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Background: Premenopausal women with early hormone-receptor positive (HR+) breast cancer receive 5–10 years of adjuvant endocrine therapy (ET) during which pregnancy is contraindicated and fertility may wane. The POSITIVE study investigates the impact of temporary ET interruption to allow pregnancy. Methods: POSITIVE enrolled women with stage I-III HR + early breast cancer, ≤42 years, who had received 18–30 months of adjuvant ET and wished to interrupt ET for pregnancy. Treatment interruption for up to 2 years was permitted to allow pregnancy, delivery and breastfeeding, followed by ET resumption to complete the planned duration. Findings: From 12/2014 to 12/2019, 518 women were enrolled at 116 institutions/20 countries/4 continents. At enrolment, the median age was 37 years and 74.9 % were nulliparous. Fertility preservation was used by 51.5 % of women. 93.2 % of patients had stage I/II disease, 66.0 % were node-negative, 54.7 % had breast conserving surgery, 61.9 % had received neo/adjuvant chemotherapy. Tamoxifen alone was the most prescribed ET (41.8 %), followed by tamoxifen + ovarian function suppression (OFS) (35.4 %). A greater proportion of North American women were, SCOPUS: ar.j, info:eu-repo/semantics/published

Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C

Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed in various cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from a variety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, we evaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistry use, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated intense PMab-241 staining within Bowman's capsules and glomeruli of the ferret kidney, and lymphatic endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of PDPN in ferret tissues.

CC chemokine receptor 9 (CCR9) belongs to the beta chemokine receptor family and is mainly distributed on the surface of immature T lymphocytes and enterocytes. This receptor is highly expressed in rheumatoid arthritis, colitis, type 2 diabetes, and various tumors. Therefore, more sensitive monoclonal antibodies (mAbs) need to be developed to predict the prognosis of many high CCR9 expression diseases. Because CCR9 is a structurally unstable G protein-coupled receptor, it has been difficult to develop anti-CCR9 mAbs using the traditional method. This study developed anti-human CCR9 (hCCR9) mAbs for flow cytometry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with hCCR9-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hCCR9), and hybridomas showing strong signals from CHO/hCCR9 and no signals from CHO-K1 cells were selected by flow cytometry. We established an anti-hCCR9 mAb, C

AIM Anxiety and its correlates in parents of patients with breast cancer have rarely been studied. We explored anxiety among parents of postoperative patients with breast cancer and its relationship with parents' social support and care needs and patients' anxiety. METHODS A cross-sectional survey using self-report questionnaires and medical records was conducted among patients with breast cancer after surgery and their parents at four designated cancer care hospitals between September 2015 and June 2016. Anxiety was measured using the Hospital Anxiety and Depression Scale (HADS). Parents provided information about social support and care needs. Multilevel analysis was performed on patient-parent paired data controlling for patient-level variation. RESULTS Participants included 107 patients, 83 mothers, and 51 fathers. The mean HADS anxiety scores reported by mothers and fathers were 7.2 and 6.5, respectively, which were higher than patients' HADS anxiety scores. Fulfillment of important care needs was related to lower anxiety among mothers and fathers (estimate = -1.38, p = .01). Lower family support and higher patient anxiety were associated with higher anxiety in mothers, but not fathers. CONCLUSIONS Parents of patients with breast cancer had high anxiety. Communication, providing cancer-related information, and fulfilling care needs can alleviate anxiety in parents of patients with breast cancer after surgery. Furthermore, increasing family support and decreasing patients' anxiety are essential to alleviating mothers' anxiety.

