High platelet-to-lymphocyte ratios in triple-negative breast cancer associates with immunosuppressive status of TILs.

BACKGROUND: Rating lymphocytes (TILs) are a prognostic marker in breast cancer and high TIL infiltration correlates with better patient outcomes. Meanwhile, parameters involving immune cells in peripheral blood have also been established as prognostic markers. High platelet-to-lymphocyte ratios (PLRs) and neutrophil-to-lymphocyte ratios (NLRs) are related to poor outcomes in breast cancer, but their mechanisms remain unknown. To date, TILs and these parameters have been examined separately. METHODS: We investigated the relationship between TILs and the peripheral blood markers, PLR and NLR, in the same patients, using surgical specimens from 502 patients with invasive breast carcinoma without preoperative chemotherapy. For analysis of triple-negative breast cancer (TNBC) patient outcomes, 59 patients who received preoperative chemotherapy were also examined. For immune cell profiling, multiplexed fluorescent immunohistochemistry (mfIHC) of CD3, CD4, CD8, FOXP3 and T-bet, was conducted. RESULTS: A positive correlation between PLR and TIL was observed in TNBC (P = 0.013). On mfIHC, tumors in patients with high PLR and NLR contained more CD3+CD4+FOXP3+ T-cells (P = 0.049 and 0.019, respectively), while no trend was observed in CD8+ T-cells. TNBC patients had different patterns of outcomes according to TIL and PLR, with the TIL-high/PLR-low group having the lowest rate of disease relapse and death, and the longest distant metastasis-free and overall survivals, while the TIL-low/PLR-high group had the shortest survivals. CONCLUSIONS: Our data suggest that the combination of PLR with TIL assessment may enable more accurate prediction of patient outcomes with TNBC.

Involvement of kallikrein-PAR2-proinflammatory pathway in severe trastuzumab-induced cardiotoxicity.

Trastuzumab-induced cardiotoxicity interferes with continued treatment in approximately 10% of patients with ErbB2-positive breast cancer, but its mechanism has not been fully elucidated. In this study, we recruited trastuzumab-treated patients with ≥30% reduction in left ventricular ejection fraction (SP) and noncardiotoxic patients (NP). From each of these patients, we established three cases of induced pluripotent stem cell-derived cardiomyocytes (pt-iPSC-CMs). Reduced contraction and relaxation velocities following trastuzumab treatment were more evident in SP pt-iPSC-CMs than NP pt-iPSC-CMs, indicating the cardiotoxicity phenotype could be replicated. Differences in ATP production, reactive oxygen species, and autophagy activity were observed between the two groups. Analysis of transcripts revealed enhanced kallikrein5 expression and pro-inflammatory signaling pathways, such as interleukin-1ß, in SP pt-iPSC-CMs after trastuzumab treatment. The kallilkrein5-protease-activated receptor 2 (PAR2)-MAPK signaling pathway was more activated in SP pt-iPSC-CMs, and treatment with a PAR2-antagonist suppressed interleukin-1ß expression. Our data indicate enhanced pro-inflammatory responses through kallikrein5-PAR2 signaling and vulnerability to external stresses appear to be the cause of trastuzumab-induced cardiotoxicity in SP.

Lessons Learned in Practice with Li-Fraumeni Syndrome: LFS-Related Breast Cancer Treatment Strategy and Establishment of a Surveillance System.

We herein present the case of a 33-year-old woman with no family history of metachronous bilateral breast cancer and osteosarcoma, diagnosed with Li-Fraumeni syndrome (LFS), which is a rare autosomal dominant hereditary cancer syndrome associated with a germline TP53 variant. She was diagnosed with left distal femoral osteosarcoma at the age of 16, and metachronous bilateral breast cancer at the ages of 29 and 33. When the third cancer was diagnosed, a hereditary tumor syndrome was suspected and the patient was referred to our genetic outpatient clinic. There was no family history of the 'core' cancers for LFS, but since the patient met Chompret's criteria, germline TP53 genetic testing was performed with the patient's will. A pathogenic variant, TP53:c.216dupC (p.Val73ArgfsX76) was found in exon 4 of the gene. This case is didactic because radiotherapy was performed on the first breast cancer before the diagnosis of LFS was made; radiation should be avoided if there are other options in LFS because of the inability to repair DNA damage. As a lesson learned, oncologists reaffirmed the importance of being aware of hereditary tumors from the keywords "multiple," "young," "familial," and "rare," and consulting the genetic department. In addition, surveillance using whole-body magnetic resonance imaging is recommended in LFS. However, this system is not yet provided nationwide, but we have newly settled it in our hospital.

