Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice.

The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4-FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4-FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

Clinical significance of plasma cell-free DNA mutations in PIK3CA, AKT1, and ESR1 gene according to treatment lines in ER-positive breast cancer.

The somatic activation of PI3K/AKT pathway mutations, PIK3CA and AKT1, and ESR1 mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive procedure to quickly assess and monitor disease progression or therapeutic effect in breast cancer (BC) patients, but the clinical significance of these mutations in late treatment lines (TLs) remains unclear. The subjects of this study were a total of 251 plasma samples from 128 estrogen receptor-positive (ER+) BC patients. Of these plasma samples, 133 were from 73 primary BC (PBC) patients, and 118 plasma samples were from 68 metastatic BC (MBC) patients. We developed droplet digital PCR (ddPCR) assays to verify the clinical significance of PIK3CA, AKT1, and ESR1 mutations in these patients. cfDNA PIK3CA mutations were observed in 15.1% of the PBC patients, while a cfDNA AKT1 mutation was observed in 1.4% of patients, and cfDNA ESR1 mutations were observed in 2.7% of patients. Patients with detectable cfDNA PIK3CA mutations were not associated with clinical outcomes. According to the TL, the prevalence of the PIK3CA and ESR1 mutations in cfDNA were lower in early TLs compared with late TLs. In the early TL group, patients with cfDNA PIK3CA mutations had a shorter time to treatment failure (TTF) than patients without mutations (P = 0.035). However, there was no statistically significant difference between patients with or without cfDNA ESR1 mutations. However, in the late TL group, patients with cfDNA ESR1 mutations had a shorter TTF than patients without mutations (P = 0.048). However, there was no statistically significant difference between patients with or without cfDNA PIK3CA mutations. Since the prevalence of cfDNA AKT1 mutation is low in both PBC and MBC patients, the impact of AKT1 mutations on the prognosis remains unclear. We have demonstrated the difference in the clinical significance of the hotspot PIK3CA, AKT1, and ESR1 mutations in cfDNA for each TL in ER+ BC patients.

Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients.

BACKGROUND: ESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations. METHODS: We screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations (E380Q, Y537S, Y537N, Y537C, and D538G) using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated. RESULTS: We detected ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment. CONCLUSIONS: We have shown that there are distinct populations of ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients.

BACKGROUND: The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. METHODS: The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. RESULTS: cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. CONCLUSIONS: We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients.

BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). RESULTS: Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.

A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRYPTIC, a close homologue of CRIPTO, is not sensitive. CRIPTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRIPTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRIPTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRIPTO shedding.

Prediction of sentinel lymph node status using single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging of breast cancer.

PURPOSE: Single-photon emission computed tomography (SPECT)/computed tomography (CT) improves the anatomical identification of sentinel lymph nodes (SNs). We aimed to evaluate the possibility of predicting the SN status using SPECT/CT. METHODS: SN mapping using a SPECT/CT system was performed in 381 cases of clinically node-negative, operable invasive breast cancer. We evaluated and compared the values of SN mapping on SPECT/CT, the findings of other modalities and clinicopathological factors in predicting the SN status. RESULTS: Patients with SNs located in the Level I area were evaluated. Of the 355 lesions (94.8 %) assessed, six cases (1.6 %) were not detected using any imaging method. According to the final histological diagnosis, 298 lesions (78.2 %) were node negative and 83 lesions (21.7 %) were node positive. The univariate analysis showed that SN status was significantly correlated with the number of SNs detected on SPECT/CT in the Level I area (P = 0.0048), total number of SNs detected on SPECT/CT (P = 0.011), findings of planar lymphoscintigraphy (P = 0.011) and findings of a handheld gamma probe during surgery (P = 0.012). According to the multivariate analysis, the detection of multiple SNs on SPECT/CT imaging helped to predict SN metastasis. CONCLUSIONS: The number of SNs located in the Level I area detected using the SPECT/CT system may be a predictive factor for SN metastasis.

Docetaxel and cyclophosphamide chemotherapy induced radiation recall phenomenon in a postoperative breast cancer patient: a case report.