HER3 belongs to the epidermal growth factor receptor (EGFR) family and is known to form an active heterodimer with other three family members EGFR, HER2, and HER4. HER3 is overexpressed in lung, breast, colon, prostate, and gastric cancers. In the present study, we developed and validated an anti-HER3 monoclonal antibody (mAb), H3Mab-17 (IgG2a, kappa), by immunizing mice with HER3-overexpressed CHO-K1 cells (CHO/HER3). H3Mab-17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The KD for H3Mab-17 in CHO/HER3 and Caco-2 (a colon cancer cell line) were determined to be 3.0×10−9 M and 1.5×10−9 M via flow cytometry, respectively, suggesting high binding affinity of H3Mab-17 to HER3. Then, we assessed the H3Mab-17 antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Caco-2, and evaluated its antitumor capacity in a Caco-2 ×enograft model. In vitro experiments revealed H3Mab-17 had strongly induced both ADCC and CDC against Caco-2 cells. In vivo experiments on Caco-2 ×enografts revealed that H3Mab-17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H3Mab-17 could be a promising treatment option for HER3-expressing colon cancers.

The development of specific antibodies is essential to understand a wide variety of biological phenomena and pathophysiological analyses. Podoplanin (PDPN), a type I transmembrane glycoprotein, is known as a diagnostic marker. Anti-PDPN monoclonal antibodies (mAbs) against many species, such as human, mouse, rat, rabbit, dog, bovine, cat, tiger, horse, pig, goat, alpaca, Tasmanian devil, bear, whale, and sheep, have been established in recent studies. However, sensitive and specific mAbs against elephant PDPN (elePDPN) have not been established. Thus, this study established a novel mAb against African savanna elephant (Loxodonta africana) PDPN using the Cell-Based Immunization and Screening method. elePDPN-overexpressed Chinese hamster ovary-K1 (CHO/elePDPN) cells were immunized, and mAbs were screened against elePDPN using flow cytometry. One of the mAbs, PMab-265 (IgM, κ), specifically detected CHO/elePDPN cells by flow cytometry. These findings suggested the potential usefulness of PMab-265 for the functional analyses of elePDPN.

Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG2b, κ) was developed using a Cell-Based Immunization and Screening (CBIS) method. TrMab-6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab-6 possesses in vitro antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2-overexpressed CHO-K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468. In vitro experiments revealed that TrMab-6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHO-K1 and MCF7/TROP2-knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 ×enografts revealed that TrMab-6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO-K1 and MCF7/TROP2-knockout xenografts. The findings suggest that TrMab-6 is a promising treatment option for TROP2-expressing breast cancers.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs.

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.

The expression of human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed in several cancers, such as breast, lung, gastric, pancreatic, and colorectal cancers, and be associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H2Mab-41 (IgG2b, kappa), and the antitumor activity of H2Mab-41 was investigated using mouse xenograft models. Caco-2 cells (human colon cancer cell line), which expresses HER2, were subcutaneously implanted into the flanks of nude mice. H2Mab-41 and control mouse IgG were injected three times into the peritoneal cavity of mice. H2Mab-41 significantly reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 5, 8, 11, 15, and 19. Taken together, these results suggest that H2Mab-41 is useful for antibody therapy against HER2-expressing colon cancers.

Breast reconstruction is a favorable option for women with BRCA1 or BRCA2 mutations (BRCA1/2Mut+) who undergo risk-reducing mastectomy (RRM). We assessed characteristics of patients who underwent RRM, with or without breast reconstruction. We included 46 patients with BRCA1/2Mut+ who underwent RRM from July 2011 to December 2017. Among the 46 patients who underwent RRM, 3 had not been diagnosed with breast cancer (BC) and 43 had cancer in a single breast; 33 patients (71.7%) underwent breast reconstruction with RRM; and 13 patients (28.3%) did not undergo breast reconstruction. All of 3 patients who had not been diagnosed with BC underwent bilateral RRM with breast reconstruction. There was no significant difference of clinical characteristic between patients undergoing RRM with and without breast reconstruction. However, patients who decided to undergo RRM with the current diagnosis of BC had significantly higher rates of RRM with breast reconstruction than that of patients with a past history of BC (P = 0.043). The rate of nipple-sparing mastectomy (NSM) in patients with breast reconstruction was significantly higher (28 of the 37 breasts, 75.7%) than that in patients without reconstruction (3 of the 14 breasts, 21.4%) (P

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.