Molecular Characteristics of Lymphocyte-predominant Triple-negative Breast Cancer.

BACKGROUND/AIM: Tumor-infiltrating lymphocytes (TILs) are considered a prognostic marker for triple-negative breast cancer (TNBC). Immune checkpoint inhibitor (ICI)-based treatments are more effective for tumors with PD-L1-positive TILs, suggesting crucial roles of TILs in the local tumor immunity. However, factors attracting TILs are still largely unknown. Focusing on tumor antigenicity, we examined TNBC samples to identify the characteristics of TIL-high tumors. PATIENTS AND METHODS: Nine treatment-naïve TNBCs (TIL-high: five, TIL-low: four) were subjected to next-generation sequencing (NGS). Loss of heterozygosity (LOH) of PTEN was also analyzed. RESULTS: A variety of copy number variations were observed, and no genes differed significantly between TIL-high and -low groups. However, PTEN loss was more frequently observed in the TIL-high group: 60% compared to 25% in TIL-low tumors. NGS correlated well with LOH analysis in identifying PTEN loss. All three tumors with PTEN loss in the TIL-high group showed high PD-L1. All nine samples were microsatellite-stable. CONCLUSION: Frequent PTEN loss and high expression of PD-L1 in TIL-high TNBC suggest that PTEN mutation may be a biomarker for ICIs.

Cardiotoxicity after Additional Administration of Pertuzumab following Long-Term Trastuzumab: Report of 2 Cases.

Pertuzumab, a humanized antibody drug, has improved outcomes of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, when administered in combination with trastuzumab and other chemotherapies. Cardiotoxicity due to trastuzumab is widely recognized, while data on pertuzumab-based treatments in daily clinical practice are lacking. We herein report 2 Japanese patients, aged 72 and 49 years, who developed left ventricular dysfunction after pertuzumab administration, following long-term trastuzumab treatments. Both patients underwent curative surgery for their HER2-positive breast cancer and received anthracycline-based treatments. After developing metastatic disease, trastuzumab-based treatments were administered without cardiac toxicity, but both patients developed left ventricular dysfunction after pertuzumab administration (6 and 13 cycles, respectively). Although several large randomized trials have shown no additive effect of pertuzumab on cardiac dysfunction, careful monitoring of cardiac function appears to be necessary in daily practice, particularly for patients with prior long-term trastuzumab treatments.

High FOXA1 protein expression might predict late recurrence in patients with estrogen-positive and HER2-negative breast cancer.

BACKGROUND: Multi-gene expression assays have been developed with the aim of predicting late recurrence in patients with estrogen receptor (ER)-positive breast cancer. However, establishment of alternative markers based on immunohistochemistry is also important for achieving practical use. Based on our previous study, forkhead box A1 (FOXA1) protein was tested as a potentially useful predictive marker for late recurrence. METHODS: 117 patients with ER-positive HER2-negative invasive breast cancer who developed distant metastasis following curative surgery were retrospectively investigated. We also evaluated responsiveness to endocrine therapy according to FOXA1 expression. Furthermore, publicly available mRNA microarray data were analyzed to examine patterns of metastasis according to FOXA1 mRNA expression, employing the Kaplan-Meier plotter. RESULTS: High expression of FOXA1 was an independent factor predicting long disease-free survival (DFS), along with small tumor size (p = 0.010 and 0.016, respectively). Discrimination of DFS was improved by combining these two factors, i.e., patients with FOXA1-high small tumors had the longest DFS while those with FOXA1-low large tumors had the shortest DFS. Moreover, we revealed that risk of distant metastasis started to increase after the completion of adjuvant endocrine therapy in patients with FOXA1-high tumors. CONCLUSION: Among patients who developed distant metastasis, those with FOXA1-high tumors had significantly longer DFS. We believe our data to raise the possibility of FOXA1 being a useful predictive marker for late recurrence and to provide new insights into the biology of FOXA1-high breast cancers.

