Biosensing-based quality control monitoring of the higher-order structures of therapeutic antibody domains.

Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.

Tokophobia: Psychopathology and Diagnostic Consideration of Ten Cases.

Tokophobia is regarded as the intensive fear of childbirth that some pregnant women have. However, little is known about the psychopathological details of tokophobia (fear of childbirth). Between 2020 and 2021, a total of 10 pregnant women (nine nulliparae and one multipara) with a strong fear of childbirth were referred by obstetricians. Semi-structured psychopathological interviews were conducted, and two cases were judged to have obsession, three an overvalued idea, and one secondary delusion. Three were characterised by both obsession and overvalued idea and one by both obsession and secondary delusion. In total, six cases had features of an overvalued idea. All of the participants except one had a lifetime history of a specific phobia. In addition, their history included social phobia in two cases, panic disorder in one case, obsessive-compulsive disorder (other than tokophobia) in two cases, depressive disorder in two cases, bipolar disorder in two cases, and PTSD in six cases. To conclude, this study showed that tokophobia was not a phobic disorder but a kind of overvalued idea that requires specific assessment and treatment.

Prognostic significance of C-reactive protein in unresectable hepatocellular carcinoma treated with atezolizumab and bevacizumab.

AIM: C-reactive protein (CRP) is both an inflammatory and prognostic marker in various cancers. This study aimed to elucidate the characteristics of CRP and the prognostic factors in patients who were administered with atezolizumab plus bevacizumab (ATZ + BEV) for unresectable hepatocellular carcinoma (HCC). METHODS: A total of 213 patients who received ATZ + BEV for HCC from November 2020 to March 2023 at 15 hospitals were enrolled in this retrospective study. The prognosis was analyzed by subdividing the patients based on baseline characteristics, radiologic response, and treatment lines. Accuracy of survival prediction was assessed using CRP, alpha fetoprotein (AFP), C-reactive protein and alpha fetoprotein in immunotherapy (CRAFITY), and Glasgow Prognostic Score. RESULTS: Compared with patients with baseline CRP <1 mg/dL, those with baseline CRP ≥1 mg/dL (n = 45) had a significantly higher baseline albumin-bilirubin score and AFP levels, significantly lower disease control rate (62.2%), and significantly shorter median overall survival (hazards ratios 2.292; 95% confidence interval 1.313-5.107; log-rank test, p < 0.001). Multivariate analysis identified CRP ≥1 mg/dL, AFP ≥100 ng/mL, and modified albumin-bilirubin grade as the significant prognostic factors. The baseline CRP, AFP, CRAFITY, and Glasgow Prognostic Score demonstrated higher discrimination for 1-year survival prediction after first-line ATZ + BEV administration, compared with beyond second line, with area under the receiver operating characteristic curves of 0.759, 0.761, 0.805, and 0.717, respectively. CONCLUSIONS: CRP was a significant biomarker in patients treated with ATZ + BEV for HCC. Elevated CRP levels may indicate aggressive cancer progression and potential resistance to ATZ + BEV therapy.

COVID-19 severity is associated with the risk of gastrointestinal bleeding.

OBJECTIVE: The association between the severity of COVID-19 and gastrointestinal (GI) bleeding is unknown. This study aimed to determine whether the severity of COVID-19 is a risk factor for GI bleeding. DESIGN: A multicentre, retrospective cohort study was conducted on hospitalised patients with COVID-19 between January 2020 and December 2021. The severity of COVID-19 was classified according to the National Institute of Health severity classification. The primary outcome was the occurrence of GI bleeding during hospitalisation. The main analysis compared the relationship between the severity of COVID-19 and the occurrence of GI bleeding. Multivariable logistic regression analysis was performed to evaluate the association between the severity of COVID-19 and the occurrence of GI bleeding. RESULTS: 12 044 patients were included. 4165 (34.6%) and 1257 (10.4%) patients had severe and critical COVID-19, respectively, and 55 (0.5%) experienced GI bleeding. Multivariable analysis showed that patients with severe COVID-19 had a significantly higher risk of GI bleeding than patients with non-severe COVID-19 (OR: 3.013, 95% CI: 1.222 to 7.427). Patients with critical COVID-19 also had a significantly higher risk of GI bleeding (OR: 15.632, 95% CI: 6.581 to 37.130). Patients with severe COVID-19 had a significantly increased risk of lower GI bleeding (OR: 10.349, 95% CI: 1.253 to 85.463), but the risk of upper GI bleeding was unchanged (OR: 1.875, 95% CI: 0.658 to 5.342). CONCLUSION: The severity of COVID-19 is associated with GI bleeding, and especially lower GI bleeding was associated with the severity of COVID-19. Patients with severe or critical COVID-19 should be treated with caution as they are at higher risk for GI bleeding.

