Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer

Biphosphonates (BPs) are widely used to inhibit osteoclastic activity in malignant diseases such as bone metastatic breast and prostate carcinoma. Recent studies reported that BPs could also cause a direct antitumor effect, probably due to their ability to interfere with several intracellular signalling molecules. The enzyme cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) play an important role in the control of cancer cell growth and inhibitors of COX-2 and EGFR have shown antitumor activity in vitro and in vivo in several tumor types. We, and others, have previously shown that EGFR and COX-2 may be directly related to each other and that their selective inhibitors may have a cooperative effect. In the present study we have evaluated the combined effect of zoledronic acid, the most potent nitrogen-containing BP, with the COX-2 inhibitor SC-236 and the selective EGFR-tyrosine kinase inhibitor gefitinib, on breast and prostate cancer models in vitro and in xenografted nude mice. We show that combination of zoledronic acid with SC-236 and gefitinib causes a cooperative antitumor effect accompanied by induction of apoptosis and regulation of the expression of mitogenic factors, proangiogenic factors and cell cycle controllers both in vitro and in xenografted nude mice. The modulatory effect on protein expression and the inhibitory effect on tumor growth is much more potent when the three agents are used together. Since studies are ongoing to explore the antitumor effect of zoledronic acid, our results provide new insights into the mechanism of action of these agents and a novel rationale to translate this feasible combination treatment strategy into a clinical setting.     

Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy,induces complete responses in triple-negative breast cancer models

Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G₂-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-κB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.     

Pathologic complete response to neoadjuvant anti-HER2 therapy is associated with HER2 immunohistochemistry score in HER2-positive early breast cancer

To evaluate whether pathologic complete response (pCR) to neoadjuvant anti-human epidermal growth factor receptor 2 (HER2) therapy is dependent on the HER2 immunohistochemistry (IHC) score.A total of 181 HER2-positive early breast cancer patients who had received neoadjuvant anti-HER2 therapy were included in this study. Associations were examined between IHC score and tumor pCR status (commonly defined by ypT0+ypN0, ypT0/is+ypN0, or ypT0/is).In trastuzumab-based neoadjuvant-treated patients, ypT0+ypN0 was achieved in 46.0% of patients with HER2 IHC 3+ tumors but only 25.0% of patients with HER2 IHC 2+/fluorescence in situ hybridization (FISH)-positive tumors (P = .016). When pCR was defined as ypT0/is+ypN0 or ypT0/is, 54.7% and 61.3% of patients with HER2 IHC 3+ tumors had a pCR, whereas only 29.5% and 38.6% with HER2 IHC 2+/FISH-positive tumors achieved pCR (P = .004 and P = .008, respectively). The association between dual HER2 blockade and pCR was almost exclusively confined to HER2 IHC 3+ tumors (ypT0+ypN0: 61.9% vs 38.9%, P = .013; ypT0/is+ypN0: 71.4% vs 47.4%, P = .009; and ypT0/is: 81.0% vs 52.6%, P = .002) and was absent in HER2 IHC 2+/FISH-positive tumors. Multivariate logistic regression revealed that HER2 IHC 3+ tumors had a significantly higher probability of achieving ypT0+ypN0 (odds ratio [OR], 0.265; 95% confidence interval [CI], 0.109–0.645; P = .003), ypT0/is+ypN0 (OR, 0.221; 95% CI, 0.094–0.521; P = .001), and ypT0/is (OR, 0.254; 95% CI, 0.111–0.583; P = .001) than HER2 IHC 2+/FISH-positive tumors. A significantly better pCR rate was also found in patients with T1 tumors and patients with dual HER2 blockade.The pCR rate was highly correlated with the HER2 IHC score in neoadjuvant anti-HER2 treatment. The addition of pertuzumab to a neoadjuvant trastuzumab-based regimen improved pCR rates, but there was no significant difference in pCR rates in the IHC 2+/FISH-positive group. This suggests that HER2 IHC scores can predict the effectiveness of treatment.     

