Advances in diagnostic liquid-based cytology.

Liquid-based cytology (LBC) has changed the landscape of gynaecological cytology. A growing demand exists for LBC in diagnostic cytology, particularly for ancillary testing, such as immunocytochemistry and molecular testing. Ancillary testing solely based on conventional preparation (CP) methods remains challenging. Recently, the increased demand for specialist testing and minimally invasive techniques, such as endoscopic ultrasonography fine-needle aspiration, to obtain cellular samples has led to an increasing demand for ancillary testing on cytology LBC supernatant, slides and cell block (CB). This facilitates the diagnosis and prognosis in cytology samples enabling personalized treatment. An understanding of the history and future prospects of LBC is crucial for its application in routine diagnostics by cytopathologists and cytotechnologists. In this review, we initiated an internet search using the keyword 'liquid-based cytology', and we conducted a literature review to discuss the usefulness of combined diagnosis of LBC and CP, immunocytochemistry and molecular testing and assessed the quality of nucleic acids in diagnostic LBC. High-quality and cell-rich diagnostic LBC surpassed the CP method alone in terms of reliability and versatility of ancillary testing in cytological diagnosis. Conclusively, diagnostic LBC lends itself to various new technologies and is expected to continue evolving with innovations in the future.

Induction of SGK1 via glucocorticoid-influenced clinical outcome of triple-negative breast cancer patients.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive breast malignancy. Glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays a pivotal role in the cellular responses to various stresses including chemotherapy. Serum- and glucocorticoid-induced kinase-1 (SGK1) is known as an important downstream effector molecule in the GR signaling pathway, we attempted to explore its clinicopathological and functional significance in TNBC in which GR is expressed. METHODS: We first immunolocalized GR and SGK1 and correlated the results with clinicopathological variables and clinical outcome in 131 TNBC patients. We also evaluated the effects of SGK1 on the cell proliferation and migration in TNBC cell lines with administration of dexamethasone (DEX) to further clarify the significance of SGK1. RESULTS: The status of SGK1 in carcinoma cells was significantly associated with adverse clinical outcome in TNBC patients examined and was significantly associated with lymph node metastasis, pathological stage, and lymphatic invasion of the patients. In particular, SGK1 immunoreactivity was significantly associated with an increased risk of recurrence in GR-positive TNBC patients. Subsequent in vitro studies also demonstrated that DEX promoted TNBC cell migration and the silencing of gene expression did inhibit the cell proliferation and migration of TNBC cells under DEX treatment. CONCLUSIONS: To the best of our knowledge, this is the first study to explore an association between SGK1 and clinicopathological variables and clinical outcome of TNBC patients. SGK1 status was significantly positively correlated with adverse clinical outcome of TNBC patients and promoted carcinoma cell proliferation and migration of carcinoma cells.

Intraductal papilloma with atypical ductal hyperplasia and neuroendocrine differentiation as a possible precursor lesion of solid papillary carcinoma.

Breast papillary neoplasms include a wide range of tumor types, and their pathological diagnosis is sometimes difficult. Furthermore, the etiology of these lesions is still not fully understood. We report the case of a 72-years-old woman referred to our hospital with bloody discharge from the right nipple. An imaging study detected a cystic lesion, including a solid component contiguous with the mammary duct, in the subareolar region. The lesion was then removed by segmental mastectomy. Pathological examination of the resected specimen revealed an intraductal papilloma with atypical ductal hyperplasia. Moreover, the atypical ductal epithelial cells expressed neuroendocrine markers. The presence of an intraductal papillary lesion with neuroendocrine differentiation suggests solid papillary carcinoma. Thus, this case suggests that intraductal papilloma could be a precursor of solid papillary carcinoma.

Tumor-infiltrating B cells and T cells correlate with postoperative prognosis in triple-negative carcinoma of the breast.

