Kazufumi Honda

An 80‐year‐old female was referred to our hospital 6 years earlier with an abnormal plasma apolipoprotein A2 isoform (ApoA2‐i) index, which is a promising biomarker for pancreatic cancer. Endoscopic ultrasonography (EUS) revealed a single small cystic lesion in the pancreatic head. Subsequently, the patient was followed up regularly. Two years after the initial visit, her ApoA2‐i index was 50.2 μg/mL, and her plasma CA19‐9 level was 42.5 U/mL. Magnetic resonance cholangiopancreatography (MRCP) performed 3 years after the initial visit also demonstrated small cystic lesions in the pancreatic head, consistent with the findings of the initial EUS, and a signal defect in the main pancreatic duct (MPD) in the pancreatic body, with a normal upstream MPD diameter. Her ApoA2‐i index was 52.4 μg/mL, and her plasma CA19‐9 level was 42.0 U/mL. Follow‐up MRCP indicated similar cystic lesions in the pancreatic head and a signal defect in the MPD in the pancreatic body, with a normal upstream MPD diameter. However, her serial ApoA2‐i indices gradually decreased from 52.1 to 35.7 μg/mL, correlated with an increase in plasma CA19‐9. The latest MRCP demonstrated a similar signal defect and dilatation of the upstream MPD diameter compared with the previous MPD diameters. Her ApoA2‐i index slightly decreased to 35.2 μg/mL, whereas her CA19‐9 level remarkably increased to 1320 U/mL. Endoscopic retrograde pancreatography with cytology demonstrated adenocarcinoma cells. In the present case, we observed longitudinal changes in ApoA2‐i indices correlated with CA19‐9 and MRCP findings.

Background: Pancreatic ductal adenocarcinoma (PDAC) has one of the poorest prognoses among malignant tumors, mainly due to the difficulty of early diagnosis. Therefore, it is crucial to identify reliable blood markers for a highly sensitive diagnostic system. We previously developed a highly sensitive extracellular vesicle (EV)-counting system, which can quantify the absolute number of specific EVs in serum. In this study, a multiplex assay using lectins that recognize specific glycans on EVs in the serum of PDAC patients was performed to select the optimal lectin combination. Methods: The glycan alteration signature of serum EVs from patients with PDAC was analyzed using a lectin-based multiplex assay combined with the EV-counting system. The optimal lectin combination that recognizes PDAC-specific changes was selected using machine learning analyses (support vector machine) for high diagnostic performance across independent patient cohorts. Results: An optimal lectin combination, Jacalin and Agaricus bisporus agglutinin (ABA), for PDAC detection was identified using machine learning analysis. This lectin-based system, reflecting changes in Jacalin/ABA binding, showed significantly higher diagnostic performance (area under the curve [AUC] = 0.890 and 0.971) than that of the conventional diagnostic marker carbohydrate antigen 19-9 (CA19.9; AUC = 0.752). Notably, the system achieved an AUC of 0.870 in patients with the stage I disease. Conclusions: These findings highlight the potential of a serum EV-based diagnostic system leveraging Jacalin and ABA glycan recognition for the early detection of PDAC.

Actinin‐4 (gene name: ACTN4 ) is an actin‐bundling protein implicated in cancer invasion and metastasis. This study evaluated whether ACTN4 amplification and actinin‐4 protein expression were associated with osimertinib efficacy in epidermal growth factor receptor‐mutant non‐small cell lung cancer. We retrospectively analyzed 63 patients with epidermal growth factor receptor‐mutant non‐small cell lung cancer treated with osimertinib as first‐line treatment. Immunohistochemistry was performed for pretreatment tumor tissues. Actinin‐4 immunohistochemistry positivity was defined as positive staining of ≥ 30% tumor cells. In positive cases, ACTN4 amplification was assessed via fluorescence in situ hybridization. Progression‐free survival and overall survival were compared across groups. Among 63 patients (median age: 73 years, 52 with Eastern Cooperative Oncology Group performance status 0–1, 63 with adenocarcinoma; epidermal growth factor receptor mutations: 19del/L858R/uncommon = 32/24/7), there were 33 and 30 actinin‐4 immunohistochemistry‐positive and actinin‐4 immunohistochemistry‐negative cases, respectively. The propensity score‐weighted overall survival and progression‐free survival were significantly shorter for actinin‐4 immunohistochemistry‐positive patients than for actinin‐4 immunohistochemistry‐negative patients (overall survival: hazard ratio, 2.76; 95% confidence interval, 1.02–7.45; progression‐free survival: hazard ratio, 1.91; 95% confidence interval, 1.03–3.54). Among the 33 actinin‐4 immunohistochemistry‐positive cases, four showed positivity in ACTN4 fluorescence in situ hybridization. Overall survival and progression‐free survival were numerically shorter for patients with ACTN4 positivity than for those with ACTN4 negativity in fluorescence in situ hybridization. The findings suggest that ACTN4 amplification and actinin‐4 protein expression are prognostic markers for poor osimertinib efficacy in epidermal growth factor receptor‐mutant non‐small cell lung cancer.