575 Background: COVID-19 vaccination-related lymphadenopathy is a frequent imaging finding that may be indistinguishable from malignant nodes and can lead to diagnostic difficulties in patients with cancer or healthy individuals on cancer screening. However, no prospective trials regarding COVID-19 vaccination-related lymphadenopathy following a booster shot have been conducted. The purpose of this study was to determine the incidence and imaging characteristics of COVID-19 vaccination-related axillary lymphadenopathy and assess the recovery period following a booster shot. Methods: We prospectively enrolled healthy women working at St. Luke’s International Hospital, who would receive the third shot of the Pfizer-BioNTech COVID-19 vaccine between December 6 and 28, 2021. Women with a history of cancer, atopic dermatitis, auto-immune disease, or axillary surgery were excluded. All participants underwent ultrasound (US) examinations for the bilateral axilla at baseline (prior to the third shot), early phase (1–3 days after the shot), and late phase (6 weeks after the shot) if lymphadenopathy was detected at the early phase. We evaluated the incidence and US characteristics of lymphadenopathy. As for US characteristics mimicking a malignant node, focal cortical thickening, absence of the echogenic hilus, and vascularity were examined. In this study, abnormal lymphadenopathy was defined as [1] an increase in the short-axis size by more than 2 mm compared with the baseline, [2] an increase in the number of nodes with short-axis diameter more than 5 mm, and [3] demonstrating US characteristics mimicking malignant nodes. Results: A total of 100 women were enrolled in this study. The median age was 41 years (range 23–63). Abnormal axillary lymphadenopathy on the vaccinated side was observed in 59% of participants in the early phase and 8% in the late phase. In the contralateral axilla, abnormal lymphadenopathy was observed in 1% of participants in the early phase and 2% in the late phase. The median short-axis size of ipsilateral abnormal lymphadenopathy was 7.6 mm in the early phase and 5.7 mm in the late phase. In the early phase, US characteristics mimicking malignant nodes were observed, including focal cortical thickening in 54% of participants, absence of the echogenic hilus in 16%, and hypervascularity in 33%. Conclusions: COVID-19 vaccination-related axillary lymphadenopathy indistinguishable from malignant nodes was observed in more than half of the participants compared with the baseline, which improved in most cases within 6 weeks after the latest booster shot. To avoid a diagnostic conundrum, patients with breast cancer should be vaccinated on the arm contralateral to the cancer side. It is recommended that non-urgent imaging screening for the axilla should be scheduled after 6 weeks following the latest vaccination.

Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1 (PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2, resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than their single use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new anti-human TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG

There are few studies have conducted digital breast tomosynthesis (DBT) in addition to digital mammography (2DDM) and ultrasound (US) for screening. The purpose of this study is to determine the possibility of synergistic effects of DBT and US screening for Japanese.5023 examinations of the opportunistic screening using 2DDM and US (2D group: 2581) or 2DDM and US plus DBT (3 group: 2442) were performed at our facility from May 1, 2017 to March 31, 2019. This study was not RCT, and the backgrounds of the two groups were different.The recall rate was 3.1% in the 2D group and 2.6% for the 3D group (p = 0.27). The number of detected cancer cases was 6 (0.23%) in the 2D group and 12 (0.49%) in the 3D group (p = 0.16). The positive predictive value (PPV) was 7.4% for the 2D group and 19.0% for the 3D group (p = 0.045). There was one invasive ductal carcinoma case which had no findings in 2DDM and US, but had a slight distortion in the images of DBT.We examined and reported whether DBT was useful for breast cancer screening combined with mammography and US. Compared to the 2D group, the 3D group showed better results of PPV with significant difference. However, due to the non-randomized design and difference between the two groups, the results should be interpreted in caution. Adding DBT in 2DDM and US screening would be acceptable only if the benefits and disadvantages are explained to the women undergoing the screening.