BACKGROUND: In this study, we investigated CD20+ TILs in triple-negative breast cancer (TNBC) and their relationship with T lymphocyte subsets (CD4+, CD8+, CD25+, and FOXP3+), including their combined prognostic value using an immunohistochemical staining method. METHODS: We investigated 107 patients with TNBC for whom a full-face section stained by hematoxylin and eosin between 2006 and 2018 at Dokkyo Medical University Hospital was available. RESULTS: The strongest association of infiltrating CD20+ TILs was with CD4+ TILs. There was a significant relationship between CD20+ and CD4+ TILs (r = 0.177; p < 0.001), CD8+ TILs (r = 0.085; p = 0.002), and FOXP3+ TILs (r = 0.0043; p = 0.032). No significant relationships were observed between the CD20+ and CD25+ TILs (r = 0.012; p = 0.264). Multivariate analysis revealed that only the CD20+/FOXP3 ratio was an independent factor for relapse-free survival (p < 0.001) and overall survival (p < 0.001). Patients with tumors highly infiltrated by CD4+, CD8+, and CD20+ TILs had a good prognosis. In contrast, those with tumors weakly infiltrated by CD20+ TILs but highly infiltrated by CD25+ and FOXP3+ TILs had a poor prognosis. CONCLUSIONS: CD20+ TILs may support an increase in CD4+ and CD8+ TILs, which altered the anti-tumor response, resulting in a positive prognosis. CD20+ TILs correlated with FOXP3+ Treg lymphocytes, which were reported to be correlated with a poor prognosis. Our study suggested that TIL-B cells have dual and conflicting roles in TIL-T immune reactions in TNBC.

Metaplastic breast cancers frequently express immune checkpoint markers FOXP3 and PD-L1.

BACKGROUND: Metaplastic breast carcinoma encompasses a heterogeneous group of tumours with differentiation into squamous and/or spindle, chondroid, osseous or rhabdoid mesenchymal-looking elements. Emerging immunotherapies targeting Programmed Death Ligand 1 (PD-L1) and immune-suppressing T cells (Tregs) may benefit metaplastic breast cancer patients, which are typically chemo-resistant and do not express hormone therapy targets. METHODS: We evaluated the immunohistochemical expression of PD-L1 and FOXP3, and the extent of tumour infiltrating lymphocytes (TILs) in a large cohort of metaplastic breast cancers, with survival data. RESULTS: Metaplastic breast cancers were significantly enriched for PD-L1 positive tumour cells, compared to triple-negative ductal breast cancers (P < 0.0001), while there was no significant difference in PD-L1 positive TILs. Metaplastic breast cancers were also significantly enriched for TILs expressing FOXP3, with FOXP3 positive intra-tumoural TILs (iTILs) associated with an adverse prognostic outcome (P = 0.0226). Multivariate analysis identified FOXP3 iTILs expression status as an important independent prognostic factor for patient survival. CONCLUSIONS: Our findings indicate the clinical significance and prognostic value of FOXP3, PD-1/PD-L1 checkpoint and TILs in metaplastic breast cancer and confirm that a subset of metaplastics may benefit from immune-based therapies.

Androgen receptor expression is useful to predict the therapeutic effect in HER2-positive breast carcinoma.

PURPOSE: Although HER2-positive (HER2+) invasive breast carcinomas (BC) have a different clinical therapeutic responsiveness according to estrogen and progesterone receptor expression, the relationship with androgen receptors (AR), which are the same family of steroid hormones, is poorly understood. We investigated the relationship between AR expression in HER2 BCs and therapeutic responsiveness and prognosis in this study. METHODS: We evaluated patients with HER2 (H) + invasive BC undergoing surgery after neoadjuvant chemotherapy (± HER2-targeted therapies) from 2007-2017, classified as hormone receptor-positive (Allred score: 2-8) (luminal B: LH) and receptor-negative groups (Allred: score 0) (non-luminal: NLH). AR expression was assessed by immunostaining pre-neoadjuvant chemotherapy biopsy specimens, positive with Allred score ≥ 4. The pathological complete response, disease-free survival, and overall survival rates were compared between AR-positive and AR-negative groups. RESULTS: We classified 82 patients with HER2 + invasive BC into LH (n = 45, 54.9%) and NLH groups (n = 37, 45.1%), and AR + was observed in 43 patients (52.4%) (LH: 23, 51.1%; NLH: 20, 54.1%; p = 0.79). Quasi-pathological complete response was observed in 40 patients (48.8%) (LH: 18, 40%; NLH: 22, 59.5%; p = 0.08) overall, and in 31 AR + patients (72.1%) (LH: 15, 34.9%; NLH: 16, 37.2%), significantly higher than in the AR - group for both subgroups (p < 0.001). Regarding prognosis, disease-free survival was relatively better in the AR + group in all HER2 + BCs (p = 0.085), and overall survival was significantly better in the AR + group for NLH (p = 0.029). CONCLUSIONS: High AR expression may be a useful predictor of therapeutic effects and prognosis in both subgroups of HER2 + BCs.

