Factors associated with liver injury and prognosis in advanced cancer patients treated with immune checkpoint inhibitors.

AIM: The use of immune checkpoint inhibitors (ICIs) has increased remarkably, and immune-related adverse events (irAEs) have also increased. This study aimed to identify factors associated with immune-related liver injury (irLI), and the relationship between the grades of irLI and overall survival (OS) in patients treated with ICIs. METHODS: A total of 571 patients who had been treated for advanced malignancies with ICIs between January 2015 and March 2022 were retrospectively recruited. The presence of liver injury was determined by the aspartate aminotransferase and alanine aminotransferase elevation. The irLI grading was based on Common Terminology Criteria for Adverse Events version 5.0. RESULTS: A total of 50 (8.8%) patients had grade ≥2 irLI and 24 (4.2%) had grade ≥3 irLI. Treatment with anti-cytotoxic T-lymphocyte-associated protein-4 agents and baseline grade 1 aspartate aminotransferase/alanine aminotransferase elevation were independent predictive factors of grade ≥2 irLI. Treatment with anti-cytotoxic T-lymphocyte-associated protein-4 was the only independent predictive factor of grade ≥3 irLI. The median OS for patients who experienced any irAEs was significantly longer than of those without irAEs (hazard ratio 0.503, 95% CI 0.398-0.636, p < 0.001). The median OS in patients with grade ≥2 irLI was significantly longer (HR 0.570, 95% CI 0.387-0.838, p = 0.022). There was no significant difference between the median OS in patients with grade ≥3 irLI and the others (p = 0.11). CONCLUSION: The incidence of irLI was significantly higher in patients treated with anti-cytotoxic T-lymphocyte-associated protein-4 agents. Even in patients with pre-existing grade 1 aspartate aminotransferase/alanine aminotransferase elevation, appropriate follow-up and control of the irLI can improve the prognosis.

The Efficacy of Immunotherapy and Clinical Utility of Comprehensive Genomic Profiling in Adenoid Cystic Carcinoma of Head and Neck.

Background and Objectives: Adenoid cystic carcinoma (ACC) of the head and neck is generally slow-growing but has a high potential for local recurrence and metastasis to distant organs. There is currently no standard pharmacological treatment for recurrent/metastatic (R/M) ACC, and there are cases in which immune checkpoint inhibitors (ICIs) are administered for ACC according to head and neck squamous cell carcinoma (HNSCC). However, the efficacy of ICIs for ACC remains unclear, and the predictive biomarkers need to be elucidated. Materials and Methods: The Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database enabled the retrospective but nationwide analysis of 263 cases of ACC of the head and neck. Then, we examined and reported four cases of ACC that received ICIs and comprehensive genomic profiling (CGP) in our institution. Results: The C-CAT database revealed that 59 cases out of 263 received ICIs, and the best response was 8% of objective response rate (ORR) and 53% of disease control rate (DCR) (complete response, CR 3%, partial response, PR 5%, stable disease, SD 44%, progressive disease, PD 19%, not evaluated, NE 29%). The tumor mutational burden (TMB) in ACC was lower overall compared to HNSCC and could not be useful in predicting the efficacy of ICIs. Some cases with MYB structural variants showed the response to ICIs in the C-CAT database. A patient with MYB fusion/rearrangement variants in our institution showed long-term stable disease. Conclusions: ICI therapy is a potential treatment option, and the MYB structural variant might be a candidate for predictive biomarkers for immunotherapy in patients with R/M ACC.

[Malignant paraganglioma mimicking multiple myeloma].

We here present a 33-year-old woman who was referred to our hospital with a complaint of back pain and was found to have elevated IgG and hypercalcemia, as well as osteolytic lesions of pelvis and spines. 18F-FDG-PET/CT scan revealed numerous uptakes in the bones. An examination of the bone marrow revealed increased plasma cells (10.2%). Despite clinical similarities to multiple myeloma, any evidence of plasma cell clonal proliferation, including serum M-protein and light chain restriction, was not found. A reexamination of the bone marrow with a biopsy revealed the proliferation of abnormal cells with chromogranin A and synaptophysin expression but no expression of hematopoietic and epithelial cell markers. Based on these results together with extra-adrenal lesions, a diagnosis of malignant paraganglioma was made. Malignant paraganglioma is known to frequently cause bone metastasis and skeletal related events, whose clinical manifestations are similar to those of multiple myeloma. Since patients with osteolytic lesions, hypercalcemia, and hypergammaglobulinemia are likely to be referred to hematologists, malignant paraganglioma should be considered as a differential diagnosis.