[Significance of neutrophil-to-lymphocyte ratio in atezolizumab plus bevacizumab treatment for unresectable hepatocellular carcinoma].

This study aimed to investigate the significance of neutrophil-to-lymphocyte ratio (NLR) as a prognostic predictor by reporting 21 patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab plus bevacizumab (Atezo/Bev) as the first line of treatment. The optimal cut-off value of NLR was 2.25 with Atezo/Bev, and patients with NLR of ≥2.25 had a shorter progression free survival (PFS) (199 vs. 393 days, p=0.009) compared to patients with NLR of <2.25. NLR was positively correlated with C-reactive protein (r=0.525, p=0.016). The high NLR group demonstrated a shorter PFS than the low NLR group. NLR may be a useful predictive biomarker of the first-line Atezo/Bev treatment for unresectable HCC.

[A Long-Term Survival Case of Multiple Liver Metastases from Colon Cancer Treated with Intensive Multimodality Therapy].

A 60-year-old man diagnosed with sigmoid colon cancer was admitted to our hospital. A CT scan revealed multiple liver metastases. The patient was administered 15 courses of FOLFIRI chemotherapy and 15 courses of FOLFIRI plus Cmab chemotherapy. After this treatment, multiple liver metastases disappeared, and laparoscopic resection of the sigmoid colon was performed. Two months later, a recurrent lesion was found in the liver segment(S1), and 5 courses of FOLFIRI plus Cmab chemotherapy were performed. Although the CEA level decreased, the tumor size remained unchanged. Therefore, partial resection of the liver was performed, followed by 18 courses of FOLFIRI chemotherapy. After that, the patient was followed for a year without chemotherapy. However, about 1 year later, recurrence was observed in liver segments S5 and S6. A right lobectomy was performed for these 2 lesions, and then 16 more courses of FOLFIRI chemotherapy were performed. The chemotherapy was discontinued, and the patient was then followed up as an outpatient without chemotherapy; there has been no recurrence.

Tokophobia: Case Reports and Narratives of Ten Japanese Women.

Intense fear of childbirth by expectant women is called tokophobia. Because there are no qualitative studies targeting women with an intense fear of childbirth in Japan, it is unknown whether there is any link between the type of fear of objects/situations among tokophobic women and their psychological/demographic background. Furthermore, there is no available summary of the lived experience of Japanese women with tokophobia. This study aims to identify the intensity patterns of various types of fear among the participants and summarize the lived experience of having intense fear of childbirth. A qualitative descriptive study was conducted using a semi-structured interview. Pregnant women with an intense fear of childbirth participated in individual interviews facilitated by a psychiatrist and a midwife. Audio recordings of the interviews were transcribed and analyzed using a content analysis approach. The number of participants was ten. The types of feared objects varied individually and these were categorized as being related to either prospective or retrospective fear. The participants' experiences were grouped into three categories: difficulty in daily life, preoccupied negative expectation towards childbirth, and psychological adaptation to the upcoming childbirth. The results imply that women with tokophobia continuously suffer from fear in their daily life; hence, a special approach is needed to detect and reduce their fear.

Cystic degeneration during neo-adjuvant chemotherapy predicts squamous metaplasia of triple negative breast cancer: report of two cases.