Significance of glucocorticoid signaling in triple-negative breast cancer patients: a newly revealed interaction with androgen signaling.

PURPOSE: Chemotherapy is the only current effective systemic treatment for triple-negative breast cancer (TNBC) patients. Therefore, the identification of active biological pathways that could become therapeutic targets is crucial. In this study, considering the well-reported biological roles of glucocorticoid and androgen receptors (GR, AR) in TNBC, we attempted to explore the effects of glucocorticoids (GCs) on cell kinetics as well as the potential interaction between GR and AR in TNBC. METHODS: We first explored the association between the status of GR, AR, and/or GCs-metabolizing enzymes such as 11ß-hydroxysteroid dehydrogenase (11ßHSD) 1 and 2 and the clinicopathological variables of the TNBC patients. Thereafter, we also studied the effects of dexamethasone (DEX) with/without dihydrotestosterone (DHT) on TNBC cell lines by assessing the cell proliferation, migration and GC response genes at the transcriptional level. RESULTS: GR positivity in carcinoma cells was significantly associated with adverse clinical outcome of the patients and AR positivity was significantly associated with lower histological grade and Ki-67 labeling index of the cases examined. In particular, AR positivity was significantly associated with decreased risks of developing recurrence in GR-positive TNBC patients. The subsequent in vitro studies revealed that DEX-promoted cell migration was inhibited by the co-treatment with DHT in GR/AR double-positive HCC38 cells. In addition, DHT inhibited the DEX-increased serum and glucocorticoid-regulated kinase-1 (SGK1) mRNA expression. CONCLUSION: This is the first study to reveal that the interaction of GR and AR did influence the clinical outcome of TNBC patients and GCs induced cell migration in TNBC cells.

Diverse histomorphology of HER2-positive breast carcinomas based on differential ER expression.

AIMS: HER2-positive (HER2+) breast carcinoma (BC) cases are often treated similarly; however, they can be classified as either luminal B (LH) or non-luminal type (NLH) BC, which have different prognoses. In this study, we investigated the clinicohistomorphological features of each HER2+ BC subgroup. METHODS AND RESULTS: We classified 166 patients with HER2+ invasive BC into LH (n = 110, 66.3%) and NLH groups (n = 56, 33.7%). We further subclassified LH into patients with carcinomas expressing high levels of hormone receptors [LH-high; Allred score, oestrogen receptor (ER) and/or progesterone receptor (PgR) 4-8, n = 89, 53.6%] or low levels (LH-low; Allred score, ER and/or PgR 2 or 3, n = 21, 12.7%) for clinicohistomorphological characterisation. Morphological review showed that NLH included a percentage of patients with comedo necrosis, while LH patients had significantly more central scarring. In terms of immune responsiveness, NLH showed significantly higher rates of tumour-infiltrating lymphocytes and healing. The LH-high and NLH groups showed distinct characteristics (by both models, P < 0.05) and the LH-low group appeared to demonstrate intermediate characteristics according to multinomial analyses using covariates reflecting tumour morphology and immune response outcomes. CONCLUSIONS: These results support the classification of HER2+ BC into two major subgroups, LH-high and NLH, based on tumour morphology and immune response; LH-high proliferates via scirrhous and/or spiculated growth with a central scar, while the primary proliferation pattern of NLH is based on in-situ carcinomas containing comedo necrosis with noticeable TILs and healing.

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multiplex PCR analysis of apocrine lesions shows frequent PI3K-AKT pathway mutations in both benign and malignant apocrine breast tumors.