The radiation recall phenomenon (RRP) is an acute inflammatory reaction at a site previously treated with radiation, and is triggered by anti-cancer therapies such as chemotherapy or antibiotics. A 48-year-old Japanese woman with primary breast cancer underwent partial mastectomy and sentinel lymph node biopsy followed by postoperative radiotherapy. Subsequent to breast-conserving surgery, adjuvant chemotherapy, including docetaxel in combination with cyclophosphamide (TC), was administrated after 16 days of radiotherapy involving the right breast. The patient experienced the RRP with erythema and burning pain at the site of the irradiation fields at 6 days after the administration of TC. The skin symptoms resolved after treatment with topical corticosteroid therapy over a few days. After the second course of TC, the patient had only mild symptoms relative to the first course. She successfully completed four cycles of TC without dose reduction and treatment delay. We report this case involving the RRP induced by TC together with a review of the literature.

Clinical significance of pretherapeutic Ki67 as a predictive parameter for response to neoadjuvant chemotherapy in breast cancer: is it equally useful across tumor subtypes?

BACKGROUND: Ki67 has been identified as a prognostic and predictive marker for breast cancer and it was suggested that it may contribute to pathologic complete response (pCR) after neoadjuvant chemotherapy. It is unclear whether expression of Ki67 is particularly helpful for prediction of pCR across tumor subtypes. METHODS: Pretherapeutic Ki67 was evaluated in a series of 121 breast cancer core biopsies. After neoadjuvant chemotherapy, we used postoperative specimens to evaluate the pCR status. Several parameters predictive of pCR were identified using logistic regression analysis. We investigated subgroups defined by estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2, in which predicting pCR with Ki67 might be feasible. RESULTS: Ki67 was found to be an independent predictor of pCR in multivariate analysis (odds ratio [OR], 3.62; 95% CI, 1.21-10.8). When stratified by ER, the above significance was exclusive to ER-positive tumors (OR, 6.24; 95% CI, 1.40-27.7). Using an receiver-operating characteristic curve, we obtained moderate discriminative accuracy with an area under the curve of 0.7752 for Ki67 prediction of pCR in ER-positive tumors. In subgroup analysis, patients with high Ki67 showed significantly improved pCR rate in luminal-type disease, with a median Ki67 value of 43% in the patients who achieved pCR, versus 29% for those without pCR (P = .018), whereas no associations were observed in other subtypes. CONCLUSION: Our results suggest that stratification according to Ki67 levels might improve predictive significance of the response in hormone-responsive breast cancer. Even in these subtypes assumed to be less chemosensitive, some patients with highly proliferative tumors derive a significant benefit from chemotherapy, and consequently it is important to identify them.

Clinical significance of CYLD downregulation in breast cancer.

Cylindromatosis (CYLD) is a tumor suppressor gene that is mutated in familial cylindromatosis, a rare autosomal dominant disorder associated with numerous benign skin adnexal tumors. CYLD is now known to regulate various signaling pathways, including transforming growth factor-ß signaling, Wnt/ß-catenin signaling, and NF-κB signaling by deubiquitinating upstream regulatory factors. Downregulation of CYLD has been reported in several malignancies; however, the clinical significance of CYLD expression in many malignancies, including breast cancer, remains to be elucidated. This study investigated the clinical significance of CYLD in breast cancer and its roles in tumor progression. We evaluated CYLD expression in matched normal breast tissue samples and tumor breast tissue samples from 26 patients with breast cancer and in a series of breast cancer cell lines. In addition, by means of immunohistochemistry, we investigated CYLD protein expression and its clinical significance in 244 breast cancer cases. We also analyzed the effects of CYLD repression or overexpression on breast cancer cell viability, cell migration, and NF-κB activity with or without receptor activator of NF-κB ligand (RANKL) stimulation. Breast cancer tissues demonstrated significantly reduced CYLD mRNA expression compared with normal breast tissues. Downregulation of CYLD promoted cell survival and migratory activities through NF-κB activation, whereas CYLD overexpression inhibited those activities in MDA-MB-231 cells. As an important finding, CYLD overexpression also inhibited RANKL-induced NF-κB activation. Our immunohistochemical analysis revealed that reduced CYLD protein expression was significantly correlated with estrogen receptor negativity, high Ki-67 index, high nuclear grade, decreased disease-free survival, and reduced breast cancer-specific survival in primary breast cancer. Moreover, reduced CYLD expression was an independent factor for poor prognosis in breast cancer. CYLD downregulation may promote breast cancer metastasis via NF-κB activation, including RANKL signaling.

High survivin mRNA expression is a predictor of poor prognosis in breast cancer: a comparative study at the mRNA and protein level.