INTRODUCTION: High-risk stigmata (HRS) and worrisome features (WFs) are imaging findings that reflect malignant potential of intraductal papillary mucinous neoplasms (IPMNs). The aim of this study was to determine the potential utility of stratifying malignant potential of IPMNs using apolipoprotein-A2 isoforms (apoA2-i) as a blood biomarker. METHODS: A total of 212 patients with IPMNs diagnosed by either magnetic resonance cholangiopancreatography or contrast-enhanced computed tomography, and endoscopic ultrasound were retrospectively enrolled. ApoA2-i and CA19-9 of all patients and 295 healthy individuals were measured. The distributions of apoA2-i and CA19-9 were analyzed in association with the malignant potential evaluated by imaging. RESULTS: In 212 patients with IPMN, 17 had HRS (HRS group), 70 had WFs (WF group), and 125 had no endoscopic ultrasound findings of HRS or WFs (non-WF group). The median of the apoA2-i Index in the HRS, WF, non-WF, and healthy groups were 57.7, 72.4, 87.5, and 87.9 μg/mL, respectively, with significantly lower levels in the HRS and WF groups (P < 0.001). By contrast, CA19-9 showed no significant difference among the 4 groups. The area under the curve of the apoA2-i Index to differentiate patients in the HRS and WF groups from the non-WF group was significantly higher than that of the CA19-9 (0.676 vs 0.554, P = 0.029). DISCUSSION: The diagnostic performance of apoA2-i for detecting IPMN with malignant potential was superior to that of CA19-9. The apoA2-i Index could serve as a useful biomarker for risk stratification and surveillance of IPMNs.

Pancreatic cancer is a rare and refractory cancer, and the development of blood biomarkers for the enrichment of high-risk individuals who have risk factors for pancreatic cancer from the asymptomatic population is an unmet medical need. We identified abnormalities in the C-terminal truncation of the apolipoprotein A2 dimer (apoA2-isoforms: apoA2-i) in the blood of pancreatic cancer patients through proteomic analysis, and we have reported the potential for diagnosing resectable pancreatic cancer by detecting these abnormalities. We successfully developed enzyme-linked immunosorbent assay (ELISA) reagents for measuring apoA2-i for research use only, and then the basic data for diagnosing pancreatic cancer were accumulated by several studies using these reagents. In 2023, ELISA for measuring apoA2-i was regenerated by the regulation under the Japanese Quality Management System (QMS), it received marketing approval in Japan as an in vitro diagnostic (IVD) kit to aid in the diagnosis of pancreatic cancer, and it is now used in clinical practice. This review chronicles the journey from the initial discovery through omics research, to demonstrating clinical utility via multicenter studies in Japan and international collaborative research using the research reagent and validating the clinical performance of the IVD ELISA kit through a regulatory, science-guided, clinical trial in Japan, and finally to recent activities in the USA.

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular‐targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (Kras^muOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Kras^mu/p53^muOR) from murine epithelia of the pancreatic duct in Kras^LSL‐G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Kras^mu/p53^muOR compared with Kras^muOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation.

