Alteration of HER2 Status During Breast Cancer Progression: A Clinicopathological Analysis Focusing on HER2-Low Status.

Recent studies have shown that novel antibody-drug conjugates (ADCs) can improve clinical outcomes in patients with HER2-low breast cancers. This study aimed to investigate alteration of HER2 status during breast cancer progression with an emphasis on HER2-low status. Using 386 paired samples of primary and recurrent breast cancers, HER2 discordance rate between primary and matched recurrent samples, the relationships between HER2 discordance and clinicopathological characteristics and clinical outcomes of the patients were analyzed. HER2 discordance rate between primary breast cancer and first recurrence was 25.9% (κ = 0.586) with mostly zero-to-low (10.6%) or low-to-zero (9.3%) conversion. There was no significant difference in the discordant rates according to type or location of the recurrence. Of 70 cases with a second recurrence, HER2 discordance rate between the primary tumor and the second recurrence was 27.1% (κ = 0.554). HER2 discordance was associated with lower HER2 level, lymphovascular invasion, and progesterone receptor positivity of the primary tumor. In further analyses, HER2-zero-to-low conversion was associated with lymph node metastasis and hormone receptor (HR) positivity, whereas HER2-low-to-zero conversion was associated with HR negativity and triple-negative subtype. In survival analyses, HER2 discordance was associated with decreased overall survival of patients in the HR-positive group but not in the HR-negative group. Furthermore, patients with HER2-low-to-zero converted tumors showed worse overall survival compared with those with HER2-low concordant tumors. In conclusion, HER2 status changes during breast cancer progression in significant proportions, mostly between zero and low status. As HER2 instability increases during progression and affects clinical outcome, HER2 status needs to be reevaluated in recurrent settings.

Alteration of PD-L1 (SP142) status after neoadjuvant chemotherapy and its clinical significance in triple-negative breast cancer.

PURPOSE: The tumor immune microenvironment can change after neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC). We aimed to investigate the effects of NAC on PD-L1 (SP142) status and its clinical significance in TNBC. METHODS: Paired samples of biopsy and resection specimens were collected from 182 patients with TNBC before and after NAC. PD-L1 (SP142) expression in immune cells in pre- and post-NAC breast cancer samples and the changes between them were analyzed, along with their relationships with the clinicopathological features and clinical outcomes of the patients. RESULTS: Of the 182 patients, 61 (33.5%) achieved pathologic complete response (pCR) after NAC. PD-L1 (SP142) positivity, defined as immune cell staining in ≥ 1% of tumor area, was a predictor for pCR. PD-L1-positive immune cells significantly increased after NAC (2.8% to 5.2% on average) in 109 patients with measurable residual disease. Alteration of PD-L1 status was observed in 24 (22.0%) of the 109 patients with measurable residual tumors after NAC, and all PD-L1 status-converted patients, except one, revealed negative-to-positive conversion. Regarding chemotherapeutic agents, the use of platinum agents was associated with a significant increase in PD-L1-positive immune cells after NAC. In survival analyses, a positive PD-L1 status after NAC and increase of PD-L1-positive immune cells after NAC were associated with better recurrence-free survival of the patients. CONCLUSION: PD-L1 (SP142) status changes after NAC, mostly as a positive conversion. As PD-L1 (SP142) status can convey prognostic and predictive information, it needs to be tested before and after NAC.

Prognostic significance of S100A8-positive immune cells in relation to other immune cell infiltration in pre-invasive and invasive breast cancers.

Myeloid-derived suppressor cells (MDSCs) play an important role in tumor progression through both immunologic and non-immunologic mechanisms. This study was conducted to evaluate the expression of S100A8, a well-known MDSC marker, and the significance of its expression in pre-invasive and invasive breast cancers. S100A8 expression in tumor cells (TCs) and immune cells (ICs) was assessed by immunohistochemistry, and its association with clinicopathologic features and infiltration of other IC subsets including CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs) and PD-L1+ ICs was evaluated. S100A8 expression in TCs and ICs showed a positive correlation in pre-invasive carcinoma and invasive carcinoma. S100A8+ ICs, but not S100A8+ TCs, were significantly higher in number in invasive carcinoma than in pre-invasive carcinoma. Infiltration of S100A8+ ICs was revealed as a poor prognostic indicator in pre-invasive and invasive carcinomas, especially in hormone receptor-positive subgroup. Infiltration of CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ ICs was significantly higher in S100A8+ IC (+) group than in S100A8+ IC (-) group. Combined analyses of IC subset infiltration revealed that infiltration of S100A8+ ICs was associated with poor clinical outcome in the PD-L1+ IC (-), CD8+ TIL-low, and FOXP3+ TIL-low subgroups. In conclusion, S100A8+ ICs seem to undergo a dynamic change during breast cancer progression in association with other IC subset infiltration. The prognostic impact of S100A8+ IC infiltration was greater in less immunogenic tumors.

