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Metastasis is responsible for the majority of deaths of patients with cancer. However, mechanisms governing metastasis in colorectal cancer remain largely unknown. Here we investigated how colorectal cancer cells acquire metastatic potential using a novel mouse model of colorectal cancer that spontaneously develops liver metastasis, generated by introducing sporadic mutations of Ctnnb1, Kras, Trp53, and Smad4 (CKPS) genes. Proteomic analyses revealed elevated expression of colorectal cancer stem cell markers ALCAM (CD166) and PROM1 (CD133) in colorectal cancer cells from the metastatic model compared with those from a nonmetastatic model. Spleen-to-liver metastasis assays using colorectal cancer cells derived from the CKPS model (CKPS cells) demonstrated the functional importance of ALCAM and PROM1 in initiating metastasis. Genetic and pharmacologic analyses using CKPS cells in 2D and spheroid culture revealed that expression of ALCAM and PROM1 is regulated positively and negatively by the cAMP/PKA/CREB and TGFβ/SMAD4 pathways, respectively. Consistently, phospho-CREB was expressed in both primary and metastatic lesions of CKPS mice and patients with colorectal cancer, and knockout of CREB in CKPS cells reduced their spheroid-forming and metastasis-initiating abilities. Treatment with a CREB inhibitor potentiated the effect of irinotecan in suppressing liver metastasis by CKPS cells. These results reveal the essential roles of ALCAM and PROM1, as well as their upstream regulators, the cAMP/PKA/CREB and TGFβ/SMAD4 pathways, in maintaining the stemness and metastatic potential of colorectal cancer cells and indicate that CREB inhibition may be a potential therapeutic strategy against metastatic colorectal cancer. Significance: This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

The prognostic significance of peritoneal lavage cytology (PLC) in patients with pancreatic ductal adenocarcinoma (PDAC) remains controversial. The purpose of this study was to evaluate the prognostic impact of PLC status in PDAC patients.Patients intending to undergo resection for PDAC between 2007 and 2020 were included. Survival was compared among patients who underwent resection with negative or positive PLC status and those who did not undergo resection. Univariable and multivariable analyses were conducted to evaluate the prognostic impact of positive PLC status. A systematic literature review was performed to evaluate the correlation between prognosis and the positive PLC rate.A total of 480 patients formed the study cohort and were divided as follows: 438 in the negative PLC group, 18 in the positive PLC group, and 24 in the no resection group. Although the median survival time significantly differed between the negative and positive PLC groups (35.7 vs. 13.6 months, P0.001), it did not significantly differ between the positive PLC and no resection groups (13.6 vs. 12.2 months, P = 0.605). Multivariable analyses demonstrated that positive PLC status (hazard ratio = 3.54, 95% confidence interval = 1.97-6.38, P0.001) was the strongest poor prognostic factor. Based on statistical analyses for the systematic review, the prognostic impact of positive PLC status weakened significantly as the institutional positive PLC rate increased (P = 0.044).Resection did not improve the prognosis of patients with positive PLC status in our cohort. The institutional positive PLC rate may be a good reference for surgical indication in these patients.

Adoptive cancer immunotherapy can induce objective clinical efficacy in patients with advanced cancer; however, a sustained response is achieved in a minority of cases. The persistence of infused T cells is an essential determinant of a durable therapeutic response. Antitumor T cells undergo a genome-wide remodeling of the epigenetic architecture upon repeated antigen encounters, which inevitably induces progressive T-cell differentiation and the loss of longevity. In this study, we identified PR domain zinc finger protein 1 (PRDM1) ie, Blimp-1, as a key epigenetic gene associated with terminal T-cell differentiation. The genetic knockout of PRDM1 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) supported the maintenance of an early memory phenotype and polyfunctional cytokine secretion in repeatedly stimulated chimeric antigen receptor (CAR)-engineered T cells. PRDM1 disruption promoted the expansion of less differentiated memory CAR-T cells in vivo, which enhanced T-cell persistence and improved therapeutic efficacy in multiple tumor models. Mechanistically, PRDM1-ablated T cells displayed enhanced chromatin accessibility of the genes that regulate memory formation, thereby leading to the acquisition of gene expression profiles representative of early memory T cells. PRDM1 knockout also facilitated maintaining an early memory phenotype and cytokine polyfunctionality in T-cell receptor-engineered T cells as well as tumor-infiltrating lymphocytes. In other words, targeting PRDM1 enabled the generation of superior antitumor T cells, which is potentially applicable to a wide range of adoptive cancer immunotherapies.

