Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing.

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

[Total Gastrectomy for Gastric Invasion of Multiple Myeloma-A Case Report].

A 65-year-old woman was referred to our hospital for lumbago. The patient was diagnosed with multiple myeloma in 2020. She underwent chemotherapy and radiation therapy, and the disease progression stabilized. In 2022, the patient presented with severe anemia(Hb 4.9 mg/dL), and upper gastrointestinal endoscopy revealed a type 1 tumor in the middle body of the stomach. Computed tomography showed masses in the stomach and pancreas. The patient required a large volume of blood transfusion and underwent total gastrectomy to control the bleeding. Histological examination of the resected specimen indicated infiltration of myeloma cells. The patient died from invasive lesions in other organs, a year after surgery. Usually, extramedullary multiple myeloma lesions occur in the liver, spleen, and lymph nodes. Gastric invasion of multiple myeloma is very rare. Because of poor prognosis, surgery for gastric invasion of multiple myeloma is even rarer. We report a case of gastric invasion of multiple myeloma with a literature review.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures.

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

Factors related to self-care behaviors among hospitalized patients with heart failure in Japan, based on the European Heart Failure Self-Care Behaviour Scale.

[Purpose] The characteristics of heart failure in hospitalized patients with poor self-care behaviors are unknown. We investigated factors associated with self-care behaviors by using the European Heart Failure Self-Care Behaviour Scale (EHFScBS) in heart failure patients based on three comprehensive concepts. [Participants and Methods] This was a cross-sectional single-center study of heart failure patients hospitalized at a tertiary-care hospital. We investigated age, gender, family living together/apart, employment, and the Specific Activity Scale (SAS). A physical therapist provided the EHFScBS one time to determine the patients' pre-hospital self-care behavior status. The 12 items of the EHFScBS were classified into the following three categories: Maintenance, Monitoring, and Management. [Results] The median age of the 39 consecutive patients was 81 years. A multiple regression analysis revealed that the factors exhibiting significant associations were the SAS score (ß=0.504) for Management and age (ß=-0.403) for the total EHFScBS score (adjusted by the number of hospitalizations for heart failure). Maintenance and Monitoring were not significantly associated with the survey items. [Conclusion] These data indicate that self-care education for hospitalized patients with heart failure leads to individualized approaches based on characteristics such as age and physical activity capacity.

Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display.

Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.

Reconstitution of an Anti-HER2 Antibody Paratope by Grafting Dual CDR-Derived Peptides onto a Small Protein Scaffold.

Target-binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design strategy to create small proteins called fluctuation-regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity-determining region (CDR) loops of mAbs (CDR-derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target-binding capability. Here, a strategy to create FLAPs bearing dual CDR-derived peptides (D-FLAPs) using the anti-human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR-derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D-FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D-FLAP with HYTTPP and GDGFYA peptides from CDR-L3 and CDR-H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.

L-type amino acid transporter 1 expression is upregulated and associated with cellular proliferation in colorectal cancer.

Previous studies have shown that the L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancer. Upregulated LAT1 expression is considered to be associated with cancer cell proliferation. In the present study, we investigated LAT1 expression in 210 patients with colorectal cancer (CRC) and 40 patients with colonic adenoma using an immunohistochemical method, and analyzed the associations between LAT1 expression and clinicopathological factors and prognosis. The biological significance of LAT1 was also examined under conditions with sub-normal amounts of essential amino acids using colon cancer cell lines. High expression of LAT1 was observed in 152 of 210 CRC patients (72.4%) and 12 of 40 patients with colonic adenoma (30%), and this difference in the frequency of LAT1 expression between CRC and adenoma was significant (P<0.001). High expression of LAT1 was associated with venous invasion (P=0.027). The restriction of amino acids suppressed cell proliferation in CRC cells with higher LAT1 expression, while cellular proliferation was not suppressed in the cells expressing lower levels of LAT1. Mammalian target of rapamycin (mTOR) expression was also downregulated under restricted availability of amino acids, suggesting that the restriction of amino acids induced an antitumor effect through inhibition of the LAT1/mTOR pathway. In summary, the present study demonstrated that LAT1 expression is frequently upregulated in CRC and is associated with cancer cell proliferation via the mTOR pathway.

[Primary Malignant Melanoma of the Gallbladder].

Primary malignant melanoma of the gallbladder is a rare disease, and 37 cases have been reported in the literature.The current patient was a 78-year-old man who was admitted with a pelvic tumor and left leg edema due to compression of the external iliac vein by the pelvic tumor.The edema improved following resection of the tumor, which was diagnosed at pathology as a malignant melanoma.After surgery, the patient became anorexic and complained of discomfort in the upper right abdomen.A whole body FDG-PET scan demonstrated significant uptake in the gallbladder and in the lymph nodes of the lower abdomen.The patient underwent open cholecystectomy, and the pathological diagnosis was malignant melanoma. Junctional activity was seen in the gallbladder, suggesting that this was the primary site.No melanocytic lesions of the skin or eyes were detected, further supporting the diagnosis of primary malignant melanoma of the gallbladder.Chemotherapy was initiated, but the patient died on February 28, 2016.