BACKGROUND: Survivin plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. The purpose of this study was to examine the expression of survivin and its role in predicting clinical outcome in a series of human breast cancer cases both at the mRNA and protein level. METHODS: Formalin-fixed paraffin-embedded tumor tissues from 245 female patients with invasive breast cancer and 13 patients with ductal carcinoma in situ were examined for survivin mRNA by quantitative real-time RT-PCR (RT-qPCR). In addition, 237 of these tumors with invasive breast cancer were available for immunohistochemistry (IHC). The relationship between survivin status and clinicopathological characteristics and prognosis was evaluated. RESULTS: RT-qPCR revealed that high levels of survivin mRNA were strongly associated with high nuclear grade, positive axillary lymph nodes, negative hormone receptor status, positive Her2 amplification, higher Ki67 labeling index, and presence of vascular invasion. In the Cox proportional regression model analysis, survivin mRNA was shown to be a significant univariate parameter for relapse-free survival (RFS), distant relapse-free survival (DRFS), and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for RFS, DRFS, and BCSS. In hormone receptor (HR)-positive/Her2-negative subtype cases, survivin mRNA expression was also an independent predictor in terms of DRFS. Immunohistochemically, positive staining was seen in the cytoplasm and/or nucleus of cancer cells, although this did not correlate with the mRNA level, and harbored no prognostic value. CONCLUSIONS: High mRNA expression of survivin was an independent marker of poor prognosis both in the entire cohort and in the HR-positive/Her2-negative subtype, whereas the protein expression of survivin was not. These findings suggest that RT-qPCR can provide more reliable data than IHC in validating the prognostic significance of survivin for breast cancer patients.

[A case of estrogen receptor-positive metastatic breast cancer in a postmenopausal woman treated with ethinyl estradiol].

The patient was 77-year-old woman whose breast cancer had metastasized to the bone and soft tissue 5 years after surgery. Although she had been sequentially treated with endocrine therapies following chemotherapies, new metastatic lesions in the pleura and skin appeared 8 years after recurrence. The biopsied skin tissue showed high positivity for estrogen receptor(ER), was negative for human epidermal growth factor receptor 2(HER2), and had a low Ki-67 labeling index. Following the treatment with exemestane(EXE)for 3 months, ethinyl estradiol(EE2)was administered at 3mg/day. After 4 months of treatment, the lymph nodes shrunk to 35% of their size and pleural effusion disappeared. The efficacy of EE2 was observed for 10 months. Subsequently, fulvestrant was administered because the skin lesions showed progressive disease. Adverse events such as nausea and general fatigue were observed at the beginning of EE2 therapy. Pigmentation of the nipple and areola and cystic swelling of the cervical canal were observed after a few months. This therapy can be considered to be effective in patients with ER-positive metastatic breast cancer who have been heavily treated with endocrine therapies and chemotherapies.

Clinical relevance of Ki67 gene expression analysis using formalin-fixed paraffin-embedded breast cancer specimens.

BACKGROUND: Ki67 is a protein associated with cell cycle activity and shows a good correlation with the growth fraction, which has been proposed as a prognostic or predictive marker in breast cancer. In this study, we aimed to analyze the expression levels of Ki67 (MKI67) messenger RNA (mRNA) derived from formalin-fixed paraffin-embedded (FFPE) tissues for comparison with the immunohistochemical Ki67 labeling index, and investigate the correlation coefficients with clinical outcomes. METHODS: We analyzed the data of Ki67 mRNA from FFPE and matched fresh-frozen (FF) tissues based on a real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay system in 203 cases of primary invasive breast cancer. RESULTS: The correlation between Ki67 mRNA expression of either FFPE or FF specimens and Ki67 labeling index was positive, as was the correlation between the FFPE and FF results (P < 0.0001). Ki67 mRNA expression of FFPE specimens was significantly associated with clinicopathological characteristics: tumor size, lymph node status, nuclear grade, hormone receptors, human epidermal growth factor receptor 2 (Her2) status, and tumor subtype. In prognostic results, Ki67 gene expression in the FFPE specimens revealed almost similar patterns of significance in Kaplan-Meier curves and univariate and multivariate relapse-free survival results as the Ki67 labeling index. CONCLUSIONS: Gene expression analysis of Ki67 of FFPE specimens could be successfully performed using RT-qPCR, closely resembling the significant clinical characteristics of Ki67 labeling index. These results confirm that Ki67 gene expression of FFPE specimens has potential for evaluation of cell cycle activity of breast cancer specimens.

Evaluation of tumor stiffness by elastography is predictive for pathologic complete response to neoadjuvant chemotherapy in patients with breast cancer.