Objectives DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. Methods We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. Results A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). Conclusions To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Supplementary Table S3. Factors related to overall survival

Supplementary figureS1 - Representative cases of pancreatic NEN-G3

Supplementary Data from The cAMP/PKA/CREB and TGFβ/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells

Metastasis is responsible for the majority of deaths of patients with cancer. However, mechanisms governing metastasis in colorectal cancer remain largely unknown. Here we investigated how colorectal cancer cells acquire metastatic potential using a novel mouse model of colorectal cancer that spontaneously develops liver metastasis, generated by introducing sporadic mutations of Ctnnb1, Kras, Trp53, and Smad4 (CKPS) genes. Proteomic analyses revealed elevated expression of colorectal cancer stem cell markers ALCAM (CD166) and PROM1 (CD133) in colorectal cancer cells from the metastatic model compared with those from a nonmetastatic model. Spleen-to-liver metastasis assays using colorectal cancer cells derived from the CKPS model (CKPS cells) demonstrated the functional importance of ALCAM and PROM1 in initiating metastasis. Genetic and pharmacologic analyses using CKPS cells in 2D and spheroid culture revealed that expression of ALCAM and PROM1 is regulated positively and negatively by the cAMP/PKA/CREB and TGFβ/SMAD4 pathways, respectively. Consistently, phospho-CREB was expressed in both primary and metastatic lesions of CKPS mice and patients with colorectal cancer, and knockout of CREB in CKPS cells reduced their spheroid-forming and metastasis-initiating abilities. Treatment with a CREB inhibitor potentiated the effect of irinotecan in suppressing liver metastasis by CKPS cells. These results reveal the essential roles of ALCAM and PROM1, as well as their upstream regulators, the cAMP/PKA/CREB and TGFβ/SMAD4 pathways, in maintaining the stemness and metastatic potential of colorectal cancer cells and indicate that CREB inhibition may be a potential therapeutic strategy against metastatic colorectal cancer.Significance:This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

Purpose: Patients with pancreatic neuroendocrine neoplasm grade-3 (PanNEN-G3) show variable responses to platinum-based chemotherapy. Recent studies indicated that PanNEN-G3 includes well-differentiated neuroendocrine tumor with G3 (NET-G3). Here, we examined the clinicopathologic and molecular features of PanNEN-G3 and assessed the responsiveness to chemotherapy and survival.Experimental Design: A total of 100 patients with PanNEN-G3 were collected from 31 institutions, and after central review characteristics of each histologic subtype [NET-G3 vs. pancreatic neuroendocrine carcinoma (NEC-G3)] were analyzed, including clinical, radiological, and molecular features. Factors that correlate with response to chemotherapy and survival were assessed.Results: Seventy patients analyzed included 21 NETs-G3 (30%) and 49 NECs-G3 (70%). NET-G3 showed lower Ki67-labeling index (LI; median 28.5%), no abnormal Rb expression (0%), and no mutated KRAS (0%), whereas NEC-G3 showed higher Ki67-LI (median 80.0%), Rb loss (54.5%), and KRAS mutations (48.7%). Chemotherapy response rate (RR), platinum-based chemotherapy RR, and prognosis differed significantly between NET-G3 and NEC-G3. Chemotherapeutic outcomes were worse in NET-G3 (P < 0.001). When we stratified PanNEN-G3 with Rb and KRAS, PanNENs-G3 with Rb loss and those with mutated KRAS showed significantly higher RRs to platinum-based chemotherapy than those without (Rb loss, 80% vs. normal Rb, 24%, P = 0.006; mutated KRAS, 77% versus wild type, 23%, P = 0.023). Rb was a predictive marker of response to platinum-based chemotherapy even in NEC-G3 (P = 0.035).Conclusions: NET-G3 and NEC-G3 showed distinct clinicopathologic characteristics. Notably, NET-G3 does not respond to platinum-based chemotherapy. Rb and KRAS are promising predictors of response to platinum-based chemotherapy for PanNEN-G3, and Rb for NEC-G3. Clin Cancer Res; 23(16); 4625–32. ©2017 AACR.