Neo-adjuvant chemotherapy (NAC) has become a standard treatment for advanced breast cancer because of the advantage of monitoring drug sensitivity and enabling breast-conserving therapy. The changes during NAC are also important to know the biological characteristics of the tumor. We experienced two cases with cystic degeneration and enhancement of the cyst wall during NAC for triple negative breast cancer (TNBC). They were diagnosed to have breast cancer with squamous metaplasia. In case 1, a 37-year-old woman with right breast cancer diagnosed as TNBC, T3N3M0, Stage 3b was treated with NAC. MRI showed a cystic degeneration with a diameter of 3.5 cm and enhancement of the cyst wall, and the other nodules were extinguished. The histopathological finding of the surgical specimen revealed solid tubular carcinoma with squamous metaplasia. In case 2, a 58-year-old woman with right breast cancer diagnosed as HER2 enriched subtype, T2N0M0 stage 2 was treated with NAC containing trastuzumab. The post-NAC MRI showed extinguishment of the mass in the right breast, but showed a cystic lesion with 24 mm in diameter and enhancement of its wall in the left breast. She underwent breast conserving surgery for bilateral breast cancer, and histopathological finding of the surgical specimen indicated complete remission of right breast cancer and squamous cell carcinoma developed in the left breast. These changes are impressive and remind us that there are metaplastic changes (especially for squamous metaplasia) with resistance to chemotherapy.

Chip-scale high-peak-power semiconductor/solid-state vertically integrated laser.

Compact lasers capable of producing kilowatt class peak power are highly desirable for applications in various fields, including laser remote sensing, laser micromachining, and biomedical photonics. In this paper, we propose a high-peak-power chip-scale semiconductor/solid-state vertically integrated laser in which two cavities are optically coupled at the solid-state laser gain medium. The first cavity is for the intra-pumping of ytterbium-doped yttrium aluminum garnet (Yb:YAG) with an electrically driven indium gallium arsenide (InGaAs) quantum well, and the second cavity consists of Yb:YAG and chromium-doped yttrium aluminum garnet (Cr:YAG) for passive Q-switching. The proposed laser produces pulses as short as 450 ps, and an estimated peak power of 57.0 kW with a laser chip dimension of 1 mm3. To the best of our knowledge, this is the first monolithic integration of semiconductor and solid-state laser gain mediums to realize a compact high-peak-power laser.

TP53-positive clones are responsible for drug-tolerant persister and recurrence of HER2-positive breast cancer.

PURPOSE: The prognosis of HER2-positive breast cancer has improved with the development of anti-HER2 therapies. In order to further improve the prognosis of HER2-positive breast cancer, it is essential to elucidate the cells that survive during the therapy (drug-tolerant persister DTP). METHODS: Of the 2022 breast cancer patients operated at our institution during 2004-2018, 240 (12%) had HER2-positive breast cancer. Neo-adjuvant chemotherapy including trastuzumab (Tr-NAC) was administered to 94 of them. Forty-six of them were complete remission (CR), and 48 were non-CR. After 6.9 ± 3.7 years of follow-up, all 46 CR cases showed no recurrence (Cohort A), and 48 non-CR cases were divided into 31 cases with no recurrence (Cohort B) and 17 cases with recurrence (Cohort C). In addition to clinical backgrounds, we compared genomic profiles for 27 patients (Cohort A; 15/48, B; 7/31, and C; 5/17) who consented to genomic analysis. RESULTS: Genomic abnormalities of TP53 and PIK3CA were frequently observed in biopsy samples pre Tr-NAC, but we found no differences between CR (Cohort A) and non-CR (Cohorts B + C). Then, we examined both of pre and post Tr-NAC samples of Cohort B (7) and C (5) to see the relationship between recurrence and genomic abnormalities. TP53 mutations were significantly more prevalent in Cohort C (5/5, 100%) than cohort B (3/7, 43%) in the surgical sample after treatment (p = 0.04). PyClone analysis of TP53 mutations showed that the cellular frequency of TP53 clones increased in 4 of 5 patients in Cohort C and none of B. On the other hand, we found no enhancement of PIK3CA mutant clones in Cohort C. CONCLUSIONS: The DTP after Tr-NAC associated with subsequent relapse had TP53 mutations, suggesting that overcoming DTP with TP53 mutations is the most important clinical challenge. TRIAL REGISTRATION: Not applicable.