Immune microenvironment in ductal carcinoma in situ: a comparison with invasive carcinoma of the breast.

BACKGROUND: The immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. This study was conducted to evaluate the immune microenvironment of DCIS including the composition of tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells and to compare it with that of invasive breast cancer. MATERIALS AND METHODS: A total of 671 cases including three different disease groups of pure DCIS, DCIS with microinvasion (DCIS-M), and invasive carcinoma were included in this study. CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ immune cells were detected with immunohistochemistry using tissue microarrays and were analyzed in relation to clinicopathologic characteristics and different disease groups. RESULTS: In pure DCIS, high infiltrations of CD4+, CD8+, and FOXP3+ T cells and the presence of PD-L1+ immune cells were associated with high nuclear grade, comedo-type necrosis, hormone receptor (HR) negativity, and high Ki-67 proliferation index. All immune cell infiltrations were higher in invasive carcinoma than in pure DCIS regardless of the HR status. While CD4+ T cells were more abundant than CD8+ T cells in pure DCIS, CD8+ T cells were dominant in invasive carcinoma, especially in HR-negative tumors. Within individual cases of invasive carcinoma with DCIS component, all immune cell subset infiltration was higher in the invasive component than in the DCIS component; however, CD4+ TIL infiltration did not differ between the two components in HR-negative tumors. Comparing pure DCIS, DCIS-M, and DCIS associated with invasive carcinoma (DCIS-INV), CD4+ TIL infiltration revealed a gradual increase from pure DCIS to DCIS-M and DCIS-INV in the HR-negative group, whereas FOXP3+ TIL infiltration was significantly increased in DCIS-INV than in pure DCIS in the HR-positive group. The high infiltration of FOXP3+ TIL and the presence of PD-L1+ immune cells were associated with tumor recurrence in patients with pure DCIS. CONCLUSIONS: Our study showed that the immune microenvironment differs significantly not only between DCIS and invasive carcinoma but also between pure DCIS, DCIS-M, and DCIS-INV depending on the HR status.

PD-L1 (SP142) Expression in Primary and Recurrent/Metastatic Triple-Negative Breast Cancers and Its Clinicopathological Significance.

PURPOSE: The programmed death-ligand 1 (PD-L1) SP142 assay identifies patients with triple-negative breast cancer (TNBC) who are most likely to respond to the anti-PD-L1 agent atezolizumab. We aimed to compare PD-L1 (SP142) expression between primary and recurrent/metastatic TNBCs and elucidate the clinicopathological features associated with its expression. MATERIALS AND METHODS: Primary and recurrent/metastatic TNBCs tested with PD-L1 (SP142) were collected, and clinicopathological information of these cases was obtained through a review of slides and medical records. RESULTS: PD-L1 (SP142) positivity was observed in 50.9% (144/283) of primary tumors and 37.8% (31/82) of recurrent/metastatic TNBCs with a significant difference. Recurrent or metastatic sites were associated with PD-L1 positivity, with high PD-L1 positivity in the lung, breast, and soft tissues, and low positivity in the bone, skin, liver, and brain. When comparing PD-L1 expression between primary and matched recurrent/metastatic TNBCs using 55 paired samples, 20 cases (36.4%) showed discordance; 10 cases revealed positive conversion, and another 10 cases revealed negative conversion during metastatic progression. In primary TNBCs, PD-L1 expression was associated with a higher histologic grade, lower T category, pushing border, and higher tumor-infiltrating lymphocyte infiltration. In survival analyses, PD-L1 positivity, especially high positivity, was found to be associated with favorable prognosis of patients. CONCLUSION: PD-L1 (SP142) expression was lower in recurrent/metastatic TNBCs, and substantial cases showed discordance in its expression between primary and recurrent/metastatic sites, suggesting that multiple sites may need to be tested for PD-L1 (SP142) when considering atezolizumab therapy. PD-L1 (SP142)-positive TNBCs seems to be associated with favorable clinical outcomes.

miR-145, miR-205 and miR-451: potential tumor suppressors involved in the progression of in situ to invasive carcinoma of the breast.

