BACKGROUND/AIM:Neurotrophic tropomyosin receptor kinase (NTRK) fusion is one of the druggable driver genes of thyroid cancer, which is confirmed in surgical specimens. However, there is a risk that certain fusion patterns may not be detected using gene panel testing. This study aimed to identify patients harboring NTRK fusion who might be overlooked by panel testing. PATIENTS AND METHODS:Two patient groups were analyzed. Group A included patients with advanced thyroid cancer who underwent panel testing. Immunostaining for pan-tropomyosin receptor kinase (TRK) was performed, and RNA sequencing was added to cases without mitogen-activated protein kinase alterations. Group B, including surgical cases with papillary thyroid carcinoma, was screened by immunohistochemistry for BRAF mutation and pan-TRK. RNA sequencing was performed on BRAF-negative and pan-TRK-positive cases. RESULTS:Among the 100 cases with advanced thyroid cancer in group A, gene panel testing detected mitogen-activated protein kinase alterations in 88. The most common alteration was the BRAF mutation (n=72), followed by RET fusion (8), RAS mutation (7), and NTRK fusion (1). Additional RNA sequencing for eight cases did not detect pathological NTRK fusions. Group B included 76 cases, in which immunostaining identified two cases with NTRK fusions: TFG-NTRK1 fusion was detected by panel testing, whereas SQSTM1-NTRK3 fusion was overlooked. In immunohistochemistry, NTRK1 fusion showed relatively strong staining, whereas NTRK3 showed weak staining. Notably, one case with the ETV6-NTRK3 fusion had no staining. CONCLUSION:This study identified a case of thyroid carcinoma harboring NTRK fusion that was overlooked by panel testing. Active searching for NTRK fusions is desirable in cases without mitogen-activated protein kinase abnormalities or with pan-TRK staining.

Background:Mixed ductal-lobular carcinoma (MDL) of the breast exhibits considerable molecular complexity. The pathways leading to the glycogen-rich clear cell morphology of the breast tumors, and its clinical relevance, currently remain unclear. Herein, we report a case of MDL, predominantly composed of invasive lobular carcinoma with a glycogen-rich clear cell pattern (gILC), accompanied by classic invasive lobular carcinoma and invasive ductal carcinoma (IDC). Case presentation:A 70-year-old woman presented with a 3.5 cm mass in the left breast, for which total mastectomy was performed. The pathological diagnosis was MDL predominantly comprising gILC. Tissue samples from the gILC and IDC areas were subjected to whole-exome and RNA sequencing. The gILC region had a higher tumor mutation burden than the IDC. Three stop-gain single nucleotide variations (SNVs) in CDH1, SETD2, and USP9 and two nonsynonymous SNVs in PIK3CA were identified in the gILC region, whereas only two nonsynonymous SNVs in SMAD4 and PIK3CA were identified in the IDC region. Phylogenetic analysis revealed a common ancestor of gILC and IDC, sharing a pathogenic PIK3CA p.H1047L mutation. Reduced SETD2 protein and H3K36me3 levels and the DNA mismatch repair-microsatellite instability-associated mutational signatures SBS6 and SBS26 were uniquely demonstrated in gILC. Further, a structural variant involving HNF1B and elevated HNF1B transcript levels was detected in gILC. The predominant gILC component was estrogen receptor-positive. Adjuvant endocrine therapy was administered postoperatively, and the patient currently remains disease-free at 51 months. Conclusion:In this case, the gILC and IDC components of an MDL shared a common origin, but exhibited marked genomic divergence. This experience also shows that SETD2 functional impairment may underlie gILC hypermutation, while HNF1B overexpression could contributes to a glycogen-rich clear cytoplasm. Overall, this case emphasizes the complexity of MDL with gILC, and highlights the need for further studies to clarify the underlying molecular mechanisms and their prognostic implications.

