Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment.

BACKGROUND: Alpha-2-glycoprotein 1, zinc-binding (ZAG), a secreted protein encoded by the AZGP1 gene, is structurally similar to HLA class I. Despite its presumed immunological function, little is known about its role in tumor immunity. In this study, we thus aimed to determine the relationship between the expression of AZGP1/ZAG and the immunological profiles of breast cancer tissues at both the gene and protein level. METHODS: Using a publicly available gene expression dataset from a large-scale breast cancer cohort, we conducted gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1. We analyzed the correlation between AZGP1 expression and immune cell composition in breast cancer tissues, estimated using CIBERSORTx. Previously, we evaluated the infiltration of 11 types of immune cells for 45 breast cancer tissues using flow cytometry (FCM). ZAG expression was evaluated by immunohistochemistry on these specimens and analyzed for its relationship with immune cell infiltration. The action of ZAG in M1/M2 polarization models using primary cultures of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) was analyzed based on the expression of M1/M2 markers (CD86, CD80/CD163, MRC1) and HLA class I/II by FCM. RESULTS: AZGP1 expression was negatively correlated with multiple immunological processes and specific immune cell infiltration including Mφ M1 using GSEA and CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in tumor tissues assessed using FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I/II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. CONCLUSION: ZAG is suggested to be a novel immunoregulatory factor affecting the Mφ phenotype in breast cancer tissues.

Expression of hormone receptors is associated with specific immunological profiles of the breast cancer microenvironment.

BACKGROUND: Elucidating the unique immunoregulatory mechanisms in breast cancer microenvironment may help develop new therapeutic strategies. Some studies have suggested that hormone receptors also have immune regulatory functions, but their mechanisms are not fully understood. In this study, we have comprehensively analyzed the relationship between the expressions of estrogen (ER), progesterone (PgR), and androgen receptors (AR), and the immunological profile in breast cancer. METHODS: Using publicly available gene expression profile datasets, METABRIC and SCAN-B, the associations between the expressions of hormone receptors and the immune cell compositions in breast cancer tissue, estimated by CIBERSORTx algorithm, were analyzed. We histologically evaluated tumor-infiltrating lymphocytes (hTIL), PD-L1 (hPD-L1) expression, and the infiltration of 11 types of immune cells by flow cytometry (FCM) for 45 breast cancer tissue samples. The relationships between them and the expressions of ER, PgR, and AR of tumor tissues, evaluated immunohistochemically, were analyzed. RESULTS: Expressions of ESR1, PGR, and AR were negatively correlated with overall immune composition. Expressions of ER and AR, but not that of PgR, were inversely associated with hTIL and hPD-L1 expression. FCM analysis showed that the expressions of ER and AR, but not that of PgR, were associated with decreased total leukocyte infiltration. Both CIBERSORTx and FCM analysis showed that ER expression was associated with reduced infiltration of macrophages and CD4+ T cells and that of AR with reduced macrophage infiltration. CONCLUSION: Hormone receptor expression correlates with specific immunological profiles in the breast cancer microenvironment both at the gene and protein expression levels.

[Two Cases of Pneumocystis Pneumonia during Adjuvant Chemotherapy for Breast Cancer].

In recent years, human immunodeficiency virus(HIV)-negative Pneumocystis pneumonia(PCP)onset has been occasionally seen in breast cancer. In particular, dose-dense epirubicin and cyclophosphamide(EC: ddEC)therapy, in which EC is administered every 2 weeks, has been generally used in clinical practice for early stage breast cancers. PCP may develop before and during postoperative chemotherapy. We report the cases of 2 patients who developed PCP during postoperative adjuvant chemotherapy. Case 1: A 62-year-old woman, who underwent postoperative EC therapy, developed PCP during the 4th EC cycle. During EC therapy, steroids(prednisolone[PSL])were administered at an average dose of 11.4mg/day, and the number of lymphocytes at the initiation of the 4th EC cycle was 516/mL. Case 2: After receiving 4 cycles of postoperative ddEC, a 27-year-old woman developed PCP after 1 cycle of docetaxel(DTX)administration. During ddEC therapy, PSL was administered at a dose of 17.14mg/day, and the number of lymphocytes at DTX administration was 311/mL. The onset of PCP is presumed to be related to the steroid dose administered and the number of lymphocytes. Therefore, determining effective indicators in patients at a high risk of PCP onset is important.

Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients.

The presence of tumor-infiltrating lymphocytes (TILs) is associated with favorable long-term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor-2 (HER2+ , n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin-eosin-stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD-L1), PD-L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t-test, P = 0.004) and that of CD8+ and CD4+ T cells significantly decreased from primary to metastatic tumors (paired t-test, P = 0.008 and P = 0.026, respectively). The PD-L1, PD-L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of TILs and CD8+ and CD4+ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.

Feasibility and pharmacokinetics of combined therapy with S-1 and trastuzumab in patients with human epidermal growth factor receptor 2-positive metastatic or recurrent breast cancer.

BACKGROUND: To clarify the tolerance and pharmacokinetics of combined therapy with S-1 and trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic or recurrent breast cancer. METHODS: From January 2008 through to September 2009, combined therapy with S-1 and trastuzumab was given to 7 patients with HER2-positive metastatic or recurrent breast cancer. The incidence of adverse events and the pharmacokinetics of tegafur, 5-fluorouracil, and gimeracil in plasma were studied. RESULTS: One patient had grade 3 leukopenia, and another had a grade 3 elevation of alanine aminotransferase. All other adverse events were grade 2 or lower. The combination of S-1 and trastuzumab did not cause any new adverse events. The incidence of adverse events was similar to those associated with S-1 alone. The median number of treatment cycles was 11. The pharmacokinetics of tegafur, 5-fluorouracil, and gimeracil after treatment with S-1 plus trastuzumab did not markedly differ from those after S-1 alone. CONCLUSIONS: Combined therapy with S-1 and trastuzumab did not cause any new adverse events, administration continuity was good, and the therapy was well tolerated.

Efficacy of Nursing Interventions Using Motivational Interviewing Aimed at Weight Loss in Overweight/Obese Breast Cancer Patients Undergoing Endocrine Therapy.

OBJECTIVE: Obesity adversely impacts breast cancer treatment and outcomes. This study assessed the efficacy of nurses' motivational interviews (MI) in promoting weight loss among breast cancer patients. METHODS: Motivational Interviewing was performed at 4, 8, and 12 weeks from baseline in 27 overweight/ obese breast cancer patients receiving adjuvant endocrine therapy. An average weight loss rate of 5% at week 12 was the threshold for determining whether MI intervention was clinically meaningful. Clinical and sociodemographic variables were gathered from medical records and self-administered questionnaires. Body weight, body mass index (BMI), physical activity time, sedentary time, self-efficacy for weight loss, and mood scores were evaluated at baseline, 4, 8, 12, and 24 weeks. RESULTS: Significant reductions in body weight were observed throughout compared with baseline; 51.9% of participants attained the 5% weight loss target, but the average weight loss rate was 3.9% at week 12. BMI notably decreased at 8, 12, and 24 weeks compared with baseline. Physical activity increased significantly at 12 weeks, while sedentary time decreased at 8 and 24 weeks. CONCLUSIONS: Nursing-administered MI did not achieve the goal of 5% weight loss at week 12. However, it increased physical activity and reduced sedentary time, showing potential for promoting healthier habits.

Ataxia Telangiectasia in a Patient with Breast Cancer: A Case Report.

Ataxia telangiectasia (AT) is a rare autosomal recessive disorder caused by the pathological variants of the ATM gene. Owing to i ts r arity a nd n ature, complications of AT, such a s malignant tumors, a re often difficult to manage with standard imaging studies and treatments, and there are no established management strategies. We report the case of a woman who had AT in childhood and developed breast cancer in her 20s; the disease was successfully managed by the decision-making of multidisciplinary physicians professionals with ethics support. She was immunocompromised, ataxic, and mentally impaired. The patient's mother noticed a tumor in her right breast and subsequently brought her to our department. Although preoperative testing and surgical procedures were limited as AT is extremely radiosensitive, the patient was diagnosed with cT2N0M0 breast cancer and underwent right mastectomy and axillary lymph node sampling. The final diagnosis was pT2N0M0 pStage IIA mucinous carcinoma, and immunohistochemistry of the tumor specimen was estrogen receptor-positive, progesterone receptor-positive, and HER2-negative. Tamoxifen was administered as postoperative adjuvant therapy, and the patient has survived to date without recurrence. Here, we report our experience with breast cancer treatment for AT, along with a review of the literature.