Pathological diagnosis of intraductal apocrine lesions can be challenging, because even benign apocrine lesions often show atypical cytology, and immunohistochemistry is of little assistance. A new diagnostic method for apocrine lesions is desirable. The mutations present in apocrine lesions have not been well studied. We performed a MassARRAY multiplex polymerase chain reaction (PCR) study of benign and malignant apocrine lesions, which included 152 mutations of 18 genes. We found that four of 11 benign lesions showed AKT1 or PIK3CA mutations, one of four noninvasive apocrine carcinomas showed a FBX4 mutation, two of 15 invasive apocrine carcinomas showed a PIK3CA mutation, and one invasive apocrine carcinoma showed both PIK3CA and TP53 mutations. The mutation frequency did not differ significantly between benign and malignant lesions (p = 0.683). We demonstrated that both benign and malignant apocrine lesions may contain mutations of genes in the PI3K-AKT pathway, this pathway could be a good therapeutic target of these diseases.

CD1d- and PJA2-related immune microenvironment differs between invasive breast carcinomas with and without a micropapillary feature.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology and frequent nodal metastasis. However, the mechanism for development of this unique subtype has not been clearly elucidated. The aim of this study was to obtain a better understanding of IMPC. METHODS: Using representative cases of mixed IMPC, mRNA expression in the micropapillary area and usual invasive area was compared. Then, immunohistochemical analyses for 294 cases (76 invasive carcinomas with a micropapillary feature [ICMF] and 218 invasive carcinomas without a micropapillary feature [ICNMF]) were conducted. Clinicopathological analyses were also studied. RESULTS: DNA microarray analyses for mixed IMPC showed that BC-1514 (C21orf118) was commonly upregulated in the micropapillary area. CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP were commonly downregulated in the micropapillary area. Immunohistochemically, we confirmed that BC-1514 was more upregulated in ICMF than in ICNMF. CD1d and PJA2 were more downregulated in ICMF than ICNMF. All patients with cases of PJA2 overexpression survived without cancer recurrence during the follow-up period, although the differences for disease-free (p = 0.153) or overall survival (p = 0.272) were not significant. CONCLUSIONS: The CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary features is warranted.

Comparison of Claudin 18.2 expression in primary tumors and lymph node metastases in Japanese patients with gastric adenocarcinoma.

BACKGROUND: The monoclonal antibody zolbetuximab (formerly IMAB362), which is being developed as a potential treatment for gastric cancer (GC), targets Claudin 18.2 (CLDN18.2), a GC biomarker. This study aimed to determine the prevalence of CLDN18.2 in primary tumors and lymph node (LN) metastases of Japanese patients with GC. METHODS: CLDN18.2 expression was investigated in tissue samples from patients with gastric adenocarcinoma archived at Kurume University Medical Center, Japan, between 2000 and 2012. Expression of CLDN18.2 in tumor samples was evaluated by immunohistochemistry using the same detection antibody (43-14A) and assay used in the FAST clinical trial (NCT01630083), a phase 2 randomized trial that compared the safety and antitumor activity of the zolbetuximab-chemotherapy combination with chemotherapy alone. Samples showing any specific staining with ≥1+ intensity were defined as CLDN18.2-positive. RESULTS: Of 263 samples analyzed (134 primary gastric tumors and corresponding LN metastases; 128 primary tumors only; one LN metastases only), CLDN18.2 was detected in 87% (n = 228/262) of all primary tumors and 80% (n = 108/135) of LN metastases. Moderate-to-strong CLDN18.2 expression (≥2+ membrane staining intensity in ≥40% of tumor cells [FAST eligibility criterion]) was observed in 52% (n = 135/262) of primary tumors and 45% (n = 61/135) of (LN) metastases. CLDN18.2 expression was significantly higher in GCs of the diffuse histological subtype per Lauren classification and in high grade (G3) tumors. CONCLUSIONS: The high prevalence of CLDN18.2 among Japanese patients with GC supports the therapeutic assessment of zolbetuximab in this population.

Insulinoma-associated protein 1 (INSM1) is a useful marker for pancreatic neuroendocrine tumor.