Background: COVID-19 vaccination-related lymphadenopathy is a frequent imaging finding which may be indistinguishable from malignant nodal involvement and lead to diagnostic difficulties in patients with cancer or healthy individuals on cancer screening. An expert panel of the leading cancer centers in United States recommended routine imaging examinations should be scheduled at least 6 weeks after the final vaccination to allow for any reactive lymphadenopathy to resolve. However, there were no prospective trials regarding COVID-19 vaccination-related lymphadenopathy and the evidence was so limited. The purpose of this study was to determine the incidence and imaging characteristics of COVID-19 vaccination-related axillary lymphadenopathy and assess the recovery period. Methods: We prospectively enrolled healthy women working at the St. Luke’s International Hospital who received Pfizer COVID-19 vaccination within 8 weeks before enrollment between May 10th and 27th, 2021. Women with a history of any type of cancer or axillary surgery, active atopic dermatitis and auto-immune disease were excluded. Participants underwent ultrasound examinations for bilateral axilla at the enrollment. Lymphadenopathy was defined as demonstrating an enlarged node(s) with more than 5mm in short axis by ultrasound imaging in this trial. As for imaging characteristics, status of cortical thickening, echogenic hilus and vascularity of lymph nodes were evaluated. Other side effects by vaccination were assessed by a questionnaire. If lymphadenopathy was detected, we followed the participant by ultrasound examination every three weeks until the lymphadenopathy was resolved. We evaluated the incidence rate and imaging characteristics of lymphadenopathy detected by ultrasound examination, and the recovery period required for improvement of the lymphadenopathy. We also validated the association of the lymphadenopathy with the participant characteristics and other side effects. Results: A total of 135 women were enrolled in this study. Participants' median age was 37 years (range 23-63). Median time from the latest vaccination to the enrollment was 45 days (range 8-56). In the ultrasound examination at enrollment, axillary lymphadenopathy was observed in 67 participants (50%) on the injected (ipsilateral) side. In the contralateral axilla, 13 participants (10%) showed lymphadenopathy. In the ipsilateral axilla, the number of enlarged node(s) was 1 node in 25 cases (19%), 2 nodes in 24 cases (18%), 3 nodes in 13 cases (10%), 4 nodes in 4 cases (3%) and 5 nodes in 1 case (1%). Regarding the ipsilateral enlarged lymph node, focal cortical thickening was observed in 58 cases (43%) and the absence of the echogenic hilus was observed in 15 cases (11%). Hypervascularity was observed in 15 cases (11%). Incidence of the lymphadenopathy was not statistically correlated with participant’s age or incidence of fever due to vaccination. At 6 weeks after the latest vaccination, the rate of ipsilateral axillary lymphadenopathy was 48%, 40% at 8 weeks, and 6% at 12 weeks. In participants with lymphadenopathy, median recovery period to resolve the lymphadenopathy was 75 days from the latest vaccination. Conclusion: A half of participants showed COVID-19 vaccination-related axillary lymphadenopathy and the imaging characteristics were often indistinguishable from malignant nodal involvement. Therefore, patients with breast cancer should be vaccinated on the contralateral arm to the cancer side to avoid diagnostic conundrum. The lymphadenopathy was commonly observed even in 8 weeks, and mostly resolved after 12 weeks from the vaccination. Therefore, non-urgent imaging examinations such as screening would be recommended to be scheduled at least 12 weeks following the latest vaccination. Citation Format: Kumiko Kida, Hiroko Tsunoda, Risa Kasahara, Chika Tsutsumi, Akiko Numata, Yuri Takehara, Atsushi Yoshida, Junko Takei, Naoki Hayashi, Daiki Kobayashi, Hideko Yamauchi. Prospective ultrasonographic surveillance study for incidence and recovery period of COVID-19 vaccination-related axillary lymphadenopathy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-01-01.