BACKGROUND: Breast elastography (EG), which can objectively evaluate tumor stiffness, has been useful for differentiation of benign and malignant breast lesions. However, the value of EG for prediction of response to systemic therapy is poorly understood. METHODS: The baseline evaluations of EG in 55 patients who received neoadjuvant chemotherapy were reviewed. We investigated the correlation between tumor stiffness and response to neoadjuvant chemotherapy. Tumor stiffness was evaluated by the Tsukuba elasticity scoring system. RESULTS: The mean EG scores were significant lower for the clinical and pathologic complete response (pCR) groups than for the others. When we categorized patients into two groups according to tumor stiffness, 26 patients were assigned to the low EG group (soft, scores from 1 to 3) and 29 patients were assigned to the high EG group (hard, score 4 and 5). The low EG group had significantly higher clinical complete response and pCR rates than the high EG group (clinical complete response, low EG group 38 % vs. high EG group 10 %, P = 0.024; pCR, low EG group 50 % vs. high EG group 14 %, P = 0.003, respectively). Furthermore, multivariate analysis indicated that estrogen receptor, human epidermal growth factor receptor 2, and low EG (odds ratio 13.04, 95 % confidence interval 1.19-458.28, P = 0.035) were independent predictive factors of pCR. CONCLUSIONS: Tumor stiffness evaluated by EG bears predictive potential for response to neoadjuvant chemotherapy. Stiffness evaluated by EG may be recognized as a clinically significant tumor characteristic, comparable to other data obtained by functional imaging techniques.

Quantitative determination of insulin-like growth factor 1 receptor mRNA in formalin-fixed paraffin-embedded tissues of invasive breast cancer.

BACKGROUND: Insulin-like growth factor 1 receptor (IGF1R) has recently received much attention due to its role in initiation and progression of breast cancer. Previously analysis of its gene expression has been restricted to fresh-frozen (FF) samples, but application of this technique to routinely processed formalin-fixed paraffin-embedded (FFPE) samples could facilitate larger retrospective studies correlating IGF1R expression with prognosis and therapeutic response. METHODS: A series of 77 paired FFPE and FF specimens of breast tumors was used to evaluate the possibility of quantifying IGF1R gene expression with FFPE samples and to compare the results obtained from FFPE and FF samples. The feasibility and prognostic value of analyzing IGF1R gene expression using FFPE samples was evaluated in a cohort of 260 primary breast tumors. RESULTS: Total RNA was extracted from 95.4% of the FFPE samples with concentration at least 30 ng/µL. Real-time PCR based on Taqman methodology was successful in 90% of the FFPE samples. IGF1R gene expression showed strong correlation not only between FFPE and FF (Spearman ρ = 0.74), but also with IGF1R protein expression in both types of specimen. Kaplan-Meier analysis showed that higher IGF1R mRNA expression was associated with longer recurrence-free survival (P = 0.009) and breast cancer-specific survival (P = 0.0002). CONCLUSIONS: Quantitative analysis of IGF1R gene expression in FFPE tissues can be feasibly and reliably conducted, and provides information relevant to the characteristics and outcome of invasive breast cancer.

Insulin-like growth factor-1 receptor gene expression is associated with survival in breast cancer: a comprehensive analysis of gene copy number, mRNA and protein expression.

Insulin-like growth factor-1 receptor (IGF1R) plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. In a series of 325 primary invasive breast cancer patients, we performed a comprehensive analysis of IGF1R at the levels of gene copy number, mRNA expression and protein expression. The relationship between the IGF1R status and the clinicopathological characteristics and prognosis was evaluated. IGF1R mRNA levels not only correlated with protein expression, but also were significantly associated with several clinicopathological parameters and prognosis. Patients with low nuclear grade, negative axillary lymph nodes, positive hormone receptor, negative Her2, negative Ki67, and luminal subtype tumors showed higher expression levels of IGF1R mRNA, which was shown to be a significant univariate parameter for both relapse-free survival and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for BCSS. IGF1R protein expression showed an association with a prolonged BCSS in univariate analysis. In contrast, IGF1R gene copy number was not correlated with mRNA and protein expression, and harbored no prognostic value. When studied in the luminal tumor subtype groups, IGF1R mRNA level was still significantly associated with a better BCSS. Overall, our data indicated a correlation between IGF1R mRNA expression and protein expression in primary breast cancer. In particular, IGF1R mRNA expression appeared to be a good prognostic marker both in the entire cohort and in the luminal subtype group. These data may serve as background information for IGF1R-targeted therapy.