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Background Schizophyllum commune is a basidiomycete that lives in the environment and can cause infections, mainly those of the respiratory system. Although S. commune is increasingly reported as a cause of allergic bronchopulmonary mycosis and sinusitis, cases of fungal ball formation are extremely uncommon. Identification of S. commune is difficult using routine mycological diagnostic methods, and in clinically suspicious cases, internal transcribed spacer sequencing should be used for diagnosis. Here, we report a first case of lung cancer with a fungal ball formation of S. commune, confirmed by analyzing the internal transcribed spacer. Case presentation A 76-year-old man with diabetes and hypertension was admitted to the hospital with a chief complaint of hemosputum, which he had for about 19 months. A computed tomography image of the patient’s chest showed a cavity and internal nodule in the left upper lobe of his lung. A left upper lobectomy was performed, and histopathological examination revealed squamous cell carcinoma of the lung and a fungal ball. The isolate from the surgical specimen was identified as S. commune by analyzing the internal transcribed spacer. The patient had no recurrence of the infection during 5 months of follow-up. Conclusions Only three cases of lung fungal balls caused by S. commune have been previously reported, and this is the first case of lung cancer cavity with a fungal ball formation. In cases of fungal ball formation in the lung, S. commune should be considered a possible causative microorganism.

Invasive mucinous adenocarcinoma (IMA) is a rare subtype of invasive lung adenocarcinoma. However, the clinical course and prognostic outcomes following IMA resection, particularly postoperative recurrence, remain unclear.We pathologically reevaluated 1362 lung adenocarcinoma resections performed at our institution, categorizing cases into the IMA group (72 cases) and non-IMA group (1290 cases). The IMA group was further classified into pneumonia and nodular types based on preoperative computed tomography.Overall, the IMA group had lower carcinoembryonic antigen levels (3 vs 8 ng/mL; P.01), fewer lymph node metastasis (4% vs 24%; P.01), and more KRAS mutations (56% vs 7%; P.01) than the non-IMA group. Although postoperative recurrence rates did not differ between both groups (32% vs 27%; P = 0.35), lung recurrence occurred more frequently in the IMA group (83% vs 17%; P.01). Propensity score-matched pair analysis showed that the IMA group had fewer lymph node metastasis (3% vs 35%; P.01), more KRAS mutations (56% vs 9%; P.01), and higher intrapulmonary recurrence rate (84% vs 31%; P.01) than the non-IMA group. The 5-year overall survival rates did not differ between both groups (74% vs 81%; P = 0.26). However, among patients with intrapulmonary recurrence, those in the IMA group had significantly worse prognosis than those in the non-IMA group (35% vs 77%; P.01).Intrapulmonary recurrence, which induced significantly worse prognosis, was more likely to occur in the IMA than non-IMA group.

Background Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1–negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1–negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1–negative LUAD. Methods Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1–negative and 4 TTF-1–positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. Results SRGN was markedly overexpressed at mRNA and protein levels in TTF-1–negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1–positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase–mediated impairment of methionine metabolism. Conclusions Our findings suggest that SRGN plays a pivotal role in tumor–stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1–negative LUAD.

Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from −273 to −10 base pairs upstream of the transcription start site identified by 5'‐RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB‐downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression., Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT.

Few reports have demonstrated robotic surgery for large tumors in the upper esophagus. We report a case of a 52-year-old woman with a giant submucosal tumor in the upper esophagus successfully enucleated using robotic surgery. She presented with odynophagia and dysphagia, with subsequent evaluation revealing a submucosal mass measuring approximately 10 cm in diameter in the upper esophagus. The mass was compressing the trachea and enlarged over 3 years. Endoscopic ultrasound fine needle aspiration of the tumor was non-diagnostic. Robot-assisted esophageal submucosal tumor enucleation was performed for diagnosis and treatment. Flexible forceps control allowed for a multi-directional approach to dissect the tumor and stable forceps handling was critical in this delicate procedure. Subsequent pathological review revealed a well-differentiated esophageal liposarcoma. While surgical margins were not entirely negative, the local recurrence rate of the tumor is low. At the patient's request, we decided to observe the patient without additional resection.