Immunological profiles of the breast cancer microenvironment represented by tumor-infiltrating lymphocytes and PD-L1 expression.

Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) are established prognostic and predictive biomarkers for certain breast cancer subsets. However, their association with the immune response complexity is not fully understood. Therefore, we analyzed the association between the immune cell fractions in breast cancer tissues and histologically assessed TIL (hTIL) and PD-L1 (hPD-L1). Forty-five tumor and eighteen blood samples were collected from patients with breast cancer. Total leukocyte counts, frequency of 11 immune cell populations, and PD-L1 expression in each cell fraction were evaluated by flow cytometry. TILs and PD-L1 were assessed by hematoxylin and eosin staining and immunohistochemistry, respectively. A higher hTIL score showed association with increased leukocyte infiltration, higher CD4+ and CD8+ T cell proportions, and lower natural killer and natural killer T cell proportions. PD-L1 was highly expressed in nonclassical monocytes, monocyte/macrophages, myeloid-derived suppressor cells, myeloid dendritic cells, dendritic cells, and other lineages in tumors. hPD-L1 positivity reflected PD-L1 expression accurately in these fractions, as well as increased leukocyte infiltration in tumors. These results indicate that hTILs reflect differences in the immune responses in the tumor microenvironment, and certain immune cell fractions are favorably expressed in the PD-L1 pathway in breast cancer microenvironments.

Post-marketing safety evaluation of S-1 in patients with inoperable or recurrent breast cancer: especially in patients treated with S-1 + trastuzumab.

OBJECTIVE: The purpose of this study was to assess the safety of S-1 in Japanese in inoperable or recurrent breast cancer patients. METHODS: A prospective post-marketing surveillance was performed at 313 sites in Japan in patients with inoperable or recurrent breast cancer treated with S-1. We examined 1361 patients between January 2006 and December 2007 with regard to the incidence of adverse drug reactions graded by the Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. RESULTS: At least one adverse drug reaction was encountered by 858 patients, with an overall incidence of 63.0% (858/1361). The incidence of Grade 3 or higher adverse drug reactions in a descending order was 14.7% (200/1361). In this study, the most common combination drug was trastuzumab. The overall incidence of adverse drug reactions was 63.5% (431/679 patients) in patients treated with S-1 alone, and 55.9% (66/118 patients) in patients treated with S-1 + trastuzumab. CONCLUSIONS: Monotherapy with S-1 or combination therapy with S-1 + trastuzumab was well tolerated for inoperable or recurrent breast cancer patients.

[Breast cancer].

Almost all breast cancer patients are treated with chemotherapy, hormone therapy, molecular targeted therapy, radiotherapy, and surgery alone or in combination. It is reasonable and standard to choose a combination according to clinical practice guidelines. One of the objectives of clinical practice guidelines is to standardize medical care and provide equal quality of care for patients wherever they live in Japan. Recently, many clinical trials have been conducted internationally. Therefore, it is necessary to standardize medical care from an international viewpoint. In this paper, the breast cancer clinical practice guideline developed by the Japanese Breast Cancer Society was compared with other guidelines which have been already developed and are frequently utilized all over the world as well as in Japan.

Diagnosis of oligometastasis.