Pten loss promotes MAPK pathway dependency in HER2/neu breast carcinomas

Loss of the tumor suppressor gene PTEN is implicated in breast cancer progression and resistance to targeted therapies, and is thought to promote tumorigenesis by activating PI3K signaling. In a transgenic model of breast cancer, Pten suppression using a tetracycline-regulatable short hairpin (sh)RNA cooperates with human epidermal growth factor receptor 2 (HER2/neu), leading to aggressive and metastatic disease with elevated signaling through PI3K and, surprisingly, the mitogen-activated protein kinase (MAPK) pathway. Restoring Pten function is sufficient to down-regulate both PI3K and MAPK signaling and triggers dramatic tumor regression. Pharmacologic inhibition of MAPK signaling produces similar effects to Pten restoration, suggesting that the MAPK pathway contributes to the maintenance of advanced breast cancers harboring Pten loss.The PTEN (phosphatase and tensin homolog) tumor suppressor gene is mutated or silenced in a wide range of tumor types (1). PTEN encodes a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase that counters the action of the phosphatidylinositol 3-kinases (PI3Ks), which otherwise transmit growth factor signals from receptor tyrosine kinases to downstream mediators such as the AKT family of serine/threonine-specific protein kinases (2). AKT, in turn, activates a series of downstream effectors that promote cellular proliferation and survival. Consequently, PTEN loss leads to hyperactivation of the PI3K pathway, and it is widely believed that this is the primary mechanism whereby PTEN loss drives tumorigenesis (3). Although cross-talk and feedback signaling makes the situation more complex (4), this molecular framework provides a strong rationale to target PI3K pathway components in PTEN-deficient tumors, and indeed, a variety of small-molecule antagonists with such activities are currently in clinical trials (5, 6).Deregulation of the PI3K pathway is common in breast cancer and most frequently occurs through mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (7). By contrast, PTEN mutation or loss is less frequent at diagnosis but instead is associated with disease progression (8, 9). For example, PTEN inactivation often arises in oncogenic receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2/neu) amplified tumors in patients who acquire resistance to the HER2/neu targeting agent trastuzumab (10–14). Similarly, PTEN mutations were recently reported in a patient harboring PIK3CA mutations that developed resistance to the PI3Kα inhibitor BYL719 (15). Thus, PTEN inactivation occurs in advanced disease in patients with poor prognosis, defining a breast cancer subtype for which there is an unmet clinical need.Studies using mouse models have confirmed the importance of PI3K signaling in breast cancer (16). Transgenic mice that overexpress mutant PIK3CA in conjunction with HER2/neu recapitulate resistance to anti-HER2/neu therapies (17), and conditionally overexpressed mutant PIK3CA in the mammary gland gives rise to tumors at long latency that regress upon oncogene withdrawal (18). Although these observations contribute to the rationale for targeting PI3K pathway components in breast cancer, they use a model in which mutant PIK3CA is expressed at unphysiological levels and serves as the initiating event. Furthermore, studies using conditional knockout mice indicate that deregulation of the endogenous PI3Ks indirectly through Pten inactivation can promote advanced disease in combination with HER2/neu (19, 20). Still, whether sustained PTEN inactivation is needed for the maintenance of advanced cancers remains unknown. Resolving this issue may reveal cellular dependencies and, as such, instruct the clinical use of molecularly targeted agents attacking the PTEN network. In this study, we explore the impact of genetic and pharmacologic manipulation of the Pten pathway in breast cancer. Unexpectedly, our results reveal that Pten loss is required to maintain advanced disease by enhancing signaling through both the PI3K and mitogen-activated protein kinase (MAPK) cascades.     

COX-2抑制剂联合放疗对人乳腺癌裸鼠移植瘤作用的研究

目的观察环氧化酶-2（COX-2）抑制剂塞来昔布联合放疗对人乳腺癌MDA-MB-231细胞裸鼠移植瘤生长的影响。方法建立人乳腺癌裸鼠移植瘤模型,观察塞来昔布联合放疗治疗后裸鼠移植瘤的生长情况,测定移植瘤中COX-2、血管内皮生长因子（VEGF）的表达情况。结果塞来昔布联合放疗治疗后移植瘤的体积和重量减小（P〈0.01）,COX-2、VEGF表达下降（P〈0.05）。结论塞来昔布联合放疗能抑制人乳腺癌裸鼠移植瘤的生长。     

CRINEPT-TROSY NMR reveals p53 core domain bound in an unfolded form to the chaperone Hsp90

The molecular chaperone Hsp90 sequesters oncogenic mutants of the tumor suppressor p53 that have unstable core domains. It is not known whether p53 is bound in an unfolded, partly folded, or distorted structure, as is unknown for the structure of any bound substrate of Hsp90. It is a particularly difficult problem to analyze in detail the structures of large complexes in which one component is (partly) unfolded. We have shown by transverse relaxation-optimized NMR spectroscopy combined with cross-correlated relaxation-enhanced polarization transfer (CRINEPT-TROSY) that p53 core domain bound in an approximately 200-kDa complex with Hsp90 was predominantly unfolded lacking helical or sheet secondary structure. This mode of binding might be a general feature of substrates of Hsp90.     

HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2^YVMA) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2^YVMA is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2^YVMA transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.     

Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis

Development of the pathologies associated with Helicobacter pylori infection, most seriously gastric adenocarcinoma, are a consequence of chronic inflammation, which both host and pathogen go to some lengths to minimize. Recently, we presented evidence that H. pylori can suppress the development of inflammation in its immediate microenvironment in the gastric mucosa of 129/Sv mice.

We have now extended this study by showing that H. felis, a gastric colonizing Helicobacter closely related to H. pylori, does not possess the same ability to suppress Helicobacter-induced gastritis in mice. Differences between these bacterial species may provide clues as to the mechanism behind the inflammation-regulating ability of H. pylori. Moreover, our demonstration that H. pylori but not H. felis can locally suppress inflammation in vivo may explain why H. felis infection induces superior levels of gastritis as compared with H. pylori infection of mice.     

Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis

Development of the pathologies associated with Helicobacter pylori infection, most seriously gastric adenocarcinoma, are a consequence of chronic inflammation, which both host and pathogen go to some lengths to minimize. Recently, we presented evidence that H. pylori can suppress the development of inflammation in its immediate microenvironment in the gastric mucosa of 129/Sv mice. We have now extended this study by showing that H. felis, a gastric colonizing Helicobacter closely related to H. pylori, does not possess the same ability to suppress Helicobacter-induced gastritis in mice. Differences between these bacterial species may provide clues as to the mechanism behind the inflammation-regulating ability of H. pylori. Moreover, our demonstration that H. pylori but not H. felis can locally suppress inflammation in vivo may explain why H. felis infection induces superior levels of gastritis as compared with H. pylori infection of mice.     

Palb2 synergizes with Trp53 to suppress mammary tumor formation in a model of inherited breast cancer

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.     

Hsp90 inhibitors cause G2/M arrest associated with the reduction of Cdc25C and Cdc2 in lung cancer cell lines

Purpose: Hsp90, a molecular chaperone, is involved in folding, assembly, maturation, and stabilization of the client proteins which regulate survival of cancer cells, and thus Hsp90 inhibitors may be potential molecular targeting agents for cancer treatment. We investigated whether Hsp90 inhibitors have therapeutic value in lung cancer. Methods: First, expression levels of Hsp90 in lung cancer cells were examined by western blotting and immunohistochemical analyses. Next, the effect of Hsp90 inhibitors, geldanamycin and 17-allylaminogeldanamycin (17-AAG), on lung cancer cell growth was examined. Results: Remarkable high expression of Hsp90 protein in lung cancer cell lines and a more intense signal for Hsp90 by immunohistochemistry in males, patients with smoking index over 600, and squamous cell carcinoma were observed. Both Hsp90 inhibitors dose dependently inhibited the growth of lung cancer cell lines and induced G2/M arrest concomitant with decreased protein levels of Cdc25C and Cdc2. Moreover, combination of an Hsp90 inhibitor and irradiation had an additive effect on cell growth inhibition and reduction of Cdc25C and Cdc2 protein levels. Conclusion: Hsp90 inhibitor is thus a therapeutic tool for lung cancer based on its target proteins, which are involved in tumor progression and antiproliferative activity in lung cancer cells.     

An activated receptor tyrosine kinase,TEL/PDGFbetaR,cooperates with AML1/ETO to induce acute myeloid leukemia in mice

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML/ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19/19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.     

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.     

Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti–cytotoxic T-lymphocyte–associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti–DEC-205 (dendritic and epithelial cells, 205 kDa)–HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.Promoting a robust CD8 T-cell response is vital for the generation of an effective antitumor immune response. However, immunosuppressive mechanisms used by the tumor result in the exhaustion of tumor-infiltrating lymphocytes (TIL), leading to cytotoxic T-cell anergy and dysfunction. To overcome this dysfunction, investigators have had considerable success using immune checkpoint inhibitors, such as aCTLA-4 (cytotoxic T-lymphocyte–associated protein 4) mAbs, to promote T-cell function. CTLA-4, a negative regulatory molecule on the surface of T cells, is indispensable for preventing the expansion and activation of autoreactive T cells. Inhibition of this surface receptor using antagonist aCTLA-4 mAb–augmented effector CD4 and CD8 T-cell responses and inhibited or reduced the suppressive function of Treg cells (1–5). However, only a subset of patients treated with aCTLA-4 mAb exhibit an objective clinical response (6).Checkpoint blockade targets T-cell coinhibitory molecules, but other strategies targeting costimulatory molecules, such as the TNF receptor family member OX40 (CD134), have demonstrated success in promoting tumor regression in a wide variety of preclinical models. Treatment with an agonist aOX40 mAb directly stimulated CD4 and CD8 T cells and induced their expansion, differentiation, and up-regulation of prosurvival molecules (7–12). Moreover, OX40 ligation promoted the generation of long-lived memory CD8 T cells and enhanced their function. Recent data indicate that combined aOX40/aCTLA-4 therapy induced a robust effector CD4 and CD8 T-cell response necessary for tumor regression and significantly improved the survival of tumor-bearing hosts relative to therapy with either agent alone (13). Despite this success, the response to combination aOX40/aCTLA-4 treatment was greatly diminished in more established tumors. This therapy may be unable to overcome T-cell anergy in more established immunosuppressive tumor microenvironments, possibly because it is ineffective at specifically targeting and expanding tumor-reactive T cells and relies on limited or defective endogenous priming by dendritic cells (14). Currently, clinically tested vaccines targeting cross-presenting dendritic cells [i.e., anti–DEC-205 (dendritic and epithelial cells, 205 kDa) mAb conjugated to tumor antigens] have demonstrated promise by priming a more robust cytotoxic T-cell response (15–19). The possibility remains that increased Th2-cytokine production by CD4 T cells following combination therapy may reduce its therapeutic efficacy, because inhibition of IL-4 with aOX40/aCTLA-4 treatment significantly improved survival (13). Others have shown that a dominant Th2 cytokine response reduced the efficacy of treatment, whereas a Th1-skewed immune response resulted in more favorable outcomes (20–23).We hypothesized that dendritic cell-targeted vaccination against a tumor-associated antigen in conjunction with combination aOX40/aCTLA-4 mAb immunotherapy would be sufficient to promote a cytotoxic antitumor T-cell response, redirect a Th2 bias in CD4 T cells, and improve survival in mice with established tumors. Here, we report that vaccination using anti–DEC-205/HER2 (human epidermal growth factor receptor 2) mAb along with combination aOX40/aCTLA-4 mAbs significantly expanded effector CD8 T cells, resulting in a more favorable Th1 cytokine profile and inducing a critical accumulation of effector T cells in the tumor, with increased tumor-free survival relative to either therapy alone. Moreover, combination therapy with vaccination reversed T-cell anergy and promoted a robust effector T-cell response to a tumor-associated antigen in a spontaneous adenocarcinoma model.     

Lymphocyte-activating gene-3 expression is associated with tumor-infiltrating lymphocyte levels in HER2-positive breast cancers

Lymphocyte-activating gene-3 (LAG-3, CD223) is the third inhibitory receptor targeted for immunotherapy. Several clinical trials investigating the use of interventions targeting LAG-3 are underway. The exact signaling mechanism downstream of LAG-3 is largely unknown, especially in breast cancer. The prognostic significance of tumor-infiltrating lymphocytes (TILs) in breast cancer has been previously determined.Among 167 human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, 90 and 78 patients were positive and negative for the hormone receptor, respectively. LAG-3 mRNA and protein expression levels in TILs were evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, among 12 and 167 HER2-positive breast cancer samples, respectively.High expression of LAG-3 in TILs was significantly correlated with high levels of TILs (P = .003) and an abundance of tertiary lymphoid structures around invasive components (P = .014). In addition, high expression of LAG3 was significantly associated with positivity for programmed death-ligand 1 (PD-L1) in tumor cells, a high immunostaining score of PD-L1 in TILs, and a high total immunostaining score for PD-L1 in tumor cells and TILs (all, P < .001). High expression levels of LAG-3 mRNA were associated with high levels of TILs (P = .091).LAG-3 protein expression was not a prognostic factor in HER2-positive breast cancers, and LAG-3 expression in TILs was significantly associated with the levels of TILs in HER2-positive breast cancer, although it was not a prognostic factor.