Objectives The purposes of this study are to clarify the details of the ALK tyrosine kinase inhibitor (TKI) resistance mechanism in rebiopsy cases and to predict novel resistance gene alterations using molecular dynamics simulation. Methods A total of 21 patients with ALK-positive NSCLC who underwent a rebiopsy after ALK TKI failure were included in this analysis. ALK fluorescence in situ hybridization and reverse transcription polymerase chain reaction were performed with paired initial and rebiopsy tumor specimens. Results Nine patients had no known ALK resistance mechanisms. Four had ALK amplification. L1196M, I1171N, and G1269A, mutations that are known to indicate resistance to ALK TKIs, were detected in one patient each. Small cell carcinoma and sarcomatoid transition were found in one case each. L1196Q, P1094H, and exon 24 76-base pair insertion were detected after the second-generation ALK TKIs. Conclusions The combination of a genetic analysis and a computational simulation model may make a prediction of resistance mechanisms for overcoming ALK TKI resistance, and the construction of a genomic and simulation fused database is important for the development of personalized medicine in this field.

Background/objectives Pancreatic neuroendocrine carcinoma (PanNEC)-G3 often presents along with genetic abnormalities such as KRAS, RB1, and TP53 mutations. However, the association between these genetic findings and response to chemotherapy and prognosis has not been clarified. This study aimed to clarify the clinicopathological features of PanNEC-G3. Methods We performed a subgroup analysis of the Japanese PanNEN-G3 study (multicenter, retrospective study), which revealed that Rb loss and KRAS mutation were predictors of the response to platinum-based regimen in PanNEN-G3. We re-classified WHO grades of PanNENs using the 2017 WHO classification and then analyzed the clinicopathological features and prognostic factors in 49 patients with PanNEC-G3. Results The rates of Rb loss and KRAS mutation in PanNEC-G3 were 54.5% and 48.7%, respectively. Patients with Rb loss and/or KRAS mutation showed a higher response rate to first-line platinum-based regimen than those without Rb loss or KRAS mutation (object response rate 70.0% vs 33.3%, odds ratio 9.22; 95% CI 1.26–67.3, P = 0.029), but tended to have shorter overall survival rates than those without Rb loss or KRAS mutation (median 239 vs 473 days, hazard ratio 2.11; 95% CI 0.92–4.86, P = 0.077). Conclusions Patients with PanNEC-G3 have varied clinical outcomes for platinum-based regimen. When grouped based on Rb loss and KRAS mutation, there seemed to be two groups with distinct prognoses and responses to the platinum-based regimen. PanNEC-G3 could, therefore, be classified into two distinct groups based on immunohistochemical and genetic findings.

Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first‐line chemotherapy to assess the benefit of anti–epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)‐based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti–EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next‐generation sequencing‐based tests are weakly recommended because these tests have not been validated in clinical trials., Update to the molecular testing guideline for colorectal cancer defined by Japanese Society of Medical Oncology. The appropriate timing of each test and degree of recommendation are summarized.

Glandular papilloma (GP) and mixed squamous cell and glandular papilloma (MP) are rare benign pulmonary tumors occurring in the bronchi. Bronchiolar adenoma (BA) was recently characterized as a pulmonary tumor exhibiting alveolar spread. Both GP/MP and BA are composed of a mixture of glandular, ciliated, squamous, and basal cells. We aimed to clarify whether GP/MP and BA represent the same tumor. We evaluated the detailed histologic characteristics of 11 cases involving pulmonary peripheral tumors that exhibited histologic features of GP/MP or BA, and performed genetic analyses using targeted panel sequencing, allele-specific polymerase chain reaction, and digital polymerase chain reaction. Histologically, 4 and 7 tumors were classified as GP/MP and BA, respectively. GP/MP showed endobronchiolar papillary growth with a pseudostratified or stratified epithelium. In contrast, 5 BAs showed a predominant flat structure with a bilayered or pseudostratified epithelium, whereas 2 BAs showed a GP/MP-like papillary architecture. The mean epithelial thickness in each tumor was significantly larger in GP/MPs and BAs with a GP/MP-like morphology (103 to 242 μm) than in flat-predominant BA (23 to 47 μm, P=0.0010). AKT1 E17K mutations were detected in all GP/MPs and BAs with GP/MP-like morphology but were absent in the 5 flat-predominant BAs. AKT1 mutations were always concurrent with BRAF or HRAS mutations, and the variant allele frequency or percentage of mutant copies of AKT1 mutations was equal to those of BRAF or HRAS mutations. GP/MPs are characterized by AKT1 mutations concurrent with BRAF or HRAS mutations. Peribronchiolar papillary tumors with AKT1 mutations may also be classified as GP/MP.