We assessed the long-term oncologic safety of nipple-sparing mastectomy (NSM) compared to skin-sparing mastectomy (SSM) for primary breast cancer patients with immediate reconstruction.Data of stage 0-III primary breast cancer patients undergoing NSM (n = 190) or SSM (n = 729) from June 2006 to December 2012 were retrospectively collected. Nipple-tumor distance (NTD) was measured on pretreatment mammography, magnetic resonance imaging, or ultrasonography findings. NSM patients with NTD 1 cm were excluded. Locoregional recurrence (LRR) rates were compared between groups. Disease-free survival (DFS) and overall survival (OS) according to surgical procedure were assessed.The median (range) follow-up period for NSM and SSM was 71 (10-131) months and 79 (9-140) months, respectively. LRR developed in 11 patients with invasive ductal carcinoma (5.8%) for NSM and 44 (42 in patients with invasive ductal carcinoma and 2 in patients with ductal carcinoma-in-situ) (6.0%) for SSM. Hormone receptor and HER2 status were not associated with LRR in either group. DFS and OS rates did not differ between groups (DFS: 89.3% for NSM, 89.3% for SSM, P = .87; OS: 98.4% for NSM, 94.5% for SSM, P = .43).NSM with immediate reconstruction was as safe as SSM for primary breast cancer with respect to prognosis and local control, regardless of the presence of invasive carcinoma or breast cancer subtype.

CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti‑CD44 monoclonal antibody (mAb), C44Mab‑5 (IgG1, kappa) was established by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab‑5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab‑5 lacks antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC), the antitumor activity of C44Mab‑5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab‑5 into an IgG2a subclass antibody, 5‑mG2a, and further produced a defucosylated version, 5‑mG2a‑f, using FUT8‑deficient ExpiCHO‑S (BINDS‑09) cells. Defucosylation of 5‑mG2a‑f was confirmed using fucose‑binding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5‑mG2a‑f against SAS and HSC‑2 oral cancer cells were determined through flow cytometry to be 2.8x10‑10 M and 2.6x10‑9 M, respectively, indicating that 5‑mG2a‑f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5‑mG2a‑f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5‑mG2a‑f showed moderate ADCC and CDC activities against SAS and HSC‑2 oral cancer cells. In vivo analysis revealed that 5‑mG2a‑f significantly reduced tumor development in SAS and HSC‑2 xenografts in comparison to control mouse IgG, even after injection seven days post‑tumor inoculation. Collectively, these results suggest that treatment with 5‑mG2a‑f may represent a useful therapy for patients with CD44‑expressing oral cancers.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in glioblastoma as well as breast, gastric, lung, colorectal, and pancreatic cancers. Its expression is associated with poor clinical outcomes. Anti-HER2 antibodies have provided significant survival benefits to patients with HER2-overexpressing breast and gastric cancers. We recently developed an anti-HER2 monoclonal antibody (mAb), H

Although a docetaxel and cyclophosphomide (TC) regimen without anthracycline as adjuvant therapy became one of the standard regimens especially for ER-positive (ERA TC regimen that included 75 mg/mThirty (71.4%) of 42 tumors had clinical response. No patient achieved pathologic complete response. At the median follow-up period of 105.2 months (range, 12.1-119.7 months), the disease-free survival rate was 81.6%, and the distant disease-free survival rate was 86.8%. In terms of survival, only 1 patient died during the study period. The overall survival rate was 97.4% during the study period. Patients who developed distant recurrence had a trend to have progesterone receptor-negative or weakly positive compared with those who did not develop any recurrence (85.7% vs. 45.2%; P = .05).Our prospective study showed that a TC regimen as NAC achieved a high clinical response rate in stage II to III ER

Trastuzumab is a humanized antibody against human epidermal growth factor receptor 2 (HER2) that offers significant survival benefits to patients with HER2-overexpressing breast or gastric cancer. HER2 is also known to be overexpressed in colon cancers. In this study, a novel anti-HER2 monoclonal antibody (mAb), H