Benefit of home parenteral nutrition (HPN) and chemotherapy in patients with invasive lobular carcinoma developed peritoneal metastases.

The benefit of home parenteral nutrition (HPN) for patients with malnutrition due to peritoneal metastasis depends on the type of cancer. During the period 1999-2020, we treated 460 patients with metastatic and stage 4 breast cancer, 23 of whom were invasive lobular carcinoma (ILC). Of the 23 patients with ILC, 13 (57%) developed peritoneal metastasis, and 11 died of progression of peritoneal metastasis. Among these 11 patients, 2 patients who underwent surgery due to bowel obstruction, had no improvement, and died 1-4 months after surgery. The prognosis of the other 7 patients under BSC alone was poor, survival time were ranging from 1 to 5 months. The remaining two patients who were able to continue outpatient chemotherapy under HPN were able to prolong their survival time by 18 months and 26 months, respectively. We need to recognize that HPN and chemotherapy may prolong survival time in patients with peritoneal metastasis of ILC, and determine the indication for HPN based on the non-peritoneal life-threatening metastasis, length of treatment, availability of support for HPN management and outpatient chemotherapy, and the patient's willingness to accept it.

Sensitivity to fungicides in isolates of Colletotrichum gloeosporioides and C. acutatum species complexes and efficacy against anthracnose diseases.

Colletotrichum species cause diseases on many plants and are among the 'top 10' fungal plant pathogens. Species of the C. gloeosporioides and C. acutatum complexes are particularly important because they infect temperate fruit crops, but their control relies largely on chemical fungicides. In this study, differences in intrinsic fungicide sensitivity were determined in vitro using isolates of the C. gloeosporioides sp. complex (C. fructicola, C. siamense, and C. tropicale) and the C. acutatum sp. complex (C. fioriniae and C. nymphaeae), which had never been exposed to fungicides. Mycelial growth of all isolates was sensitive to the QoI azoxystrobin, the SDHI benzovindiflupyr, and the new DMI fungicide mefentrifluconazole. The isolates of C. nymphaeae were highly sensitive to the phenylpyrrole fungicide fludioxonil. The isolates of C. gloeosporioides sp. complex were sensitive to the bis-guanidine fungicide iminoctadine-albesilate, whereas those of C. acutatum sp. complex were inherently insensitive. These results are valuable when sensitivity of field populations is monitored in resistance management. Although SDHI fungicides are largely not effective against diseases caused by Colletotrichum species, benzovindiflupyr controlled anthracnose disease of various crops such as kidney bean, garland chrysanthemum, and strawberry, caused by C. lindemuthianum, C. chrysanthemi, and C. siamense, respectively, demonstrating this fungicide to be unique among SDHIs and having a broad control spectrum against anthracnose. To help understanding the reason for differential activity of benzovindiflupyr and boscalid, sdhB gene sequences were analyzed but those of C. lindemuthianum, C. chrysanthemi, and C. scovillei revealed no known mutations reported to be responsible for SDHI resistance in other fungi, indicating that other mechanism(s) than target-site modification may be involved in differential sensitivity to benzovindiflupyr and boscalid, found in Colletotrichum species.

Radiation Therapy for Malignant Lumbosacral Plexopathy: A Case Series.