Metastatic breast cancer (MBC) is generally considered incurable. However, MBC which has limited number and sites of metastasis: oligometastatic breast cancer (OMBC) may have definitive remission with individualized, metastasis-directed ablative therapies and multidisciplinary management, such as surgery and radiotherapy. Earlier detection of primary metastatic breast cancer using accurate imaging technologies may allow the initiation of therapy for OMBC. In breast cancer, the lymph nodes, liver, bone, lung, and brain are common sites of metastasis. Additional studies are recommended if signs or symptoms indicate metastasis. The imaging modality should be selected for suspicious locations or original metastatic regions. The standard staging studies are ultrasonography for lymph node metastasis, abdominal and/or pelvic contrast-enhanced CT for liver metastasis, chest CT for pulmonary metastasis, and bone scan for bone metastasis. FDG-PET/CT may be helpful for bone or liver metastasis. MRI may be considered for liver, soft tissue, or brain metastasis.

Students' Input on Preparing a Weekly Calendar of Individual Students in the Breast Surgery Clinical Clerkship.

Background; Until 2018, the Breast and Endocrine surgery had no student calendar. A questionnaire survey was conducted on how students felt by creating a weekly schedule of individual students from 2019. METHOD: 6th-year elective courses, targeted at students who selected Breast and Endocrine surgery clinical clerkship. The schedule clarifies the contents of the training as follows; outpatient visits, small group study (preparation for graduation and national exams including mammography reading), simulator training, and surgery. The questionnaire adopted an anonymous five-point evaluation method (5; I think very much; 4; Somewhat I think; 3; Normal; 2; Somewhat I don't think; 1; I don't think), and provided a free text box. The following seven questions were asked; A. I was able to send a good training, B. I was useful for studying national and graduation exams, C. Time constraints were appropriate, D. I could fully experience surgery, E. Appropriately experienced outpatients, F. Assignments (presented at conference) appropriate, G. I was interested in Breast and Endocrine surgery. RESULTS: Average values were A. 4.7, B. 4.9, C. 4.6, D. 4.9, E. 4.8, F. 4.7, G. 4.7. However, C and F received low ratings of 1 and 2. In the free text box, there were favorable opinions such as the fact that it was good to prepare for the national examination and that reading mammography was helpful. Conversely, there were some negative opinions, such as a time spent outpatient was too long, a difference in enthusiasm among the instructors, and a hope to see more at the first visit and to cope with the procedure. DISCUSSION: Preparing a weekly calendar of individual students generally yielded satisfactory results, but also highlighted the potential for future improvements in clinical clerkship.

Myxofibrosarcoma that Developed Rapidly in the Breast of an Elderly Man and Recurred Early after Surgery: A Case Report.

BACKGROUND: Myxofibrosarcoma is a rare disease occurring subcutaneously in the limbs. We report a case of a rapidly growing myxofibrosarcoma in the breast of an elderly man that recurred early after surgery. CASE PRESENTATION: A 73-year-old man presented with a breast mass. Physical findings showed a large tumor in the right breast, and malignancy was suspected on ultrasonography. Computed tomography (CT) revealed tumor invasion into the pectoralis major and pectoralis minor muscles. Positron emission tomography/CT showed no abnormality in other organs. Needle biopsy results excluded breast cancer but did not provide a definitive diagnosis. However, the tumor grew rapidly before further results were available, so emergency mastectomy was performed. The final pathological diagnosis was high-grade myxofibrosarcoma. Postoperative radiotherapy was started because of remnant tumor. The wound became worsened and swollen, and needle biopsy 10 days after the start of therapy indicated recurrence. Radical resection and thoracoplasty were performed. Postoperative pathological specimens showed no residual tumor. Radical radiation therapy was resumed. The patient has shown no recurrence after an year. CONCLUSIONS: It is important to consult a soft tissue oncologist for tumors in the breast and perform appropriate examination and treatment if soft tissue tumors cannot be ruled out.

A Case of Unknown Primary Cancer with a Tumor Formed in the Retroperitoneum: A Case Report.