Formalin-fixed paraffin-embedded (FFPE) tissue is the principal substrate for cancer genomic profiling, yet pre-analytical factors affect nucleic acid quality. In our workflow, transurethral resection (TUR) specimens of bladder urothelial carcinoma are fixed promptly but exposed to electrocautery, whereas robot-assisted radical cystectomy specimens are refrigerated overnight before fixation; we compared nucleic acid quality between these specimen types. We analyzed 56 FFPE primary bladder urothelial carcinoma specimens (33 TUR, 23 cystectomy). DNA quality was assessed by the DNA Integrity Number (DIN) and short-to-long cycle threshold ratio (S/L Ct ratio), and RNA quality by the RNA Integrity Number (RIN) and percentage of RNA fragments ≥200 nucleotides (DV200), with group comparisons, Spearman correlations, and multivariable regression (specimen type, fixation duration, storage time). The median DIN (4.4 vs. 3.8, p = 0.004) and S/L Ct ratio (0.943 vs. 0.894, p < 0.001) were higher in TUR than cystectomy specimens; the RIN was also higher (1.9 vs. 1.7, p = 0.040), though low in both groups, and DV200 did not differ (50.3% vs. 47.7%, p = 0.934). In multivariable analysis, TUR specimen type independently predicted higher DIN (β = +0.460, p = 0.040) and S/L Ct ratio (β = +0.442, p = 0.009), whereas longer storage time independently decreased the S/L Ct ratio (β = -0.513, p < 0.001) and RIN (β = -0.352, p = 0.010). In our institutional workflow, TUR specimens showed higher DNA quality metrics than robot-assisted radical cystectomy specimens subjected to overnight refrigerated pre-fixation delay, whereas this workflow-associated advantage did not clearly extend to DV200-defined RNA fragment-length quality. Storage time was associated with selected aspects of nucleic acid deterioration.

Cribriform pattern and intraductal carcinoma of the prostate are recognized adverse histological features, yet their prognostic value in treatment-naïve metastatic disease remains uncertain. We conducted a single-center retrospective study of 183 biopsy-proven prostate carcinomas (105 with metastatic castration-sensitive prostate carcinoma and 78 non-metastatic high-grade cases) diagnosed between 2017 and 2024. Cribriform pattern, intraductal carcinoma of the prostate, and coagulative tumor necrosis were recorded per core and summarized as patient-level binary status and as semiquantitative proportions per cancer-positive core. Two multivariable logistic regression models (binary and semiquantitative) were fitted, and receiver operating characteristic (ROC) analysis evaluated the discriminatory performance of the cribriform proportion. Cribriform pattern and intraductal carcinoma of the prostate were more frequent in metastatic castration-sensitive prostate carcinoma. In the semiquantitative model, the cribriform proportion remained independently associated with metastatic status [odds ratio (OR) 1.29, 95% CI 1.07-1.55, p = 0.008; per 1.0 increase in the proportion, equivalent to OR 1.03 per 10%-point increase], whereas necrosis remained significant only in the binary model. The cancer-positive core rate and a lower total number of biopsy cores were predictive in both models, whereas prostate-specific antigen, intraductal carcinoma of the prostate, and Grade Group composition were not independent predictors. ROC analysis for the cribriform proportion yielded an area under the curve of 0.704, with a Youden Index cut-off of 0.445 (approximately half of cancer-positive cores), corresponding to a sensitivity of 57.1% and a specificity of 75.6%. These findings indicate that semiquantitative reporting of cribriform pattern - expressed as the proportion of cancer-positive cores - adds discriminatory information for metastatic status at presentation and could complement binary reporting in high-grade disease. From a clinical perspective, such evaluation may refine risk stratification at diagnosis and support treatment intensification strategies in very-high-risk patients.