Insulinoma-associated protein 1 (INSM1) is an important biomarker of Achaete-scute homolog-like 1-driven pathways. For diagnosis of pancreatic neuroendocrine tumors (PanNET), chromogranin A (CGA), synaptophysin (SYP), and neural cell adhesion molecule (NCAM) were also considered as potential biomarkers. However, it is often difficult to diagnose it immunohistochemically. Hence, we examined the expression pattern of INSM1 in pancreatic solid tumors. We detected INSM1, CGA, SYP, and NCAM immunohistochemically, in 27 cases of NET [pure type: 25 cases, mixed adenoneuroendocrine carcinoma (MANEC): 2 cases]. We included 5 cases of solid-pseudopapillary neoplasm (SPN), 7 cases of acinar cell carcinoma (ACC), and 15 cases of pancreatic ductal adenocarcinoma (PDAC) as the control group. Nuclear expression of INSM1 was found in all PanNET pure type cases. However, expression of INSM1 was negative in PDAC, ACC, and SPN in all cases, whereas faint expression was seen in the cytoplasm from SPN. MANEC comprises of two components: neuroendocrine carcinoma and adenocarcinoma components. The NET component was positive for INSM1 expression, whereas the PDAC component does not express INSM1, which aids in distinguishing these components. Our results suggest that INSM1 is a useful immunohistochemical marker for diagnosing pancreatic neuroendocrine tumor.

Feasibility study of personalized peptide vaccination for hepatocellular carcinoma patients refractory to locoregional therapies.

Overall survival of patients with hepatocellular carcinoma (HCC) refractory to locoregional therapy is dismal, even following treatment with sorafenib, a multikinase inhibitor. To develop a more efficacious treatment, we undertook a feasibility study of personalized peptide vaccination (PPV) for HCC, in which the peptides were selected from 31 peptide candidates based on the pre-existing immunity. Twenty-six HCC patients refractory to locoregional therapies (cohort 1) and 30 patients refractory to both locoregional and systemic therapies (cohort 2) were entered into the study. There were no severe adverse events related to PPV except for one injection site reaction. At the end of the first cycle of six vaccinations, successful CTL or IgG boosting was observed in 57% or 46% of patients in cohort 1 and in 54% or 52% of patients in cohort 2, respectively. Successful IgG boosting at the end of the second cycle was observed in the majority of patients tested. Median overall survival was 18.7 months (95% confidence interval, 12.2-22.5 months) in cohort 1, and 8.5 months (95% confidence interval, 5.9-12.2 months) in cohort 2. Based on the higher rates of immune boosting and the safety profile of PPV, further clinical studies of PPV would be warranted for patients with HCC refractory to not only locoregional therapy but also both locoregional and systemic therapies. The protocol of this study was registered with the UMIN Clinical Trials Registry (UMIN000001882 and UMIN000003590).

Phase I study of a new cancer vaccine of ten mixed peptides for advanced cancer patients.

A phase I study of a new cancer vaccine (KRM-10), consisting of a mixture of 10 different short peptides, was conducted for patients with advanced gastrointestinal cancers. Primary or secondary endpoints included the dose-limiting toxicity (DLT), or safety and immune responses, respectively. Peptide-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG), together with soluble inflammatory factors, were measured before and after vaccination. Twenty-one patients were vaccinated with KRM-10 at dose levels of 10 (n = 6), 20 (n = 8) or 30 mg (n = 7) of peptides every week for 6 weeks. No DLT were observed in the dose range evaluated. Common treatment-related adverse events were a grade 1 injection site reaction in 15 patients, and fever in three patients (grade 1 in two patients and grade 2 in one patient). CTL activity to at least one peptide at the time of the third and sixth vaccination increased in 2 and 3 of 6 (10 mg), 2 of 8 and 4 of 6 (20 mg), or 2 and 1 of 6 (30 mg) patients, respectively. IgG levels, at the third and sixth vaccination, were also increased in 1 and 1 of 6 (10 mg), 2 of 8 and 4 of 6 (20 mg), or 1 and 3 of 6 (30 mg) patients, respectively. The KRM-10 vaccine consisting of 20 mg of peptides was determined as the optimal dose for a coming phase II trial because of its safety, and also for demonstrating the most potent activity for augmenting the immune response of the three doses tested. This trial was registered at the UMIN Clinical Trials Registry as UMIN000008820.

Management of breast papillary lesions diagnosed in ultrasound-guided vacuum-assisted and core needle biopsies.