The epithelial cell adhesion molecule (EpCAM), which is a calcium-independent homophilic intercellular adhesion factor, contributes to cell signaling, differentiation, proliferation and migration. EpCAM is essential for carcinogenesis in numerous types of human cancer. The purpose of the present study was to establish an anti-EpCAM monoclonal antibody (mAb) for targeting colorectal adenocarcinomas. Thus, an anti-EpCAM mAb, EpMab-16 (IgG2a, κ), was established by immunizing mice with EpCAM-overexpressing CHO-K1 cells, and validated using flow cytometry, western blot, and immunohistochemical analyses. EpMab-16 reacted with endogenous EpCAM specifically in a colorectal adenocarcinoma cell line as determined by flow cytometry and western blot analyses. Immunohistochemical analysis demonstrated that EpMab-16 stained a plasma membrane-like pattern in clinical colorectal adenocarcinoma tissues. The dissociation constant (K D) for EpMab-16 in a Caco-2 colorectal adenocarcinoma cell line determined by flow cytometry was 1.8×10-8 M, suggesting moderate binding affinity of EpMab-16 for EpCAM. Whether the EpMab-16 induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Caco-2 or antitumor activity was then assessed in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC induction in Caco-2 cells by EpMab-16 treatment. In vivo experiments in a Caco-2 ×enograft model demonstrated that EpMab-16 treatment significantly reduced tumor growth compared with that in mice treated with the control mouse IgG. These results suggested that EpMab-16 may be a promising treatment option for EpCAM-expressing colorectal adenocarcinomas.

Panel sequencing of susceptibility genes for hereditary breast and ovarian cancer (HBOC) syndrome has uncovered numerous germline variants; however, their pathogenic relevance and ethnic diversity remain unclear. Here, we examined the prevalence of germline variants among 568 Japanese patients with BRCA1/2-wildtype HBOC syndrome and a strong family history. Pathogenic or likely pathogenic variants were identified on 12 causal genes for 37 cases (6.5%), with recurrence for 4 SNVs/indels and 1 CNV. Comparisons with non-cancer east-Asian populations and European familial breast cancer cohorts revealed significant enrichment of PALB2, BARD1, and BLM mutations. Younger onset was associated with but not predictive of these mutations. Significant somatic loss-of-function alterations were confirmed on the wildtype alleles of genes with germline mutations, including PALB2 additional somatic truncations. This study highlights Japanese-associated germline mutations among patients with BRCA1/2 wildtype HBOC syndrome and a strong family history, and provides evidence for the medical care of this high-risk population.

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKη is classified as a type II DGK. Reports have shown that DGKη is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG2b, kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKη and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKη.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo- or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti-EGFR monoclonal antibody (mAb), EMab-134, by immunizing mice with the ectodomain of human EGFR. EMab-134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) to facilitate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab-134 and 134-mG2a against EGFR-expressing CHO-K1 (CHO/EGFR) cells were deter-mined by flow cytometry to be 3.2×10−9 M and 2.1×10−9 M, respectively; these results indicate that 134-mG2a has a higher binding affinity than EMab-134. The 134-mG2a antibody was more sensitive than EMab-134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134-mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC-2, and SAS cells. Moreover, the in vivo administration of 134-mG2a significantly inhibited the development of CHO/EGFR, HSC-2, and SAS mouse xenografts in comparison to the results observed in response to EMab-134. Taken together, the findings of the present study demonstrate that the newly-formulated 134-mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR-expressing oral cancer.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast cancer, gastric, lung, colorectal, oral, and pancreatic cancers. HER2 expression is associated with poor clinical outcomes. An anti-HER2 humanized antibody, trastuzumab, has improved survival rates in patients with HER2-overexpressing breast and gastric cancers. Previously, we established a novel anti-HER2 monoclonal antibody (mAb), H

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the Cell-Based Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1, kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256 stained type I alveolar cells of lung, renal glomerulus and Bowman's capsule, and lymphatic endothelial cells of lung and colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG2a, kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.