Summary: α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MBA-MD-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

Background: We have previously reported apolipoprotein A2-isoforms (apoA2-is) as candidate plasma biomarkers for early-stage pancreatic cancer. The aim of this study was the clinical development of apoA2-is. Methods: We established a new enzyme-linked immunosorbent sandwich assay for apoA2-is under the Japanese medical device Quality Management System requirements and performed in vitro diagnostic tests with prespecified end points using 2732 plasma samples. The clinical equivalence and significance of apoA2-is were compared with CA19-9. Results: The point estimate of the area under the curve to distinguish between pancreatic cancer (n = 106) and healthy controls (n = 106) was higher for apoA2-ATQ/AT [0.879, 95% confidence interval (CI): 0.832–0.925] than for CA19-9 (0.849, 95% CI 0.793–0.905) and achieved the primary end point. The cutoff apoA2-ATQ/AT of 59.5 μg/mL was defined based on a specificity of 95% in 2000 healthy samples, and the reliability of specificities was confirmed in two independent healthy cohorts as 95.3% (n = 106, 95% CI 89.4–98.0%) and 95.8% (n = 400, 95% CI 93.3–97.3%). The sensitivities of apoA2-ATQ/AT for detecting both stage I (47.4%) and I/II (50%) pancreatic cancers were higher than those of CA19-9 (36.8% and 46.7%, respectively). The combination of apoA2-ATQ/AT (cutoff, 59.5 μg/mL) and CA19-9 (37 U/mL) increased the sensitivity for pancreatic cancer to 87.7% compared with 69.8% for CA19-9 alone. The clinical performance of apoA2-is was blindly confirmed by the National Cancer Institute Early Detection Research Network. Conclusions: The clinical performance of ApoA2-ATQ/AT as a blood biomarker is equivalent to or better than that of CA19-9.

α‐Actinin4 (ACTN4), an isoform of non‐muscular α‐actinin, is involved in enhancing cell motility and promoting cancer infiltration and metastasis in various cancers. However, information remains limited regarding the pathological significance of ACTN4 expression in upper urinary tract urothelial carcinomas (UUTUCs). We obtained tumor samples from 168 consecutive patients with newly diagnosed UUTUCs (92 with renal pelvic cancers and 76 with ureteral cancers), who were treated with nephroureterectomy or partial ureterectomy, and analyzed the expression of the ACTN4 protein and the amplification of ACTN4 using immunohistochemistry and fluorescence in situ hybridization (FISH), respectively. The median follow‐up duration was 65 months. Among 168 cases, 49 (29%) showed ACTN4 protein overexpression and 25 (15%) showed copy number gain (≥4 copies per cell) of ACTN4. The copy number gain of ACTN4 detected using FISH significantly correlated with ACTN4 protein overexpression and several adverse clinicopathological factors, including higher pathological T stage, lymphovascular invasion, lymph node metastasis, positive surgical margin, concomitant subtype histology, and non‐papillary gross finding. Cox univariate regression analyses revealed that both copy number gain of ACTN4 and ACTN4 protein overexpression were significant risk factors for extraurothelial recurrence and death (each p < 0.0001), but multivariate analysis revealed that only copy number gain of ACTN4 was an independent risk factor for extraurothelial recurrence and death (p = 0.038 and 0.027, hazard ratio = 2.16 and 2.17, respectively). This is the first study demonstrating the aberrant expression status of ACTN4 in UUTUC and indicating its putative usefulness as a prognostic indicator in patients with UUTUC.

“Liquid biopsy” is an efficient diagnostic tool used to analyse biomaterials in human body fluids, such as blood, saliva, breast milk, and urine. Various biomaterials derived from a tumour and its microenvironment are released into such body fluids and contain important information for cancer diagnosis. Biomaterial detection can provide “real-time” information about individual tumours, is non-invasive, and is more repeatable than conventional histological analysis. Therefore, over the past two decades, liquid biopsy has been considered an attractive diagnostic tool for malignant tumours. Although biomarkers for oral cancer have not yet been adopted in clinical practice, many molecular candidates have been investigated for liquid biopsies in oral cancer diagnosis, such as the proteome, metabolome, microRNAome, extracellular vesicles, cell-free DNAs, and circulating tumour cells. This review will present recent advances and challenges in liquid biopsy for oral cancer diagnosis.