Microsatellite instability and mismatch repair protein expressions in lymphocyte-predominant breast cancer.

The frequency of microsatellite instability (MSI) is reportedly extremely low in breast cancer, despite widespread clinical expectations that many patients would be responsive to immune-checkpoint inhibitors (ICI). Considering that some triple-negative breast cancers (TNBC) responded well to ICI in a clinical trial and that a high density of tumor-infiltrating lymphocytes (TILs) is frequently observed in other cancers with high levels of microsatellite instability (MSI-H), we hypothesized that some TNBC with a high density of TILs would be MSI-H. Medullary carcinoma (MedCa) of the breast, a rare histological type, is characterized by a high density of TILs. Considering that MedCa of the colon is often MSI-H, we suspected that MedCa in breast cancer might also include MSI-H tumors. Therefore, we conducted MSI tests on such breast cancers with a high density of TILs. The MSI status of 63 TIL-high TNBC and 38 MedCa tumors, all from Asian women who had undergone curative surgery, were determined retrospectively. DNA mismatch repair (MMR) proteins and PD-L1 expression were also investigated immunohistochemically. All samples were microsatellite stable, being negative for all microsatellite markers. TIL-high TNBC with low MLH1 protein had higher levels of PD-L1 in stromal immune cells (P = .041). MedCa tumors showed significantly higher PD-L1 expression in immune cells than in TIL-high TNBC (<.001). We found that MSI-H tumors were absent in TIL-high breast cancers. Examination of MMR proteins, not a purpose of Lynch syndrome screening, may merit further studies to yield predictive information for identifying patients who are likely to benefit from ICI.

Neutrophil-to-lymphocyte ratio and histological type might predict clinical responses to eriburin-based treatment in patients with metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is generally considered to be incurable. Although many options are available for treating MBC, physicians often encounter difficulties in choosing the most appropriate treatment because the MBCs of individual patients respond differently even to the same treatments. Thus, predictive markers for therapeutic efficacy are urgently needed. Neutrophil- and platelet-to-lymphocyte ratios (NLR and PLR, respectively), have been studied and established as prognostic markers for breast cancer patients but whether either or both of these markers are predictive of treatment responses is still unclear. Herein, we investigated predictive markers for eribulin-based treatment responsiveness in patients with MBC, by examining clinicopathological features, including several markers of immunocompetent cells in peripheral blood. METHODS: Clinicopathological features of the 104 patients with metastatic/Stage IV breast cancer given eribulin-based regimens were investigated in relation to clinical responses to eribulin-based treatments and progression-free-survival (PFS). RESULTS: Special histological types and high NLR at baseline were independently related to poor clinical responses to the treatments (p = 0.023 and 0.039, respectively). The Cox hazard model revealed that patients with oestrogen receptor (ER)-negative tumours and high NLR, monocyte-to-lymphocyte ratio (MLR) and PLR showed significantly shorter PFS (p = 0.021, 0.005, 0.008 and 0.030, respectively). On multivariate analysis, only ER status and NLR remained independent factors related to PFS (p = 0.011 and 0.003, respectively). CONCLUSIONS: Our data revealed that special histological types and high NLR might be factors related to low responsiveness to eribulin-based regimens in patients with MBC.

Estrogen Receptor-positive Ductal Carcinoma In Situ Frequently Overexpresses HER2 Protein Without Gene Amplification.