Introduction The influence of breast cancer (BC) subtype in discrepancies between pathologic complete response (pCR) and complete response by magnetic resonance imaging (MRI-CR) after neoadjuvant chemotherapy (NAC) have not been discussed well. We evaluated the association between BC subtype and pCR or only residual in situ lesion without invasive cancer (pCR/in situ + ) in patients with MRI-CR (positive predictive value [PPV]). Material and Methods From the data of 716 patients with primary BC who were diagnosed with invasive cancer and treated with NAC and then surgery from January 2009 to May 2014 at St. Luke's International Hospital, 180 patients were determined to have MRI-CR by retrospective chart review. BC subtypes at baseline were classified into 6 subtypes, as strong estrogen receptor (ER ++ ), moderately positive ER (ER + ), negative ER (ER − ), and HER2 status expression. Results Three subtypes had PPV (pCR) ≥ 50%: ER − /HER2 + (56.3%, 27/48), ER − /HER2 − (57.6%, 34/59), and ER + /HER2 + (56.2%, 9/16). However, PPV (pCR) for the ER ++ /HER2 − and ER ++ /HER2 + subtypes was ++ /HER2 − subtype, which was significantly low ( P ++ /HER2 − and other subtypes. PPV (pCR/in situ + ) was significantly low at 20.0% in the ER ++ /HER2 − subtype ( P + ) in other subtypes was collectively greater than 60%, and was 91.7% in the ER − /HER2 + subtype. Conclusion We should interpret carefully MRI-CR of NAC to evaluate residual disease for ER ++ /HER2 − BC.

Panel sequencing of susceptibility genes for hereditary breast and ovarian cancer (HBOC) syndrome has uncovered numerous germline variants; however, their pathogenic relevance and ethnic diversity remain unclear. Here, we examined the prevalence of germline variants among 568 Japanese patients with BRCA1/2-wildtype HBOC syndrome and a strong family history. Pathogenic or likely pathogenic variants were identified on 12 causal genes for 37 cases (6.5%), with recurrence for 4 SNVs/indels and 1 CNV. Comparisons with non-cancer east-Asian populations and European familial breast cancer cohorts revealed significant enrichment of PALB2, BARD1, and BLM mutations. Younger onset was associated with but not predictive of these mutations. Significant somatic loss-of-function alterations were confirmed on the wildtype alleles of genes with germline mutations, including PALB2 additional somatic truncations. This study highlights Japanese-associated germline mutations among patients with BRCA1/2 wildtype HBOC syndrome and a strong family history, and provides evidence for the medical care of this high-risk population.

Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues because PDPN is known as a lymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil. However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using flow cytometry. One of the mAbs, PMab-247 (IgG1, kappa), specifically detected CHO/bPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-247 for the functional analyses of bPDPN., Highlights • PDPN is known as a specific lymphatic endothelial cell (LEC) marker. • Sensitive and specific PMab-247 mAb against bear PDPN was produced. • PMab-247 strongly reacted with bear PDPN in flow cytometry. • PMab-247 is useful for IHC using paraffin-embedded cell sections.

The expression of human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed in several cancers, such as breast, lung, gastric, pancreatic, and colorectal cancers, and be associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H

In July 2018, olaparib, an oral poly adenosine diphosphate-ribose polymerase(PARP)inhibitor, was approved for the first time in Japan as a companion diagnostic drug for the treatment of recurrent breast cancer in BRCA-positive patients. Genetic testing had been offered only to clients who met the eligibility criteria for the test, and the decision whether or not to receive the test was based on their medical and family history, tumor characteristics, and the client's preference. With the approval of olaparib as a companion diagnostic drug, genetic testing has become a part of the standard treatment protocol. Modern technology has made multiple genetic testing possible in one setting. It is therefore necessary for medical practitioners to have the knowledge and ability to take care for familiar tumors. It may be difficult to understand all the available information on genetics, but an individualized approach for each patient would be necessary in the future. Medical practitioners involved in the treatment of such patients would need adequate knowledge on genetics and genetic counseling, as well as the ability to educate and provide information to the patients and their families. If a more specialized approach is necessary, referral to an institution with a clinical genetics department is essential.

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.

