hazard ratio was 5.09. The score also outperformed several commonly used markers in predicting five-year survival. Surgical experience mattered as well. Analysis of 145,523 Japanese patients who underwent distal gastrectomy showed operative mortality of 1.9 percent in low-volume hospitals compared with 0.5 percent in high-volume hospitals. After adjustment, high-volume hospitals retained a substantially lower risk of death. Similar patterns were observed for total gastrectomy, supporting greater concentration of complex gastric surgery in experienced centers. The researchers argue that future care will depend on integrating these clinical indicators with tumor genomics, transcriptomics, proteomics, metabolism, immune profiling and microbiome data. Artificial intelligence may help convert this large-scale information into individualized predictions, but the review emphasizes that prospective clinical trials will be essential before experimental biomarkers or combined treatments enter routine practice.