BACKGROUND: MicroRNAs (miRNAs) control diverse biologic processes during tumor progression. This study was conducted to identify miRNAs that are implicated in progression of in situ to invasive breast cancer (IBC) and to evaluate their association with clinicopathological features of ductal carcinoma in situ (DCIS). METHODS: We performed miRNA microarray analyses to find differentially expressed miRNAs between DCIS and IBC in a test set, and validated expression levels of selected miRNAs using a different set of tumors. Finally, we evaluated the relationship between clinicopathological features and the expression of selected miRNAs in DCIS samples. RESULTS: We found that miR-145-5p, miR-205-5p and miR-451a are significantly down-regulated in IBC compared to DCIS in the whole group, and in the estrogen receptor (ER)-positive and ER-negative subgroups. In a validation set, miR-145, miR-205, and miR-451 also showed lower expression levels in IBC than in DCIS, irrespective of ER status. Moreover, their expression levels were significantly lower in the invasive component compared to the in situ component within same tumors. MiR-145, miR-205 and miR-451 commonly showed lower expression levels in DCIS with positive HER2 status and high Ki-67 proliferation index. Especially, miR-145 and miR-205 showed lower expression levels in DCIS with microinvasion, compared to pure DCIS. In addition, lower miR-205 expression level was associated with high nuclear grade, comedo type necrosis, and hormone receptor negativity. CONCLUSIONS: Our study showed that miR-145, miR-205 and miR-451 expression decreased in IBC compared to DCIS, and their expression levels were low in DCIS with high-risk features for progression, implying their contributions in the progression of DCIS to invasive carcinoma as tumor suppressors.

Expression of HLA class I is associated with immune cell infiltration and patient outcome in breast cancer.

Downregulation of human leukocyte antigen (HLA) class I is one mechanism of escaping anti-tumor immunity by tumor cells. This study was conducted to compare HLA class I expression in ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC) and to evaluate its association with immune cell infiltration of the tumors and clinical outcome of the patients. A total of 830 cases comprising 288 DCIS and 542 IBC were included in this study. Immunohistochemistry for HLA class I expression was performed using HLA-ABC in tissue microarrays and was analyzed in relation to clinicopathologic characteristics of tumors and infiltration of CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells. As a whole, there was no difference in HLA class I expression between DCIS and IBC when dichotomized into high or low expression. However, in the HR-negative group, a high level of HLA class I expression was more frequent in IBC than DCIS. On the contrary, in the HR-positive group, a complete loss of HLA class I expression was more frequently observed in IBC than DCIS. High HLA class I expression level was generally associated with aggressive clinicopathologic features of IBC and was associated with high CD4+, CD8+, and FOXP3+ TIL and PD-L1+ immune cell infiltration in both DCIS and IBC. In survival analyses, HLA class I expression was not associated with clinical outcome in DCIS and IBC as a whole; however, low HLA class I expression was associated with poor clinical outcome in HR-negative IBC, especially in triple-negative subtype. In conclusion, this study showed that HLA class I expression increased in association with increased immune cell infiltration during in situ to invasive transition of HR-negative breast cancer, and HLA class I down-regulation had a prognostic value in HR-negative breast cancer.

Clinical Application of Next-Generation Sequencing in Patients With Breast Cancer: Real-World Data.