AIMS: To assess the outcome of breast papillary lesions diagnosed by ultrasound-guided core needle biopsy (CB) or vacuum-assisted 'mammotome' biopsy (MT), the accuracy of these diagnoses, and whether it is justified not to undertake surgical excision of non-malignant papillary lesions so diagnosed. METHODS AND RESULTS: Among 3219 (MT, 2195; CB, 1024) breast biopsies spanning 5 years, 185 (5.7%) papillary lesions [MT, 162 (88%); CB, 23 (12%)] were identified. Of these, 142 cases (77%; MT/CB, 125/17) were benign, 24 (13%, 23/1) were atypical, and 19 (10%; 14/5) were malignant. Of the 142 benign cases, 114 had imaging follow-up (FU) (FU period 2-81 months); 17 of 114 cases were excised, and four were malignant (3.5%) (FU period 4-57 months). Of the 24 atypical cases (23 had FU), 19 were excised: six were benign (32%) and 13 malignant (68%). The remaining four cases were considered to be non-malignant (FU period 7-54 months). CONCLUSIONS: Benign papillary lesions diagnosed by MT or CB might not require immediate excision, but should receive imaging FU for at least 5 years. Excision should be performed in cases showing changes in imaging features, as the possibilities of carcinoma coexisting with papilloma or carcinoma developing from papilloma cannot be excluded, as illustrated by the 4% upgrade rate at excision in this study.

Lymphocytic infiltrate is associated with favorable biomarkers profile in HER2-overexpressing breast cancers and adverse biomarker profile in ER-positive breast cancers.

The value for lymphocytic infiltration (LI) has been increasingly recognized for tumor assessment. In breast cancer, however, the overall significance of LI remains poorly defined, probably due to its heterogeneity. A large cohort of breast cancer was evaluated for the degree of LI and its association with traditional pathologic factors, biomarker expression, and cancer subtypes. The number of CD8 cytotoxic effector and FoxP3 regulatory T cell (Treg) was evaluated in those cases with high LI. High LI was associated with negative ER and PR but positive HER2 and EGFR expression (p < 0.001 for all). In ER-positive cancers, high LI was associated with poor prognostic features including higher grade, the presence of necrosis, and lymphovascular invasion (LVI) (p = 0.007 for LVI and <0.001 for the others). Conversely, LI correlated with smaller tumor size, a good prognostic feature (p = 0.046) in HER2+ ER-cancers. These observations suggested LI may show opposite prognostic values in different breast cancer subgroups. Interestingly, when the phenotype of LI in these subgroups was evaluated, a strong positive association with intratumoral accumulation of Treg was found in ER-positive cancers (p = 0.003, Rs = 0.319), while the opposite was observed in HER2+ ER-cancers (p < 0.001, Rs = -0.427). Also, in ER-positive cancers, positive associations between peri- and intra-tumoral distribution were found with both CD8 and Tregs (CD8: p < 0.001, Rs = 0.547; Treg: p = 0.001, Rs = 0.460). Nonetheless, in HER2+ ER-cancers, such strong association was found with CD8 (p < 0.001, Rs = 0.766) but not Tregs. The results may implicate a differential intratumoral migration of LI in different subtypes of breast cancer. In summary, the clinical value of LI in breast cancers could be subtype-dependent. In ER-positive cancers, high LI correlated with biologic parameters associated with poor prognosis, whereas in HER2 positive cancers, LI correlated with biologic parameters of favorable prognosis.

Immunohistochemistry in the diagnosis of papillary lesions of the breast.

AIMS: Many immunohistochemical markers have been studied for differentiating papillary lesions. However, their differentiating power has not been evaluated comprehensively. Additionally, assessment of some markers will require the difficult task of identifying different cell types. In the current study, we aimed to devise a simple papillary panel which can aid in diagnosis irrespective of architectural pattern and cell type differentiation. METHODS AND RESULTS: Immunohistochemical analysis of papillary lesions using myoepithelial markers [p63 and smooth muscle actin (SMA)], high molecular weight cytokeratins (HMWCKs: CK5, CK5/6, CK14 and 34ßE12), hormone receptors (ER and PR) and neuroendocrine markers (chromogranin and synaptophysin) was performed. Among them, neuroendocrine markers showed high specificity but low sensitivity. HMWCK specificity was better than that for myoepithelial markers. Homogeneous staining pattern for hormonal receptors rather than their percentage positivity was more effective in identifying malignant lesions. Negative staining for two or more of HMWCKs, namely CK5/6, CK14 and 34ßE12, achieved the best overall specificity and sensitivity of 87.8% and 94.1%, respectively, irrespective of the architecture. Their discriminatory power was validated with an independent cohort of core needle biopsies. CONCLUSIONS: A marker panel with HMWCKs could be used in differentiating papillary lesions irrespective of architectural pattern or cell type differentiation.