Intestinal phenotype is maintained by Atoh1 in the cancer region of intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasm (IPMN) is a precancerous lesion of pancreatic cancer. Although there are 4 types of IPMN, among which intestinal-type IPMN is likely to progress into invasive cancer known as colloid carcinoma, no information regarding the involvement of the intestinal phenotype in the carcinogenesis of IPMN exists. The present study was conducted to explore how the intestinal differentiation system is maintained during the tumor progression of intestinal-type IPMN using surgical resection specimens. Results showed that Atoh1, a critical transcriptional factor for intestinal differentiation toward the secretory lineages of intestinal epithelial cells, was expressed in an invasive-grade IPMN. To determine the function of Atoh1 in pancreatic cancer, we generated a pancreatic ductal adenocarcinoma (PDAC) cell line overexpressing Atoh1. In a xenograft model, we successfully induced an IPMN phenotype in PDAC cells via Atoh1 induction. Finally, for the first time, we discovered that GPA33 is expressed in intestinal-type IPMN, thereby suggesting a novel target for cancer therapy. In conclusion, the intestinal differentiation system might be maintained during tumor progression of intestinal-type IPMN. Further analysis of the function of Atoh1 in IPMN might be useful for understanding the molecular mechanism underlying the malignant potential during the tumor progression of IPMN.

A Pilot Study Analyzing the Clinical Utility of Comprehensive Genomic Profiling Using Plasma Cell-Free DNA for Solid Tumor Patients in Japan (PROFILE Study).

BACKGROUND: The clinical utility of plasma cell-free DNA in precision cancer medicine has not been established. A pilot study was conducted to investigate the clinical utility of comprehensive genomic profiling by liquid biopsy in a Japanese population. METHODS: In this PROFILE study, 102 patients with advanced solid tumors who showed progression with standard systemic therapy underwent liquid biopsy between August 2017 and February 2020. Liquid biopsy was performed using Guardant360. RESULTS: Of the 102 patients, 56 were women, and the median age was 65 years. Regarding the types of cancer, 31 were hepatobiliary and pancreatic cancer, 17 were gastrointestinal cancer, and 13 were breast cancer. Frequently altered genes were TP53 (53.9%, 46/102), KRAS (25.5%, 26/102), PIK3CA (19.6%, 20/102), and EGFR (17.6%, 18/102). At least one genetic aberration was detected in 92 patients (90.2%). Actionable mutation was discovered in 88 patients (86.3%), and 67 patients (65.7%) were clinical trial candidates. Of the 102 patients, 22 (21.6%) were able to receive biomarker-matched therapy. Their best responses were as follows: 1 complete response, 3 partial responses, 7 stable diseases, and 11 progressive diseases. Additionally, the treated patients were divided on the basis of matching scores (≥ 50% vs. < 50%). The patients were divided into high and low groups. The high group had a higher disease control rate (DCR) of 75% compared with 20% in the low group (P = 0.010). CONCLUSIONS: The results indicate that liquid biopsy is useful for identifying actionable mutations associated with the clinical response of selected patients.

New structural and functional insight into the regulation of Ras.

Mutations in RAS and various components of the Ras signaling pathways are among the most common causative genetic alterations in human cancers, accounting up to 25% of lung cancers and over 90% of pancreatic cancers. Ras is a small GTPase that functions as a 'molecular switch' in a number of signaling pathways that regulate vital eukaryotic cellular functions. Despite our comprehensive understanding of the molecular mechanisms governing the activity of Ras, the clinical outcome of various pharmacologic anti-cancer strategies designed to directly inactivate Ras have been less than satisfactory. In this review, the more recently uncovered mode of regulation of Ras involving non-receptor tyrosine kinase and phosphatase, which have long been suspected of contributing to the oncogenic potential of Ras, will be discussed in the context of both function and structure.

Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation.

Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.

Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis-associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non-mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF-α stabilized Atoh1 protein through the inactivation of GSK-3ß via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5-fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF-κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells.

Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.

BACKGROUND AND AIMS: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Clinical Response of Primary Malignant Pericardial Mesothelioma with Peritoneal Dissemination to Nivolumab.

Malignant pericardial mesothelioma (MPM) is extremely rare, and peritoneal dissemination has not yet been reported. There is no consensus regarding appropriate pharmacological treatment for MPM, including immune checkpoint inhibitors (ICIs). We herein report a 36-year-old man with MPM diagnosed by peritoneal dissemination and treated with an ICI. Cytology of the ascites revealed malignant peritonitis, and a re-evaluation of a pericardial biopsy performed at the previous hospital led to a diagnosis of MPM. The patient was treated with nivolumab and showed a clinical response despite several complications, such as renal dysfunction and performance status deterioration. This case report provides suggestive information for the diagnosis and ICI therapy of a rare type of mesothelioma.

Chemotherapy and COVID-19 Vaccination in Patients With Gastrointestinal Cancer.

BACKGROUND/AIM: Multiple doses of vaccines against the coronavirus disease (COVID-19) provide patients with cancer the opportunity to continue cancer treatment. This study investigated the safety and efficacy of COVID-19 vaccination in patients with cancer and the optimal timing of vaccination during chemotherapy. PATIENTS AND METHODS: A total of 131 patients with gastrointestinal (GI) cancer who received two doses of the COVID-19 vaccine were included in this study. This study combined two cohorts: an evaluation cohort of 79 patients receiving chemotherapy and a control cohort of 52 patients under follow-up after radical surgery. None of the patients had any history of COVID-19. Treatment- and vaccine-related adverse events (AEs) were recorded through outpatient interviews and self-reports. RESULTS: In the evaluation cohort, 62 patients (78.4%) experienced vaccine-related AEs after the first dose, and 62 patients (78.4%) experienced vaccine-related AEs with an increased rate of fever and fatigue after the second dose. In the control cohort, vaccine-related AEs occurred in 28 (53.8%) patients after the first dose and in 37 (71.2%) patients after the second dose, with increased fever and fatigue after the second dose. Of the 79 patients, 49 received chemotherapy before vaccination. Twelve patients (24.5%) changed their treatment schedule: four for safety reasons, four for myelosuppression, and four for convenience. Three patients discontinued the treatment because of disease progression. CONCLUSION: Systemic chemotherapy in patients with GI cancer does not have a markedly negative effect on COVID-19 vaccination, resulting in manageable vaccine-related AEs, and minimizing the need for treatment schedule changes.

Discovery of non-genomic drivers of YAP signaling modulating the cell plasticity in CRC tumor lines.

In normal intestines, a fetal/regenerative/revival cell state can be induced upon inflammation. This plasticity in cell fate is also one of the current topics in human colorectal cancer (CRC). To dissect the underlying mechanisms, we generated human CRC organoids with naturally selected genetic mutation profiles and exposed them to two different conditions by modulating the extracellular matrix (ECM). Among tested mutation profiles, a fetal/regenerative/revival state was induced following YAP activation via a collagen type I-enriched microenvironment. Mechanistically, YAP transcription was promoted by activating AP-1 and TEAD-dependent transcription and suppressing intestinal lineage-determining transcription via mechanotransduction. The phenotypic conversion was also involved in chemoresistance, which could be potentially resolved by targeting the underlying YAP regulatory elements, a potential target of CRC treatment.

The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein.

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

Strategic Insight into the Combination Therapies for Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The 5-year survival rate after curative resection is almost 80%, however, it is still less than satisfactory for metastatic CRC (mCRC). The combination approach including surgery, chemotherapy, molecular targeted therapy, and immunotherapy is a promising strategy due to its synergistic anticancer effect. Moreover, circulating tumor DNA (ctDNA) analysis has been reported to stratify the post-operative risk of recurrence, thus providing clinically valuable information for deciding to conduct adjuvant chemotherapy. Furthermore, multiple new drugs that potentially target undruggable genes, including KRAS, have been developed. In this review, we discuss the current management of patients with mCRC and future perspectives in the light of a combination therapeutic strategy.