BACKGROUND: Cancer of unknown primari (CUP) are said to account for 2% of all carcinomas. Here we report a rare case of CUP confined to the retroperitoneum. CASE PRESENTATION: A 51-year-old man consulted a nearby physician for back pain. The malignant tumor could not be denied by MRI, and she was referred to our hospital. CT and MRI revealed uneven enhanced tumor structures protruding into the L2/3 disc. Part of the tumor was continuous with the left iliopsoas muscle. A CT-guided needle biopsy was performed. Histologically, the sheet-like proliferation of atypical cells was observed. Immunohistochemistry showed that atypical cells were positive for cytokeratin AE1&3, CK7, CD10, GATA3, glypican 3, Hep Par 1, carbonic anhydrase 9 (focal), and vimentin (focal) but negative for CK20, CD56, chromogranin A, synaptophysin, TTF1, HMB45, melan A, and PSA. The pathological diagnosis was poorly differentiated carcinoma. However, it was difficult to determine the primary site from the pathological findings. Positron emission tomography (PET) scan showed no distant metastases. He was diagnosed as poorly differentiated cancer localized to the lumbar spine from the retroperitoneum. Paclitaxel plus carboplatin (TC) was started. After completing 3 kr of TC, she was hospitalized urgently due to worsening lumbago. CT and MRI at admission showed an increase in the main lesion and exacerbation of bone invasion. Radiation therapy was given for curative purposes. Eventually, he died seven months after visiting our hospital and five months after starting TC therapy. CONCLUSIONS: CUP has various disease states, and it is necessary to finish the examination immediately and shift to treatment. More effective treatment including immune checkpoint inhibitor for CUP is needed in the future.

Taste alterations in patients with breast cancer following chemotherapy: a cohort study.

BACKGROUND: Chemotherapy-induced taste and smell alterations in cancer patients are associated with multiple adverse effects, namely, malnutrition, weight loss, and a diminished quality of life. The aim of this prospective study was to identify the incidence of taste alterations following epirubicin and cyclophosphamide (EC) chemotherapy in patients with breast cancer without previous history of cancer or chemotherapy. METHODS: Forty-one patients undergoing EC chemotherapy for breast cancer at Tokai University Hospital were included. A subjective (questionnaire) and an objective (filter paper disk method) assessment for 5 basic tastes were administered on day 4 post-chemotherapy and immediately before the subsequent cycle of chemotherapy for each cycle, in addition to an olfactory evaluation and oral examination. The correlation between subjective and objective taste alterations and factors influencing these alterations were analyzed by statistical means. RESULTS: The mean incidence of subjective taste alteration on the 4th day after chemotherapy was 53%. In each of the 4 cycles, taste alterations decreased to about 9.0% immediately before the next cycle. A significant correlation between subjective and objective assessments was seen only for salty taste, suggesting important differences in subjective versus objective assessment outcomes. A multivariate analysis indicated that age and body surface area influenced taste alterations. CONCLUSIONS: EC chemotherapy induced taste alterations in more than 50% of patients, which decreased to less than 10% immediately before the next chemotherapy cycle. A combination of objective and subjective assessments is essential to evaluate taste alterations induced by EC chemotherapy. These could be used in routine clinical practice.

Distinction of IgG4-related mastitis from breast cancer: a case report.

BACKGROUND: Immunoglobulin (Ig) G4-related sclerosing disease is a pathological concept proposed in Japan during the early 2000s. This lesion-forming disease may exhibit characteristics of a systemic disease but often affects a single organ. To date, IgG4-related sclerosing disease in the mammary gland, or IgG4-related mastitis (IgG4-RM), has rarely been reported. CASE PRESENTATION: Here, we describe the case of a female patient who was admitted to our hospital with the main complaints of left breast and axillary lymphadenopathy. A careful diagnostic imaging examination led to an initial suspicion of breast cancer. However, a needle biopsy led to a diagnosis of IgG4-RM. Subsequently, the patient was successfully treated with predonin. CONCLUSIONS: The treatment requirements for breast cancer and IgG4-RM differ considerably. This is a good example of a case wherein unnecessary surgical treatment, which is indicated for breast cancer, was avoided by needle biopsy. Accordingly, the patient was appropriately treated with steroids following a correct diagnosis.

Rare Finding of Bilateral Pseudoangiomatous Stromal Hyperplasia of the Breast: A Case Report.