Association between human epidermal growth factor receptor 2 status, namely low and zero expression, and prognosis in hormone receptor-positive breast cancer: a single-center retrospective study.

Background: The recently developed anti-human epidermal growth factor receptor 2 (HER2) therapy has substantially improved the prognosis of HER2-positive breast cancer. The DESTINY-Breast04 trial results showed that trastuzumab deruxtecan (T-DXd) significantly prolonged the survival of patients with HER2-low breast cancer, thus presenting a paradigm shift in anti-HER2 therapy. This may facilitate a change in the treatment strategy for HER2-low breast cancer. However, the implication of HER2-low in hormone receptor (HR)-positive breast cancer is unclear. In this retrospective study, we aimed to reveal the association between HER2 status, namely HER2-low and HER2-zero, and prognosis in HR-positive breast cancer. Methods: We collected the data of 247 patients with estrogen receptor (ER)-positive/HER2-negative breast cancer (159 with HER2-low and 88 with HER2-zero breast cancer) who underwent surgery. Patients were divided into HER2-low and HER2-zero groups. Univariate analysis was performed to evaluate the baseline characteristics using the Wilcoxon rank sum test and Fisher's exact test. Survival analysis of the HER2-low and HER2-zero groups was performed using the Kaplan-Meier method. Results: The median observation period was 2,706 days, and the median period until recurrence was 1,380 days; 25 patients (10%) had recurrences. Age (P=0.004) and menopausal status (P=0.04) were significant variables in the univariate analysis of baseline characteristics. In the subgroup analysis of luminal A- and B-like breast cancers, there was a significant difference in overall survival (OS) only in patients with luminal A-like breast cancer, but relapse-free survival (RFS) of the HER2-low luminal B-like cancer subgroups tended to be relatively short. Conclusions: We inferred that the HER2-low and HER2-zero statuses do not affect the RFS and OS of patients with ER-positive breast cancer. The prognostic significance of HER2-low or HER2-zero status in luminal A- and B-like breast cancers might differ, and a new treatment strategy is required for the HER2-low subgroup.

Expression and localization of anti-Müllerian hormone and its receptors in bovine corpus luteum.

Although anti-Müllerian hormone (AMH) is involved in the regulation of granulosa cell function in female animals, its role in tissues other than ovarian follicles remains poorly understood. It has also been suggested that cows with high circulating AMH concentrations have increased fertility; however, the mechanism has not been elucidated. This study was conducted to identify the presence of the AMH-signaling system and its target cells in the bovine corpus luteum formed from an ovulated follicle. Immunoblotting revealed that the proteolytically cleaved C-terminal region in AMH (AMHC), a biologically active peptide, was present in trace amounts in the early corpus luteum and significantly increased during the mid to regressed stages. AMHC and cleaved N-terminal region (AMHN) in AMH generate a noncovalent isoform that improves the activity of AMH signaling. An immunohistochemical analysis revealed that AMHC, AMHN, and type II AMH receptor (AMHR2) were localized to luteal cells during the entire estrous cycle. AMH in the corpus luteum seemed to be newly synthesized since AMH expression was detected. These findings suggest that AMH signaling is involved in the regulation of luteal cell function through an autocrine and post-translational processing mechanism. The level of AMHR2 and mRNA expression of AMHR2 and type I AMH receptors (activin-like kinase 2, 3, and 6) were highest in the mid stage. Thus, AMH signaling in the corpus luteum may also be regulated by changes in the receptor levels. Since the transforming growth factor-beta superfamily, to which AMH belongs, is a multifunctional polypeptide growth factor, further studies are needed to evaluate whether AMH signaling has a role in facilitating or inhibiting luteal cell functions.