Background Malignant lumbosacral plexopathy is caused by a direct extension of an intrapelvic malignancy to involve the plexus nerves. In this report, we describe the effect of radiotherapy on patients with malignant lumbosacral plexopathy. Patients and methods We performed a retrospective review of the medical records of patients who underwent radiation therapy for pain caused by malignant lumbosacral plexopathy between 2017 and 2020 at our institution. The pain was measured using a numeric rating scale (0-10) at initiation and completion of radiotherapy or at the time when the maximum response was observed. Results A total of 12 tumor sites in 11 patients were included. Eight of the tumors invaded the iliopsoas muscle, and the remaining four invaded or abutted the piriformis muscle. The mean duration of follow-up was 215 days (31-675 days). All patients achieved pain relief at the end of radiotherapy, with complete resolution of pain in nine patients. The maximum effect was seen at a mean of three weeks (1-12 weeks) after the initiation of radiotherapy. Toxicities related to radiotherapy included grade 1 diarrhea in four patients and grade 1 frequent urination in one patient. Two patients experienced a relapse of pain at one and two months, respectively, after achieving their maximal response. Conclusion Radiotherapy provides significant pain relief for patients with the malignant lumbosacral syndrome. The recognition and diagnosis of this syndrome, and the use of radiation therapy as a therapeutic option, are important. Patients should be offered all possible therapies, regardless of curative or palliative intent.

Altered T cell subpopulations and serum anti-TYRP2 and tyrosinase antibodies in the acute and chronic phase of alopecia areata in the C3H/HeJ mouse model.

BACKGROUND: C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. OBJECTIVE: We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. METHODS: We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. RESULTS: The CD8+NKG2D+ T and CD8+ TEM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4+ TEM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8+ TEM/TRM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c+ cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8+ T cell percentages were upregulated in AA mice. CONCLUSION: These results suggest that the CD8+ and CD4+ T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

A long-term survival case with recurrent esophageal adenosquamous carcinoma.

We experienced an extremely rare case of recurrent esophageal adenosquamous carcinoma showing cutis, bone and adrenal gland metastases. Furthermore, the patient showed complete remission by chemotherapy and irradiation, with a long-term survival of over 8 years. A 46-year-old man with esophago-gastric junction cancer of clinical stage II was administered two cycles of neo-adjuvant chemotherapy with FP (5FU 800 mg/mm2 Cisplatin 80 mg/mm2 every 3 weeks), subsequently, underwent esophagectomy and mediastinal and celiac lymph node dissection. However, at 1 month after the surgery, he was admitted again due to a 1 cm cutaneous metastasis at the anterior chest wall and left side adrenal gland metastasis. Furthermore, magnetic resonance imaging showed bone metastasis at the second cervical vertebra. He was administered weekly docetaxel at 40 mg/body (3 times every 4 weeks) for 27 cycles. Irradiation therapy (liniac 36 Gy/16Fr) was performed for the vertebral metastasis. The cutaneous and adrenal metastases were diminished and complete remission persisted for over 8 years.

Local disorder of the C-terminal segment of the heavy chain as a common sign of stressed antibodies evidenced with a peptide affinity probe specific to non-native IgG.

Therapeutic antibodies have many biopharmaceutical applications; however, characterization of their higher-order structures is a major concern in quality control. We have developed AF.2A1, an artificial protein, that specifically recognizes non-native, structured IgGs. We performed binding assays using various types of IgGs and fragments to investigate the mechanisms by which AF.2A1 interacts with the non-native IgG. AF.2A1 recognized the acid-stressed IgGs from human, mouse, and rat, but not rabbit. Binding assays using the human IgG1 fragments revealed that an interface emerged by deleting five C-terminal residues. We conclude that AF.2A1 recognizes an exposed hydrophobic core centered on the Trp417. Our results concur with those of the previous studies showing that C-terminal structural changes occur early during antibody denaturation and aggregation. Our findings explain the molecular rationale for using AF.2A1 in quality control of biopharmaceutical IgGs.

Antimicrobial susceptibility surveillance of bacterial isolates recovered in Japan from odontogenic infections in 2013.