PURPOSE: Next-generation sequencing (NGS)-based tumor panel testing has been reimbursed by the Korean government since 2017. We evaluated the use of NGS-based tumor panel testing in real-world clinical practice, focusing on molecular profiling (MP)-guided breast cancer treatment. METHODS: A total of 137 breast cancer patients underwent NGS panel testing between December 2017 and July 2020 at Seoul National University Bundang Hospital (SNUBH). Samples from patients were profiled using an in-house SNUBH pan-cancer panel. Sixty-four patients were profiled on SNUBH Pan_Cancer v1.0, targeting 89 genes, while 73 patients were profiled on SNUBH Pan_Cancer v2.0, targeting 546 genes. RESULTS: Breast cancer subtypes included hormone receptor+/human epidermal growth factor receptor 2 (HER2)- (n = 87), triple-negative (n = 44), and HER2+ (n = 6). Most patients had locally advanced or metastatic cancers (92%). Approximately 92% (126/137) of the patients had significant genomic alterations (tiers I and II), and 62% (85/137) had targetable genomic alterations. The most common targetable genomic alterations were PIK3CA (39%) and ESR1 mutations (9%), followed by ERBB2 (7%), PTEN (7%), BRCA2 (6%), and BRCA1 mutations (4%). Of the 81 patients with locally advanced/metastatic breast cancer with targetable genomic alterations, 6 (7.4%) received MP-guided treatments, including PARP inhibitor (n = 4), ERBB2-directed therapy (n = 1), and PI3K inhibitor (n = 1). Among these 6 patients, 4 participated in clinical trials, 1 underwent treatment at their own expense, and 1 received drugs through an expanded access program. The remaining 66 patients (81%) with targetable genomic alteration did not receive MP-guided treatment due to lack of matched drugs and/or clinical trials, poor performance status, and/or financial burden. CONCLUSION: NGS panel testing allowed MP-guided treatment in only 4.7% (6/127) of patients with advanced breast cancer in a real-world setting. The availability of matched drugs is critical for the realistic implementation of personalized treatment.

Role of CXCL10 in the progression of in situ to invasive carcinoma of the breast.

Tumor immune microenvironment plays a crucial role in tumor progression. We performed immune profiling to compare immune-related gene expression between ductal carcinoma in situ (DCIS) and invasive carcinoma of the breast using nCounter PanCancer immune Profiling Panel and found that CXCL10 was the most significant gene that had the highest difference in expression between them. Effect of CXCL10 on breast cancer cell proliferation and invasion was examined in vitro, and expression of CXCL10 and its relationship with immune cell infiltration was assessed in breast cancer samples. CXCL10 induced cell proliferation, migration and epithelial-mesenchymal transition in MCF-7 and MDA-MB-231 breast cancer cell lines. We confirmed that CXCL10 mRNA expression was significantly higher in invasive carcinoma than in DCIS, especially in hormone receptor (HR)-negative tumors using a validation set. CXCL10 mRNA expression showed a positive correlation with tumor infiltrating lymphocyte (TIL) density in both DCIS and invasive carcinoma; CXCL10-positive tumors generally showed higher infiltration of CD8+ and FOXP3+TILs as well as PD-L1+ immune cells compared to CXCL10-negative tumors, albeit with different patterns according to HR status. In conclusion, our study showed that CXCL10 promotes tumor cell proliferation, invasion, and immune cell infiltration, implying its contribution in the progression of DCIS to invasive carcinoma of the breast.

Programmed Death Ligand 1 Immunohistochemistry in Triple-Negative Breast Cancer: Evaluation of Inter-Pathologist Concordance and Inter-Assay Variability.

PURPOSE: The programmed death ligand 1 (PD-L1) SP142 assay with a 1% immune cell (IC) cutoff is approved for the selection of advanced triple-negative breast cancer (TNBC) patients for atezolizumab treatment. We aimed to evaluate the interobserver concordance of PD-L1 scoring and inter-assay variability of various PD-L1 assays in TNBC. METHODS: Thirty patients with primary TNBC were selected, and SP142, SP263, 22C3, and E1L3N assays were performed. PD-L1 staining in ICs and tumor cells (TCs) was scored by 10 pathologists who were blinded to the assay. The interobserver concordance among pathologists and the inter-assay variability of the four PD-L1 assays were analyzed. For SP142, the intraobserver concordance among the six pathologists was analyzed after training. RESULTS: The adjusted means of PD-L1 IC scoring ranged from 6.2% to 12.9% for the four assays; the intraclass correlations showed moderate (0.584-0.649) reader concordance. The PD-L1 IC scoring with a 1% cutoff resulted in identical scoring in 40.0%-66.7% of cases and a poor to moderate agreement (Fleiss κ statistic [FKS] = 0.345-0.534) for the four assays. The SP142 assay had the widest range of positive rate (56.5%-100.0%), lowest number of cases with identical scoring, and lowest FKS at 1% cutoff. Pairwise comparison of adjusted means showed significantly decreased PD-L1 staining in SP142 compared with the other assays in both ICs and TCs. As for the intraobserver concordance in the SP142 assay, the overall percent agreement was 87.8% with a 1% IC cutoff. After training, the proportion of cases with identical scoring at a 1% IC cutoff increased to 70.0%; the FKS also increased to 0.610. CONCLUSION: The concordance of PD-L1 IC scoring among pathologists was low, at the 1% cutoff for the SP142 assay without training. SP142 showed the lowest PD-L1 expression in both IC and TC.