UHRF1 expression is upregulated and associated with cellular proliferation in colorectal cancer.

Ubiquitin-like with PHD and ring-finger domain 1 (UHRF1) binds to methylated promoters of a number of tumor-suppressor genes, including p16INK4A and p14ARF, by forming complexes with DNA methyltransferases and HDAC1, resulting in the induction of carcinogenesis. Altered UHRF1 expression has been demonstrated in various types of cancers. Previous reports indicate that UHRF1 expression is regulated by E2F-1 expression. We investigated UHRF1 expression using immunohistochemical staining in 231 colorectal cancer and 40 adenoma specimens, analyzed the relationship between UHRF1 expression and clinicopathological findings and the association between UHRF1 and E2F-1 expression. To better understand the biological function of UHRF1 in colorectal cancer, knockdown of UHRF1 expression was performed using siRNA methods. High UHRF1 expression was observed in 152 of 231 (65.8%) colorectal cancer patients, and was detected in 35 of 40 adenoma specimens samples (87.5%). UHRF1 staining was detected in the nucleus of cancer cells, while it was not detected in colonic normal mucosa. High UHRF1 expression was significantly observed in right compared with left hemicolon cancer (p=0.008). Moreover, high UHRF1 expression tended to be associated with depth of invasion (p=0.051). UHRF1 expression was significantly associated with E2F-1 expression (p<0.0001). Knockdown of UHRF1 expression suppressed cellular growth in colon cancer cell lines, HCT116 and SW620. In conclusion, we demonstrated that UHRF1 expression was upregulated in approximately two-thirds of colorectal cancer specimens and was particularly expressed in right compared with left hemicolon cancer. Moreover, knockdown of UHRF1 expression induced growth inhibition in colon cancer cell lines. UHRF1 may be involved in cellular proliferation and molecular pathogenesis of colorectal cancer in the right hemicolon.

Ectopic expression of MECA-79 as a novel prognostic indicator in gastric cancer.

The aim of this study was to clarify the clinical implications of a unique carbohydrate determinant, MECA-79, in gastric cancer specimens and cells. Immunohistochemical analysis showed that 62 of 225 (27.6%) cases were defined as positive for MECA-79. MECA-79 expression was correlated with depth of invasion, venous invasion, TNM stage, and distant metastasis. In survival analyses, patients with MECA-79 expression had worse prognosis by the log-rank test. Multivariate analysis of the Cox proportional hazard model showed that MECA-79 expression was an independent factor of a worse cancer-specific survival. Among 11 gastric cancer cells, MECA-79 was observed in only MKN7 cells, which also expressed GlcNAc6ST-2 transcript. A knockdown of GlcNAc6ST-2 in MKN7 cells showed a markedly reduced expression of MECA-79, suggesting that GlcNAc-sulfation of MECA-79 is mainly synthesized by GlcNAc6ST-2. Furthermore, real-time RT-PCR analysis revealed that GlcNAc6ST-2 was significantly increased in cancer tissues compared with paired normal mucosa. In conclusion, the expression of MECA-79 could be a useful marker for the prognosis of gastric cancer. Our results might also provide novel perspectives on the biology of MECA-79 and GlcNAc6ST-2 in cancer progression and metastasis.

CD44v6, MMP-7 and nuclear Cdx2 are significant biomarkers for prediction of lymph node metastasis in primary gastric cancer.

The aim of this study was to examine the expression of CD44v6, CD54, Cdx2, CXCL5, Cyclin B1, MMP-7, nm23, RCAS1 and Survivin in primary gastric cancer and to investigate whether these molecules were useful in predicting the lymph node status. They were selected as candidates for indicators of lymph node metastasis from various kinds of cancer-associated genes reported previously. In 135 cases of radically resected primary gastric adenocarcinoma, we investigated the association between the expression of these molecules and clinocopathologic factors by immunohistochemistry. The results revealed that the expression of CD44v6 and MMP-7 were significantly associated with lymph node status. By contrast, nuclear Cdx2 expression was found to be inversely correlated with lymph node metastasis. Moreover, multivariate analysis demonstrated that CD44v6, MMP-7 and nuclear Cdx2 were independent predictors for lymph node status. In conclusion, our results suggest that positive expression of both CD44v6 and MMP-7, and negative expression of nuclear Cdx2 may serve as powerful predictors of lymph node metastasis in gastric cancer. Combined evaluation of these markers could be further useful to predict lymph node status clinically.

[Efficacy and safety of high-dose toremifene for hormone-responsive advanced or metastatic breast cancer patients with failed prior treatment by aromatase inhibitors].