We report on the findings of the first antimicrobial susceptibility surveillance study in Japan of isolates recovered from odontogenic infections. Of the 38 facilities where patients representing the 4 groups of odontogenic infections were seen, 102 samples were collected from cases of periodontitis (group 1), 6 samples from pericoronitis (group 2), 84 samples from jaw inflammation (group 3) and 54 samples from phlegmon of the jaw bone area (group 4) for a total of 246 samples. The positivity rates of bacterial growth on culture were 85.3%, 100%, 84% and 88.9%, respectively, for groups 1, 2, 3 and 4. Streptococcus spp. isolation rates according to odontogenic infection group were 22% (group 1), 17.7% (group 3) and 20.7% (group 4). Anaerobic isolation rates were 66.9% (group 1), 71.8% (group 3) and 68.2% (group 4). Drug susceptibility tests were performed on 726 strains excluding 121 strains that were undergrown. The breakdown of the strains subjected to testing was 186 Streptococcus spp., 179 anaerobic gram-positive cocci, 246 Prevotella spp., 27 Porphyromonas spp., and 88 Fusobacterium spp. The isolates were tested against 30 antimicrobial agents. Sensitivities to penicillins and cephems were good except for Prevotella spp. The low sensitivities of Prevotella spp is due to ß-lactamase production. Prevotella strains resistant to macrolides, quinolones, and clindamycin were found. No strains resistant to carbapenems or penems were found among all strains tested. No anaerobic bacterial strain was resistant to metronidazole. Antimicrobial susceptibility testing performed on the S. anginosus group and anaerobic bacteria, which are the major pathogens associated with odontogenic infections, showed low MIC90 values to the penicillins which are the first-line antimicrobial agents for odontogenic infections; however, for Prevotella spp., penicillins combined with ß-lactamase inhibitor showed low MIC90 values.

Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein-Protein Interaction.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on Streptococcal protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.

Antitumor and Antiangiogenic Activities of Lenvatinib in Mouse Xenograft Models of Vascular Endothelial Growth Factor-Induced Hypervascular Human Hepatocellular Carcinoma.

High expression of vascular endothelial growth factor (VEGF) in patients with hepatocellular carcinoma (HCC) is associated with poor prognosis. Here, we investigated the antitumor activity of lenvatinib, a multiple receptor tyrosine kinase inhibitor, in VEGF-overexpressing HCC models. In human umbilical vein endothelial cells, lenvatinib showed potent inhibitory activities against VEGF-induced proliferation and VEGF/basic fibroblast growth factor-induced tube formation. In VEGF-overexpressing HCC xenograft models, characterized by aggressive tumor growth and hypervascularity, lenvatinib had significant antitumor and antiangiogenic activities. These results suggest that potent activity of lenvatinib against VEGF signaling underlies its antitumor and antiangiogenic activities in the hypervascular HCC models.

Lenvatinib induces death of human hepatocellular carcinoma cells harboring an activated FGF signaling pathway through inhibition of FGFR-MAPK cascades.

Lenvatinib inhibits VEGF- and FGF-driven angiogenesis, and proliferation of tumor cells with activated FGF signaling pathways in preclinical models, and we previously demonstrated antitumor activity in human HCC xenograft tumor models. Here, we examined the inhibitory activity of lenvatinib against FGF-driven survival of human HCC cell lines. First, we conducted a histological analysis of FGF19-overexpressing Hep3B2.1-7 xenograft tumors collected from mice treated with lenvatinib. Second, we examined the effects of pharmacological inhibition on survival of cultured HCC cells with an activated FGF signaling pathway under nutrient-starved culture condition to mimic tumor microenvironments induced by angiogenesis inhibition. In the first analysis, area of histological focal necrosis was greater in Hep3B2.1-7 xenograft tumors with the lenvatinib treatment than that after the treatment with sorafenib, which does not inhibit FGFRs. Lenvatinib and E7090 (a selective FGFR1-3 inhibitor), but not sorafenib, induced death of Hep3B2.1-7, and another FGF19 overexpressing HuH-7 cells. Lenvatinib and E7090 decreased phosphorylation of downstream molecules of the FGF signaling pathway (such as FRS2, Erk, and p38 MAPK), and induced PARP cleavage, even under limited nutrients. PD0325901, MEK inhibitor, caused the same changes in HCC cells as those described above for lenvatinib and E7090. These results reveal that the FGF signaling pathway through MAPK cascades plays an important role in survival of HCC cell lines with an activated FGF signaling pathway under limited nutrients, and FGFR-MAPK cascades likely contribute to survival of HCC cells with an activated FGF signaling pathway under tumor microenvironments with limited nutrients, where tumor angiogenesis is inhibited.