Prognostic implications of regression of metastatic axillary lymph nodes after neoadjuvant chemotherapy in patients with breast cancer.

Prognostic implications of therapeutic response of metastatic lymph nodes (LNs) to neoadjuvant chemotherapy (NAC) remain unclear in patients with breast cancer. We aimed to evaluate the prognostic value of axillary LN regression after NAC in locally-advanced breast cancer patients. Therapeutic response of the LNs was evaluated in 563 breast cancer patients and classified into four grades according to the regression pattern. Initial pathologic N stage was estimated from the sum of the metastatic LNs and those with complete regression. In survival analyses, LN regression grade, pathologic N stage after NAC, and presumed initial pathologic N stage stratified clinical outcome of the patients in the whole group, in both ER-positive and ER-negative subgroups, and in those with residual breast disease. On multivariate analysis, LN regression grade and presumed initial pathologic N stage were revealed as independent prognostic factors. The number of completely-responsive LNs and the ratio of non-responsive LNs also revealed a prognostic value. In conclusion, regression grade of axillary LNs and presumed initial pathologic N stage have prognostic values in breast cancer patients who receive NAC. Thus, regression of axillary LNs should be evaluated and included in pathologic reporting of post-NAC resection specimens.

HER2 status in breast cancer: changes in guidelines and complicating factors for interpretation.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and/or HER2 gene amplification is found in about 20% of invasive breast cancers. It is a sole predictive marker for treatment benefits from HER2 targeted therapy and thus, HER2 testing is a routine practice for newly diagnosed breast cancer in pathology. Currently, HER2 immunohistochemistry (IHC) is used for a screening test, and in situ hybridization is used as a confirmation test for HER2 IHC equivocal cases. Since the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines on HER2 testing was first released in 2007, it has been updated to provide clear instructions for HER2 testing and accurate determination of HER2 status in breast cancer. During HER2 interpretation, some pitfalls such as intratumoral HER2 heterogeneity and increase in chromosome enumeration probe 17 signals may lead to inaccurate assessment of HER2 status. Moreover, HER2 status can be altered after neoadjuvant chemotherapy or during metastatic progression, due to biologic or methodologic issues. This review addresses recent updates of ASCO/CAP guidelines and factors complicating in the interpretation of HER2 status in breast cancers.

The updated 2018 American Society of Clinical Oncology/College of American Pathologists guideline on human epidermal growth factor receptor 2 interpretation in breast cancer: comparison with previous guidelines and clinical significance of the proposed in situ hybridization groups.

The aim of the study was to evaluate the impact of the updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline on human epidermal growth factor receptor 2 (HER2) interpretation in breast cancer compared with that of the previous guidelines and also the significance of in situ hybridization (ISH) groups proposed by the updated guideline. HER2 ISH reports and immunohistochemistry (IHC) data from 1,348 invasive breast cancers diagnosed at a single institution were included in this study. HER2 IHC was reassessed using the 2018 guideline, and HER2 ISH status was determined by the 2007, 2013, and 2018 guidelines. When applying the updated guideline, most of the HER2 ISH-equivocal cases as per the previous guidelines were reclassified as ISH negative, and 0.8% of HER2 ISH-positive tumors as per the 2007 guideline and 2.5% of those as per the 2013 guideline were changed to ISH negative. Accordingly, the negative HER2 ISH results significantly increased in the 2018 guideline compared with the 2013 guideline. HER2 ISH-positive tumors in ISH group 3 (HER2/chromosome enumeration probe 17 [CEP17] ratio <2.0 and average HER2 copy number ≥6.0 per cell) were characterized by equivocal HER2 protein expression, CEP17 copy number gain, and low HER2 copy numbers compared with classic HER2 ISH-positive tumors in ISH group 1 (HER2/CEP17 ratio ≥2.0 and average HER2 copy number ≥4.0 per cell). HER2 ISH-negative tumors in ISH group 4 (HER2/CEP17 ratio <2.0 with average HER2 copy number ≥4.0 and < 6.0 per cell) revealed more aggressive clinicopathologic features and poorer clinical outcomes than those in ISH group 5 (HER2/CEP17 ratio <2.0 and average HER2 copy number <4.0 per cell), especially in the hormone receptor-positive subgroup. In conclusion, implementation of the updated 2018 ASCO/CAP guideline leads to a significant increase in HER2 ISH-negative results compared with the 2013 guideline, mainly via reclassification of the ISH-equivocal cases to ISH-negative ones. ISH groups proposed by the updated guideline provide additional information on the clinicopathologic characteristics of the tumors.