Clinical Utility of Comprehensive Genomic Profiling in Patients with Unresectable Hepatocellular Carcinoma.

The molecular mechanism of hepatocellular carcinoma (HCC) is partially demonstrated. Moreover, in the patients receiving multiple molecular-targeted therapies, the gene alternations are still unknown. Six molecular-targeted therapies of unresectable HCC (uHCC) and comprehensive genomic profiling (CGP) have been approved in clinical practice. Hence, the utility of CGP in patients with uHCC treated with multiple molecular-targeted agents is investigated. The data of the patients with uHCC who received CGP tests were collected, retrospectively, between February 2021 and May 2022. Gene alterations detected by foundation testing, excluding variants of unknown significance, were reported in all nine patients. The samples for CGP were derived from liver tumor biopsy (n = 2), surgical specimens of bone metastases (n = 2), and blood (n = 5). The median number of systemic therapies was four. Seven patients were candidates eligible for clinical trials. One patient with a high tumor mutation burden (TMB) could receive pembrolizumab after CGP. This study presented genomic alternations after receiving multiple molecular-targeted therapies. However, further investigation needs to be conducted to develop personalized therapies and invent newer agents for treating HCC.

Improved bioavailability of a water-insoluble drug by inhalation of drug-containing maltosyl-ß-cyclodextrin microspheres using a four-fluid nozzle spray drier.

We previously developed a unique four-fluid nozzle spray drier that can produce water-soluble microspheres containing water-insoluble drug nanoparticles in one step without any common solvent between the water-insoluble drug and water-soluble carrier. In the present study, we focused on maltosyl-ß-cyclodextrin (malt-ß-CD) as a new water-soluble carrier and it was investigated whether drug/malt-ß-CD microspheres could improve the bioavailability compared with our previously reported drug/mannitol (MAN) microspheres. The physicochemical properties of bare drug microparticles (ONO-2921, a model water-insoluble drug), drug/MAN microspheres, and drug/malt-ß-CD microspheres were evaluated. In vitro aerosol performance, in vitro dissolution rate, and the blood concentration profiles after intratracheal administration were compared between these formulations. The mean diameter of both drug/MAN and drug/malt-ß-CD microspheres was approximately 3-5 µm and both exhibited high aerosol performance (>20% in stages 2-7), but drug/malt-ß-CD microspheres had superior release properties. Drug/malt-ß-CD microspheres dissolved in an aqueous phase within 2 min, while drug/MAN microspheres failed to dissolve in 30 min. Inhalation of drug/malt-ß-CD microspheres enhanced the area under the curve of the blood concentration curve by 15.9-fold than that of bare drug microparticles and by 6.1-fold than that of drug/MAN microspheres. Absolute bioavailability (pulmonary/intravenous route) of drug/malt-ß-CD microspheres was also much higher (42%) than that of drug/MAN microspheres (6.9%). These results indicate that drug/malt-ß-CD microspheres prepared by our four-fluid nozzle spray drier can improve drug solubility and pulmonary delivery.

MYC-PDL1 axis reduces sensitivity to nivolumab in recurrent head and neck squamous cell carcinoma.

Patients with recurrent or metastatic squamous cell carcinoma of the head and neck (R/M HNSCC) have a poor prognosis. Recently, the use of immune checkpoint inhibitors (ICIs) for drug treatment has been expanding . However, the response rate to immunotherapy is low. Therefore, the identification of predictive biomarkers of response and resistance to ICIs is required for various types of malignant tumors. We report the case of a patient with recurrent and metastatic HNSCC who simultaneously showed different responses to nivolumab in metastatic lesions. After administering nivolumab, metastasis to the multiple cervical lymph node metastases showed a significant reduction, whereas a new metastasis to the right axillary lymph node occurred . Each surgical specimen was analyzed using the cancer gene panel test (FoundationOne CDx) to elucidate why treatment response is distinct among the same patient. Next-generation sequencing revealed MYC amplification and programmed cell death-1 loss in the right axillary lymph nodes but not cervical lymph nodes. Furthermore, t he histopathological findings suggested that MYC amplification regulated programmed death-ligand 1 expression and was involved in a decreased response to ICIs. This result is expected to help predict the efficacy of ICI treatment and select therapeutic agents.