Overexpression of human epidermal growth factor receptor 2 (HER2) protein is well known to be more frequent in ductal carcinoma in situ (DCIS) than in invasive ductal carcinoma (IDC). However, the reasons for this difference are poorly understood. On the basis of the high frequency of estrogen receptor-positive (ER+) and HER2-positive (HER2+) DCIS, we hypothesized that this tumor type overexpresses HER2 protein without gene amplification and retrospectively investigated the HER2/neu gene status of 71 ER(+)HER2(+) DCIS, surgically removed during the 2007 to 2017 period, employing fluorescence in situ hybridization (FISH). To compare HER2 protein expressions between in situ and invasive components of individual tumors, 86 pT1mi/1a IDC with predominantly in situ disease were also examined. Furthermore, for comparison of FISH status between in situ and coexisting invasive components, another patient cohort, 78 FISH-positive IDC cases, were employed. To elucidate biological differences among DCIS with various combinations of ER and HER2 protein expressions, we also analyzed public microarray data of mRNA. HER2 gene amplification was observed in 35% of ER(+) and HER2 protein-overexpressing specimens, significantly lower than the 94% in ER-negative (ER-) and HER2 protein-overexpressing specimens (P<0.001). HER2 protein expression was decreased in the invasive component as compared with coexisting in situ portions in 40% of individual tumors, whereas the FISH status of these 2 components was well preserved. Moreover, ER(+) and HER2 protein-overexpressing DCIS showed significantly higher hypoxia-inducible factor-1α protein expression than the ER(+) and HER2 protein-nonoverexpressing tumors (P=0.016). We revealed that ER(+) and HER2 protein-overexpressing DCIS, especially ER-high tumors, frequently overexpress HER2 protein without gene amplification. Our data may provide novel insights for understanding the biology of DCIS.

Administration of plasma-derived coagulation factor VIII during the perioperative period of mastectomy for breast cancer with acquired von Willebrand syndrome.

BACKGROUND: Acquired von Willebrand syndrome (aVWS) is a rare bleeding disorder with laboratory findings similar to those of congenital von Willebrand disease (VWD). Patients with aVWS may require prophylactic treatment to prevent excessive bleeding following surgery. To our knowledge, to date, there have been no reports on perioperative management for breast cancer patients with aVWS. CASE PRESENTATION: A 60-year-old woman with breast cancer was diagnosed with aVWS due to polycythemia vera. Pre-operative laboratory testing showed a high platelet count and low von Willebrand factor (VWF) activity. The VWF activity did not improve despite an attempt to suppress platelet count with hydroxyurea. Therefore, we decided to perioperatively supplement with plasma-derived factor VIII (FVIII) containing von Willebrand factor (FVIII/VWF concentrates) to perform curative surgery for breast cancer safely. Consequently, the patient did not develop hemorrhage during/after surgery and was discharged on postoperative day 7, as planned, without problems. CONCLUSIONS: For a patient with aVWS, which carries a high risk of hemorrhage during the perioperative period, initiation of appropriate management like supplementation of FVIII/VWF concentrates might enable safe curative surgery for breast cancer, and collaboration with the hematology department is critical.

Randomized Comparison of Subcuticular Sutures Versus Staples for Skin Closure After Open Abdominal Surgery: a Multicenter Open-Label Randomized Controlled Trial.

BACKGROUND: The incisional surgical site infection (SSI) is an extremely common complication following open abdominal surgery and imposes a considerable treatment and cost burden. METHOD: We conducted a multicenter open-label randomized controlled trial at three Tokyo Metropolitan medical institutions. We enrolled adult patients who underwent either an elective or an emergency open laparotomy. Eligible patients were allocated preoperatively to undergo wound closure with either subcuticular sutures or staples. A central Web-based randomization tool was used to assign participants randomly by a permuted block sequence with a 1:1 allocation ratio and a block size of 4 before mass closure to each group. The primary endpoint was the occurrence of a superficial SSI within 30 days after surgery in accordance with the Centers for Disease Control and Prevention criteria. This trial was registered with UMIN-CTR as UMIN 000004836 ( http://www.umin.ac.jp/ctr ). RESULTS: Between September 1, 2010 and August 31, 2015, 401 patients were enrolled and randomly assigned to either group. One hundred and ninety-nine patients were allocated to the subcuticular suture and 202 patients to the staple groups (hereafter the "suture" and "staple" group, respectively). Three hundred and ninety-nine were eligible for the primary endpoint. Superficial SSIs occurred in 25 of 198 suture patients and in 27 of 201 staple patients. Overall, the rate of superficial SSIs did not differ significantly between the suture and staple groups. CONCLUSION: Subcuticular sutures did not increase the occurrence of superficial SSIs following open laparotomies mainly consisting of clean-contaminated surgical procedures. The applicability of the wound closure material and method is likely to depend on individual circumstances of the patient and surgical procedure.