Changes and prognostic values of tumor-infiltrating lymphocyte subsets after primary systemic therapy in breast cancer.

Tumor-infiltrating lymphocyte (TIL) levels have prognostic and predictive values in treatment-naïve breast cancers. However, there have been controversies regarding TIL subset changes and their clinical implications in post-treatment breast cancers. This study aimed to explore change and prognostic significance of TIL subset infiltration after primary systemic therapy (PST) in breast cancer. One-hundred-fifty-five patients who had residual disease after anthracycline- or anthracycline plus taxane-based PST were included. The quantities of intratumoral and stromal TIL subsets (CD8+, CD4+, and FOXP3+ TILs) in pre- and post-PST breast cancer samples, as well as changes between them, were analyzed along with their correlations with clinicopathologic features and outcome of patients. As a whole, intratumoral CD8+ and CD4+ TILs increased after PST while stromal TILs decreased. Both intratumoral and stromal FOXP3+ TILs decreased after PST. The chemo-sensitive group [residual cancer burden (RCB) class I and II] showed the same pattern of change in intratumoral CD8+ TILs as the whole group, whereas the chemo-resistant group (RCB class III) showed no significant change in intratumoral CD8+ TIL infiltration after PST. Survival analyses for each TIL subset as well as their ratios revealed that high levels of intratumoral, stromal, and total CD8+ TIL infiltration after PST were independent predictors of longer patient survival. In subgroup analyses, CD8+ TIL infiltration after PST revealed prognostic significance in the chemo-resistant group but not in the chemo-sensitive group. In conclusion, infiltration of CD8+, CD4+, and FOXP3+ TIL changed after PST in the intratumoral and stromal compartments. Especially, increase of intratumoral CD8+ TILs was associated with chemo-responsiveness. Moreover, CD8+ TIL status in residual tumors after PST may be used as a potential prognostic marker in breast cancer patients who receive PST and provide additional prognostic information to chemo-resistant group.

Identification and expression pattern of a new digestive invertebrate-type lysozyme from the earthworm.

BACKGROUND: The invertebrate type (i-type) lysozyme not showing a clear homology with the known types of lysozyme was first demonstrated from a marine bivalve, conch and earthworm by N-terminal sequence. An i-type lysozyme isolated from the earthworm found to be up-regulated upon bacterial challenge, suggesting this lysozyme to function as an inducible immune factor. However, information on the i-type lysozyme related with digestive function is very limited in the earthworm. OBJECTIVE: The objective of this study is to investigate the molecular characteristics and function of the new i-type lysozyme from the earthworm. METHODS: To identify a new i-type lysozyme, multiple amino acid sequence alignment and phylogenetic analyses were employed. Its mRNA expression pattern was observed by fluorescent in situ hybridization (FISH). RESULTS: A new i-type lysozyme (Ea-iLys) from an earthworm, Eisenia andrei with the open reading frame of 678 bp (226 amino acid residues) appeared to comprise conserved 14 cysteine residues for disulfide bridges and amino acid residues for the enzyme activities of lysozyme and isopeptidase, of which mRNA expression is mainly localized in the lining of midgut epithelium. No significant expression signal was detected in immune competent sites such as chloragogue tissue, typhlosole region, body coelom and muscle layers. CONCLUSION: Our results suggest that this enzyme primarily acts as a digestive enzyme rather than an innate immune factor.

Changes in Biomarker Status in Metastatic Breast Cancer and Their Prognostic Value.