A 49-year-old woman, with a medical history of rheumatism, was admitted to our hospital with chief complaints of bilateral enlargement and redness of breasts. She underwent weekly breast examinations. Mammography findings were reported as category 3 for both breasts. Breast ultrasonography, magnetic resonance imaging, and chest contrast computed tomography revealed a massive tumor in the left BD region, however, there were no findings for suspected malignancy. Needle biopsy did not yield histologically malignant cells in both breasts. Mammary interstitium was edematous, and capillary-like slit structures were observed. The stroma stained with alcian blue and destained with hyaluronidase treatment. Since the stroma tested positive for vimentin, calponin, and CD34 and negative for CD31, the patient was diagnosed as (PASH). Because both breasts had similar diagnosis based on histopathologic findings, bilateral mastectomy was performed. Details about the origin of bilateral PASH are unknown but it may be related to the development of rheumatoid arthritis. Additionally, systemic autoimmune diseases like rheumatism may be the reason for repeated contraction and enlargement of PASH.

B-cell populations are expanded in breast cancer patients compared with healthy controls.

BACKGROUND: Historically, humoral immunity was considered unimportant in anti-tumor immunity, and the differentiation and anti-tumor activity of B cells in breast cancer are poorly understood. However, it was recently discovered that B cells participate in tumor immunity through both antibody production and immunosuppressive mechanisms. We analyzed the expression of B-cell differentiation markers in detail using fluorescence-activated cell sorting to investigate the relationship between B-cell subsets and breast cancer etiology. METHODS: Blood samples were taken from breast cancer patients and healthy donors, and peripheral blood mononuclear cells were collected. B cells at various stages of differentiation were identified by the expression of combinations of the cell surface markers CD5, CD19, CD21, CD24, CD27, CD38, CD45, and IgD. Statistical analysis of the proportions of each B-cell subtype in the different patient groups was then performed. RESULTS: Twenty-seven breast cancer patients and 12 controls were considered. The proportion of total B cells was significantly higher in cancer patients than in controls (11.51 ± 2.059 vs 8.905 ± 0.379%, respectively; p = 0.001). Breast cancer patients were then classified as High-B or Low-B for further analysis. A significantly higher proportion of memory B cells was found in the High-B group than in the Low-B or control groups (p = 0.003 and p = 0.043, respectively). CONCLUSIONS: Breast cancer patients generally have a higher proportion of B cells than healthy controls, but this is highly variable. Analysis of the major B-cell surface markers indicates that memory B cells in particular are significantly expanded, or more robust, in breast cancer patients.

Comparison of Ki-67 labeling index measurements using digital image analysis and scoring by pathologists.

BACKGROUND: Routine analysis of Ki-67 is not widely recommended for clinical decision-making because of poor reproducibility. Furthermore, counting numerous cells can be laborious for pathologists. Digital image analysis for immunohistochemical analysis was recently developed; however, the clinical efficacy of the Ki-67 index obtained using image analysis is unknown. METHODS: We retrospectively identified female patients with breast cancer with immunohistochemical Ki-67 and survival data using the pathology database at the Tokai University, Japan. Ki-67 expression was scored by three pathologists. Slides were scanned and converted to virtual slides; Ki-67-positive cells were counted using image analysis. Ki-67 indices obtained by the pathologist's scoring and image analysis were evaluated by 2 × 2 analysis. Relationships between Ki-67 index and survival outcomes were evaluated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: Based on the 2 × 2 analysis, Ki-67 index obtained using image analysis was moderately correlated with the pathologist's scoring for all patients (κ 0.41; sensitivity, 0.573; specificity, 0.878). Poorer relapse-free survival was associated with high Ki-67 index than with low Ki-67 index for estrogen receptor-positive, human epidermal growth factor receptor 2-negative, and stage I or II patients scored by pathologists (p < 0.001) and obtained using image analysis (p = 0.031). CONCLUSIONS: The Ki-67 indices obtained using image analysis were moderately correlated with those scored by pathologists. Digital image analysis can be effective for measuring Ki-67 values, because they are associated with relapse-free survival in estrogen receptor-positive, human epidermal growth factor receptor 2-negative, and patients at stage I or II.