Background RNA‐based sequencing is considered ideal for detecting pathogenic fusion‐genes compared to DNA‐based assays and provides valuable information about the relative expression of driver genes. However, RNA from formalin‐fixed paraffin‐embedded tissue has issues with both quantity and quality, making RNA‐based sequencing difficult in clinical practice. Analyzing stamp‐derived RNA with next‐generation sequencing (NGS) can address the above‐mentioned obstacles. In this study, we validated the analytical specifications and clinical performance of our custom panel for RNA‐based assays on the Ion Torrent platform. Methods To evaluate our custom RNA lung panel, we first examined the gene sequences of RNA derived from 35 NSCLC tissues with diverse backgrounds by conventional methods and NGS. Next, we moved to the clinical phase, where clinical samples (all stamp‐derived RNA) were used to examine variants. In the clinical phase we conducted an NGS analysis while simultaneously applying conventional approaches to assess the feasibility and validity of the panel in clinical practice. Results In the prerun phase, all of the variants confirmed with conventional methods were detected by NGS. In the clinical phase, a total of 80 patients were enrolled and 80 tumor specimens were sequenced from February 2018 to December 2018. There were 66 cases in which the RNA concentration was too low to be measured, but sequencing was successful in the vast majority of cases. The concordance between NGS and conventional methods was 95.0%. Conclusions RNA extraction using stamp specimens and panel sequencing by NGS were considered applicable in clinical settings. Key points Significant findings of the study Next‐generation sequencing using RNA from stamp specimens was able to detect driver gene changes in non‐small cell lung cancer including fusion genes with the same accuracy as conventional methods. What this study adds Using RNA from stamp specimens obtained from biopsy increases the number of candidate cases for RNA sequencing in clinical settings.

Secretory carcinoma (SC) of the salivary glands is a low-grade carcinoma characterized by a well-defined morphology and immunohistochemical features. ETV6-NTRK3 fusions are detected in the great majority of SCs. Recently, other partners fused to ETV6 have been documented in a small portion of SCs, suggesting the presence of alternative genetic fusion. In this study, we examined the genetic fusion of 9 SCs using fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and next-generation sequencing (ArcherDx). Classic ETV6 exon 5-NTRK3 exon 15 fusion was detected in 8 of 9 SCs. The remaining tumor was negative for the ETV6-NTRK3 fusion but harbored a novel fusion, CTNNA1 exon 11-ALK in exon 20. Immunohistochemically, pan-TRK was positive in 8 tumors with ETV6-NTRK3 fusion but negative in an ALK-rearranged SC, while ALK was positive only in the ALK-rearranged tumor. Histologically, the ALK-rearranged tumor showed dominant macrocystic architecture. In conclusion, we found a case of SC with CTNNA1-ALK fusion. Because ALK fusion after exon 20 on the ALK side (upstream of the tyrosine kinase domain) has been reported to activate a carcinogenic kinase in various ALK-rearranged tumors, ALK inhibitors may be a possible therapeutic option for ALK-rearranged SC. In addition, ALK immunohistochemistry can be a screening tool for ALK-rearranged SC. This study also expands the molecular spectrum of this tumor beyond the ETV6 gene.

PurposeNivolumab is recommended for patients with advanced esophageal squamous cell carcinoma (aESCC) who are refractory or intolerant to fluoropyrimidine-based and platinum-based chemotherapy regardless of tumor proportion score (TPS). However, the predictive role of combined positive score (CPS) to predict nivolumab efficacy is unclear.MethodsWe retrospectively collected data from patients with aESCC, who were previously treated with fluoropyrimidines and platinum, and subsequently received nivolumab monotherapy between January 1, 2014 and September 15, 2020. Next, we examined the impact of CPS and TPS on clinical response to nivolumab. The inclusion criteria were refraction or intolerance to fluoropyrimidines and platinum, absence of comorbid malignancies, availability of tissue specimen for immunohistochemistry and programmed cell death-1 analysis results before initiating nivolumab.ResultsFifty patients were included (CPS, ≥ 10/1–10/< 1, n = 23/18/9; TPS, ≥ 10/1–10/< 1%, n = 17/8/25). The median progression-free survival were 4.1, 2.5, and 1.4 months in CPS ≥ 10, 1–10 (hazard ratio [HR] vs. CPS ≥ 10, 1.05; p = 0.90), and < 1 (HR vs. CPS ≥ 10, 3.75; p = 0.003) groups, respectively. Furthermore, among the patients with CPS ≥ 10/1–10/< 1, and TPS ≥ 10/1–10/< 1%, objective response rate were 32/25/0% and 36/0/19%. In addition, disease control rate were 63/50/11% (p = 0.04) and 65/40/38% (p = 0.30).ConclusionsThis study proposes that CPS can predict responses to nivolumab in patients with aESCC better than TPS, making it especially useful for predicting negative outcomes for those with CPS < 1.