Aim Lymphocyte predominant breast cancer (BC) is associated with higher pathological complete response (pCR) rate after neoadjuvant therapy (NAT) and favorable outcome in triple negative breast cancer (TNBC) and HER2+ BC. The predictive and prognostic impact of stromal tumour-infiltrating lymphocytes (TILs) after NAT and the change of TILs before (pre-) and after (post-) NAT are not well studied. We aimed to assess the predictive and prognostic value of pre- and post-NAT TILs, as well as their pharmacodynamics modulation and their change for TNBC and HER2+ BC. Materials and methods Two-hundred and nine consecutive patients (n = 80 TNBC, n = 129 HER2+ BC) who received NAT between 2001 and 2009 in a single institution were included. We evaluated the association between pre-NAT TILs and pCR, and the association between pre- and post-NAT TILs, as well as their immunodynamics change with relapse-free survival (RFS) for patients with residual disease (RD). Results Low pre-NAT TILs compared to int/high were significantly associated with lower pCR rate (TNBC: 4.0% vs 43.6%; HER2+ BC: 26.0% vs 51.9%). The median follow-up period was 98 months. In TNBC with RD, low pre-NAT TILs showed significant association with shorter RFS (HR = 3.844 [1.190–12.421], p = 0.024) in multivariate analysis. Low post-NAT TILs showed borderline significant association with shorter RFS (HR = 2.836 [0.951–8.457], p = 0.061). The change in TILs was not associated with RFS. In HER2+ BC, low pre-NAT TILs were not associated with RFS. Conclusion In TN and HER2+ BCs, low pre-NAT TILs tumours had a low likelihood of achieving pCR. In TNBC with RD, both low pre- and post-NAT TILs were associated with shorter RFS. These results suggest that TILs information should be taken into account when additional therapies may be given in the post-neoadjuvant setting.

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques., Highlights • PDPN is known as a specific lymphatic endothelial cell (LEC) marker. • Sensitive and specific PMab-225 mAb against alpaca PDPN was produced. • PMab-225 strongly reacted with alpaca PDPN in flow cytometry. • PMab-225 is useful for IHC using paraffin-embedded cell sections.

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)–K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO–K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO–K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer., Highlights • TROP2 is overexpressed in several types of cancers. • We established a novel anti-TROP2 mAb (TrMab-29) using CBIS method. • TrMab-29 is useful for flow cytometry, western blot, and immunohistochemical analyses. • TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), L1Mab-13 (IgG1, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. L1Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether L1Mab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because L1Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of L1Mab-13 from IgG1 into IgG2a (13-mG2a), and further produced a defucosylated version (13-mG2a-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia and Pholiota squarrosa lectins. The dissociation constants (KD) for 13-mG2a-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10−9 M and 4.8 × 10−9 M, respectively, indicating that 13-mG2a-f possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-mG2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG2a-f may represent a useful therapy for patients with PD-L1-expressing oral cancers., Highlights • ・PD-L1 is expressed in many tumor cells including oral cancers. • ・We previously established an anti-PD-L1 mAb (L1Mab-13, mouse IgG1). • ・We converted the subclass of L1Mab-13 from IgG1 into IgG2a (13-mG2a), and defucosylated it (13-mG2a-f). • ・13-mG2a-f exerted ADCC, CDC, and antitumor activities against oral cancer cell lines.

Hereditary breast and ovarian cancer (HBOC) syndrome represents 5-10% of all breast cancers. In Japan, the HBOC syndrome is frequently diagnosed in patients with breast cancer. Therefore, a treatment strategy combining a plan for existing breast cancer and for reduction of future breast and ovarian cancer risk is necessary. Breast cancer risk-reducing management involves three options-surveillance, chemoprevention, and risk-reducing mastectomy (RRM). RRM can prevent >90% of new breast cancers. Ovarian cancer risk management options are more limited, and risk-reduction salpingo-oophorectomy is the only option since there is no proven effective early detection method available. The local recurrence rate following breast-conserving surgery in BRCA1/2 mutation-associated breast cancer is not significantly higher than that in sporadic breast cancer. Furthermore, there is no difference in prognosis between surgical methods. Clinicians should inform patients that there are no data on long-term monitoring and fully discuss risks of re-developing breast cancer with patients when choosing the surgical method. In HBOC, BRCA1/2 mutations lead to failure of double-strand DNA break repair, with poly ADP-ribose polymerase (PARP) playing an important role in single-strand DNA nick repair. Use of PARP inhibitors in HBOC prevents DNA repair (synthetic lethality) leading to cell death. This review summarizes management of the HBOC syndrome based on recent evidence.