PURPOSE: There is cumulative evidence that changes in biomarker status occur frequently during the metastatic progression of breast cancer and affect treatment response. The purpose of this study was to evaluate the frequency of biomarker changes in metastatic breast cancer (MBC) and its impact on prognosis. METHODS: A total of 152 patients diagnosed with MBC at the time of initial diagnosis or during post-surgical follow-up were included. Changes in biomarker status in MBCs, their frequency according to various metastatic sites, tumor characteristics, and their association with patient survival were analyzed. RESULTS: Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 status changed in 9 (6.0%), 40 (26.3%), 12 (7.9%), and 29 (19.1%) patients, respectively. ER, PR, and HER2 mainly showed positive to negative conversion, whereas Ki-67 changed mostly from a low to high index. There were no differences in the frequencies of biomarker changes according to the metastatic sites. As for ER and HER2, cases with negative conversion showed low expression levels in the primary tumor. Survival analyses indicated that a positive to negative conversion of ER was an independent poor prognostic factor in patients with primary ER-positive breast cancer. CONCLUSION: Changes in biomarker status are not rare, and usually occur in an unfavorable direction in breast cancer metastases. Negative conversion of ER status is a predictor of poor prognosis. Thus, it is beneficial to evaluate changes in biomarker status in MBC not only for the purpose of determining treatment options but also for prognostication of patients.

Preoperative Diagnostic Categories of Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features in Thyroid Core Needle Biopsy and Its Impact on Risk of Malignancy.

This study was designed to evaluate the preoperative diagnostic categories of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) using thyroid core needle biopsy (CNB) and to analyze its impact on the risk of malignancy (ROM). A total of 2687 consecutive thyroid CNBs were reviewed retrospectively and classified into six diagnostic categories using a standardized reporting system similar to the Bethesda System for Reporting Thyroid Cytopathology. Diagnostic categories of CNBs were compared with the final surgical diagnoses, and the ROM in each category was calculated both before and after excluding NIFTP from malignancy. Of 946 surgically resected cases, 683 were diagnosed as papillary thyroid carcinoma (PTC), and 32 (4.7% of PTC) were reclassified as NIFTP. The CNB diagnostic categories of NIFTP were as follows: follicular neoplasm in 20 (62.5%; 14, with nuclear atypia), indeterminate lesion in 11 (34.4%), and suspicious for malignancy in one (3.1%). When combined, NIFTP and encapsulated follicular variant of PTC (EFVPTC) were more often categorized as follicular neoplasm compared with other PTC variants including infiltrative FVPTC. Exclusion of NIFTP from malignant diagnosis led to a significant decrease in the ROM in follicular neoplasm with nuclear atypia category. Thus, thyroid CNB enables to differentiate NIFTP/EFVPTC from other PTCs, providing a useful guide for optimal treatment in patients with these tumors.

The usefulness of a mobile device-based system for patient-reported outcomes in a spine outpatient clinic.

BACKGROUND CONTEXT: Patient-reported outcomes (PROs) are typically collected using a paper form, but this format is cumbersome to incorporate into outpatient clinic visits as well as in research. Therefore, we developed a mobile device-based system (mobile system) for spinal PRO. We hypothesized that this system may improve the quality of care in an outpatient clinic. PURPOSE: This study aimed to analyze the patient-reported efficacy of a mobile system through a survey of patients' responses compared with a paper system. STUDY DESIGN/SETTING: A prospective observational study was carried out. PATIENT SAMPLE: Surveys were conducted for 103 patients who had experience using both the paper and electronic systems in the outpatient clinic. OUTCOME MEASURES: Patient-reported positive response score (PRS) was the outcome measure. METHODS: The survey included the characteristics of the patients (sex, age, use of smartphone, familiarity with smartphone applications, proficiency of typing with mobile device, site of pain, and education level) and eight questions in four domains: (1) efficacy in the waiting room, (2) efficacy during the clinic visit, (3) overall satisfaction, and (4) opinion about the use of this system. The response to each question was scored from 1 to 5 (1, negative; 5, positive response). The patient-reported PRS was calculated by adding the scores of the 8 questions and converting the total range to 0-100 (60, neutral). RESULTS: The mean PRS of the 8 questions was 79.8 (95% CI, 76.7-83.9). The mean PRS was 78.9 (75.6-82.2) at the waiting room and was 80.5 (77.1-83.9) during the clinic. The PRS for overall satisfaction and use of this system were 83.3 (79.6-87.0) and 77.1 (71.9-82.3), respectively. The use of smartphones and the proficiency of typing were independently significant predictors of PRS with an R(2) value of 0.325. CONCLUSIONS: The mobile device-based system improved the patient-reported efficacy in spine outpatient clinics. However, various factors such as the use of smartphones need to be considered when developing and applying mobile systems.