Low-grade oncocytic tumor (LOT) of the kidney is an emerging entity among renal oncocytic tumors. While the histological features of LOT of the kidney are similar to those of renal oncocytoma, LOT immunohistochemically expresses keratin 7 (KRT7) but not KIT while renal oncocytoma expresses KIT. Molecular analyses of LOTs of the kidney using next generation sequencing revealed those tumors harbor mutations of mTOR-related genes. An 80-year-old Japanese man with a history of clear cell renal cell carcinoma and prostatic cancer underwent resection of the tumor of the right kidney, 10 mm in diameter, which was monitored for six years. The tumor was histologically composed of oncocytic cells that expressed KRT7, vimentin, SDHA, SDHB and fumarate hydratase, but not KIT, GATA3 and alpha-methylacyl-CoA racemase. We diagnosed the tumor as LOT of the kidney. Whole-exome sequencing of the LOT revealed single nucleotide variants in the DNA-binding region of forkhead box I1 (FOXI1), the coil 1B domain of keratin 6 C (KRT6C) and the intracytoplasmic region of gap junction delta 2 (GJD2), which encodes connexin 36. However, there was no mutations in mTOR-related genes. No copy number alterations were detected in the tumor. We report three mutations in genes that have not been previously reported in LOT of the kidney. The genes are not related to the mTOR pathway. Therefore, LOT of the kidney might occur through several mechanisms and/or include several types of renal oncocytic tumors.

BACKGROUND:Liposarcoma and lymphoma are very rare tumors, and their combination is extremely rare. Moreover, there have been no reports of liposarcoma and lymphoma occurring in the same region. CASE:A 58-year-old man presented to Kanagawa Cancer Center with a mass in his left thigh and underwent a needle biopsy. Histological analysis showed an increase in the number of small lymphocytes and plasma cells; immunohistochemical analysis showed an increase in CD20-positive cells with Lambda light-chain restriction; therefore, the diagnosis of B-cell malignancy with plasma cell differentiation was made. A bone marrow biopsy specimen showed infiltration of atypical cells of the same phenotype and increased serum IgM-M levels; therefore, a diagnosis of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma (LPL) was made. The needle biopsy specimen showed scattered CDK4-positive cells in the background of the lymphoma cells and sporadic MDM2 signal amplification on fluorescence in situ hybridization, suggesting mixed well-differentiated liposarcoma (WDL). Tumor resection was performed. The tumor contained a mixture of WDL and LPL areas. RNA sequencing revealed upregulated expression of chemokine genes, including CCL5, CCL18, and CCL19, in WDL and that of the corresponding chemokine receptor genes CCR4, CCR6, and CCR7 in the lymphoma cells. CONCLUSION:Chemokine-chemokine receptor axes may be involved in the pathogenesis of LPL cell-infiltrating WDL. This is an extremely rare case, and we have reported some considerations regarding the tumorigenesis of LPL cell-infiltrating WDL.

Background:MDM2 protein regulates p53 protein activity; however, the prognostic impact of MDM2 expression in primary lung cancer on patient prognoses is unknown. This study investigated the prognostic implications of MDM2 protein expression among patients with epidermal growth factor receptor mutant (EGFRm) lung adenocarcinoma with pathologic lymph node metastases after surgery. Methods:Immunohistochemical analysis was conducted to determine the MDM2 expression of pN1-N2 EGFRm [exon 19 deletion mutation (Ex19) and exon 21 L858R mutation (Ex21)] lung cancer that were surgically resected between January 2010 and December 2020 (n=124). The postoperative prognosis and cumulative incidence of recurrence were retrospectively analyzed. Results:MDM2 protein expression was positive in 56 patients (45.2%) with EGFRm lung adenocarcinoma. The relapse-free survival (RFS) rate was significantly better among patients with MDM2+ vs. those with MDM2- (5-year RFS: 35.5% vs. 14.8%, P=0.04). Multivariable analysis revealed MDM2+ as an independent favorable prognostic factor for RFS [hazard ratio (HR), 0.64; 95% confidence interval (CI): 0.42-0.98; P=0.04]. Among patients with Ex21 lung cancer (n=63), significantly better RFS and overall survival (OS) were seen in patients with MDM2+ than those with MDM2- (5-year RFS: 36.4% vs. 9.2%, P=0.005; 5-year OS: 81.8% vs. 39.3%, P=0.002). Multivariable analysis revealed that among patients with Ex21 lung cancer, MDM2+ was an independent favorable prognostic factor for RFS (HR, 0.43; 95% CI: 0.23-0.80; P=0.008) and OS (HR, 0.32; 95% CI: 0.15-0.67; P=0.003), and it was also associated with a low cumulative incidence of overall distant recurrence (HR, 0.42; 95% CI: 0.20-0.87; P=0.02) and central nervous system recurrence (HR, 0.23; 95% CI: 0.07-0.80; P=0.02). Conclusions:MDM2 protein expression is a prognostic predictor of RFS and OS in patients with pN1-N2 Ex21 lung cancer after curative surgery. MDM2 can be a novel biomarker for predicting postoperative prognosis and distant metastasis, especially in the central nervous system.

Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3-4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An "all-in-one" adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors.

Placental transmogrification of the lung (PTL) is a rare, bullous lung disease with unknown pathogenesis. This is a case of PTL in AKT1-related Proteus syndrome. This overgrowth spectrum disorder was caused by a mosaic AKT1 pathogenic variant. https://bit.ly/4h6S6AG.

Formalin-fixed paraffin-embedded (FFPE) tissue samples are widely used for genomic analyses in clinical and research settings. However, prolonged storage at room temperature may compromise nucleic acid quality, potentially affecting the reliability of molecular testing. In this study, we investigated the impact of various storage temperatures and repeated temperature changes on nucleic acid quality in FFPE samples. Four surgically resected tumor cases were analyzed. Following cold ischemia times of 29-60 min, tumor tissues were sampled upon arrival at the pathology department, divided into five equal parts, and fixed in 10 % neutral buffered formalin. The resulting FFPE blocks were stored at 18 °C, 4 °C, -20 °C, -80 °C, or - 150 °C. After storage, the blocks were returned to room temperature for sectioning and analysis. DNA and RNA quality were assessed at 3, 6, and 12 months using DNA integrity number (DIN), short-to-long cycle threshold (S/L Ct) ratio, RNA integrity number (RIN), and DV200. Samples stored at 18 °C and 4 °C exhibited time-dependent deterioration in certain indicators, whereas those stored at -20 °C or below maintained stable nucleic acid quality. Despite multiple freeze-thaw cycles, no significant degradation was observed under colder conditions. Our findings suggest that -20 °C storage is a feasible and effective approach for preserving nucleic acid integrity in FFPE tissues. Although limited by a small sample size (n = 4), this study provides practical insights into tissue archiving strategies and supports selective implementation of refrigerated storage for long-term molecular analysis.

Neutrophil extracellular traps (NETs) released from neutrophils are related to cancer progression. However, the relationship between the therapeutic effects of immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-PD-L1 antibodies and plasma NET concentration in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, concentrations of citrullinated histone H3 (CitH3), a surrogate marker of NETs, in plasma before/after treatment were examined in patients with advanced or recurrent NSCLC undergoing ICI treatment (n = 185). The clinical significances of NET levels before/after treatment and posttreatment changes were statistically evaluated. As a result, multivariate Cox analysis showed that high NET levels before treatment were statistically significant predictors of unfavorable overall survival (OS; p < 0.001, HR 1.702, 95% CI 1.356-2.137) and progression-free survival (PFS; p < 0.001, HR 1.566, 95% CI 1.323-1.855). The Kaplan-Meier curves showed significant separation between the high- and low-NET groups in OS (p = 0.002) and PFS (p < 0.001). Additionally, high NET levels after treatment were also significantly associated with worse OS (p < 0.001) and PFS (p < 0.001) by multivariate Cox analysis. Notably, the pretreatment NET levels were significantly correlated with the plasma levels of NET-related inflammatory cytokines, such as IL-6 and IL-8, and with NET-related gene expression and immune-suppressive profile in peripheral blood mononuclear cells. Our findings suggest that NETs released from activated neutrophils might reduce the clinical efficacy of ICIs in patients with NSCLC.