BACKGROUND: Recently, aromatase inhibitors (AI) are widely used in postoperative adjuvant therapy for breast cancer. Nevertheless, studies of postoperative therapeutic strategies for recurrent breast cancer are insufficient. SUBJECTS AND METHOD: Data on 12 post-menopausal advanced/recurrent breast cancer patients in our department during June 2003- April 2007 were used for this study. No patient had responded to high-dose toremifene (TOR), a third-generation AI. Their therapeutic outcomes were analyzed retrospectively. The median observation period of the subjects was 16.1 months (4.0-40.9 months). Subjects were all hormone-sensitive. Overexpression of HER2 protein was found in only one case. During AI therapy immediately prior, exemestane (EXE) and anastrozole (ANA) had been given in nine and three cases, respectively. RESULTS: The complete response rate of AI therapy was 16.7% (2/12). The clinical benefit rate was 58.3% (7/12). The median of time to progression (TTP) was 33.8 weeks. Neither the presence nor absence of past history of treatment with tamoxifen (TAM) or other chemotherapies affected the anti-tumor effect. Analysis by the site of metastasis or recurrence revealed that the therapeutic effects were better for non-life-threatening cases in the lung, pleura, soft tissue, etc. The severities of adverse effects were all less than grade 2; the major ones were flushing and sweating. CONCLUSION: Results show that high-dose TOR given at an early stage can provide clinical benefits for post-menopausal advanced/recurrent breast cancer not responding to AI.

ING2 is upregulated in colon cancer and increases invasion by enhanced MMP13 expression.

Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF-kappaB binding site was found in the ING2 promoter region. We confirmed that NF-kappaB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. ING2 regulation of MMP13 expression was confirmed in both ING2 overexpression and knock down experiments. MMP13 expression was further induced by coexpression of ING2 with HDAC1 or with mSin3A, suggesting that the ING2-HDAC1-mSin3A complex members regulates expression of MMP13. In vitro invasion assay was performed to determine functional significance of ING2 upregulation. ING2 overexpressed cells exhibited greater invasive potential. Taken together, upregulation of ING2 was associated with colon cancer and MMP13-dependent cellular invasion, indicating that ING2 expression might be involved with cancer invasion and metastasis.

[Long-term results of chemoradiation therapy for the patients with locally advanced (T4) esophageal cancer].

The prognosis of patients with advanced esophageal cancer is still poor. Recently, concurrent chemoradiation therapy for esophageal cancer is being utilized with increasing frequency. In this study, we reported concurrent chemoradiation for patients with T4 esophageal cancer. From July 2000, we treated 21 consecutive patients with radiation and concurrent chemotherapy using intermittent low-dose FP chemoradiation (40 Gy radiation, 2 Gy/day, for 4 weeks 280/m(2) 5-FU intermittent 24 continuous, CDDP 8 mg/m(2)/intermittent). All patients who underwent the treatment with concurrent CRT completed the planned chemoradiation. Out of 21 patients, 2 (9.5%) showed a complete response and 9 patients (42.8%) showed a partial response. The 5-year survival rate of the T4 patients with CRT was almost the same as for those who underwent surgery alone. Concurrent chemoradiation therapy for T4 esophageal cancer patients is feasible and seems to be a standard treatment for T4 esophageal cancer patients. The results indicated that CRT is an effective therapy for advanced esophageal cancer.

Locally advanced and metastatic breast cancer with cartilaginous and/or osseous metaplasia showing excellent response to chemotherapy.

Breast cancer with cartilaginous and/or osseous metaplasia is considered a rare disease, but several cases have been reported recently. We report a case of breast cancer with cartilaginous and/or osseous metaplasia that was StageIV,(T4bN0M1b (PUL)), on the basis of the Japanese General Rules for Clinical and Pathological Recording of Breast Cancer, which responded well to chemotherapy. A 58-year-old women visited our hospital with a chief complaint of a palpable breast mass that had increased in size in March 2002. It was 20 x 15 x 14 cm and occupied the entire right breast. Chest computed tomography (CT) demonstrated multiple lung metastases. Histology of the biopsy specimens revealed a spindle-shaped cell carcinoma. It was ER(-), PgR(-), and HER2/neu Score 0. CAF was given to the patient as preoperative chemotherapy. Five cycles of treatment yielded improvement at the primary site and improvement of the metastatic lung lesions, which was judged as a partial response. Subsequently, one cycle of weekly paclitaxel 80 mg/m2 and oral administration of 5'-DFUR 800 mg/day were given. In November 2002, the patient underwent a right simple mastectomy with whole-layer skin grafting from the abdomen. The final pathological diagnosis was a rare type of breast cancer with cartilaginous and/or osseous metaplasia. Preoperative chemotherapy had caused necrosis in most of the tumor cells, and the efficacy was judged as Grade 2. From the third week postoperatively, weekly paclitaxel (80 mg/m2) was given. Six months after the operation, the multiple lung metastases were completely eliminated and new metastasis to liver or bone or local recurrence have not been observed.