No studies to date have determined the impact of pancreatic fat infiltration on postoperative pancreatic fistula (POPF) occurrence in patients undergoing invagination pancreaticojejunostomy (IV-PJ).The medical records of patients with a soft pancreas who underwent pancreatoduodenectomy followed by IV-PJ were reviewed . The pancreatic fat ratio on computed tomography (CT) images (I-PFR) was determined using preoperative CT and verified by histologic examination. The relationship between the I-PFR and POPF occurrence was determined. Patients were classified into 2 groups based on I-PFR value (fatty and non-fatty pancreas). Postoperative outcomes were compared between the two groups, and specifically among patients who developed POPF.Of 221 patients, POPF occurred in 67 (30.3%). I-PFR was positively correlated with histologic-calculated fat ratio (ρ = 0.517, p 0.001). This index was shown to be an independent predictor of POPF. Based on an I-PFR cut-off value of 3.2%, 92 patients were classified in the fatty pancreas group. Subgroup analysis of the patients who developed POPF showed that incidence of abscess formation and hemorrhage tended to be higher in patients with fatty pancreas than in those with non-fatty pancreas.Pancreatic fat infiltration is highly associated with POPF and possibly causes subsequent serious complications in patients undergoing IV-PJ.

A rare case of a squamous cell carcinoma (so-called cloacogenic carcinoma) showing extensive superficial spread to the rectum is presented. A 69-year-old woman had undergone colonoscopy for annual check-up, and a whitish, flat lesion with a central depressed area, 20 mm in size, was identified in the lower rectum. Narrow-band imaging with magnifying observation showed abnormal microvessels without the intrapapillary capillary loop patterns. Endoscopically, the margin of the lesion was unclear. Biopsy was performed, and a histological diagnosis of transitional cell carcinoma was made. Computed tomography showed no evidence of involvement of adjacent organs, lymph nodes or distant sites. Cystoscopy found no abnormality in the bladder mucosa. Owing to difficulty diagnosing this tumor accurately, local excision with transanal endoscopic microsurgery was performed. Cloacogenic carcinoma with submucosal invasion was diagnosed. A human papilloma virus (HPV) polymerase chain reaction test was positive. Judging from the histological findings and the positive HPV test, we hypothesis that the tumor was likely arising from the anal transitional zone with marked superficial spread to the rectum. Clinicians should keep in mind that this variant of squamous cell carcinoma may occur in the rectum, even if no endoscopic findings are seen in the anal transitional zone.

Background: Peritoneal metastasis occurs in approximately 7% of colorectal cancer (CRC) patients and is associated with worse prognosis compared to non-peritoneal metastasis. In CRC patients with peritoneal metastasis, administration of systemic chemotherapy only slightly improves overall survival. Therefore, a better understanding of the biology of peritoneal metastasis and the development of new molecular therapeutics for CRC patients with peritoneal metastasis are urgently needed. Materials and Methods: CT26 mouse CRC cell lines with low metastatic potentials (CT26-N5) and high metastatic potentials to peritoneum (CT26-P6) were established after several rounds of in vivo selection by orthotopic transplantation of CT26 cells into the syngeneic BALB/c mice. Transcriptomic and proteomic analyses were conducted on CT26-N5 and CT26-P6 cells. Results: Orthotopic transplantation of CT26-P6 cells exhibited significantly increased peritoneal metastasis compared to CT26-N5 cells, while growth of primary tumors was not different between these CT26 sublines. In addition, CT26-P6 cells had higher cell migration and invasion potential in vitro than CT26-N5 cells. Integrated analyses of transcriptome and proteome resulted in identification of molecular signatures associated with peritoneal metastasis. An actin-binding protein Advillin, encoded by the Avil gene, was markedly increased at both mRNA and protein levels in CT26-P6 cells compared to CT26-N5 cells. Knockdown of Avil using siRNA or shRNA in CT26-P6 cells significantly reduced cell migration and invasion in vitro and occurrence of peritoneal metastasis in vivo. Gene Set Enrichment Analysis on transcriptome datasets from CT26-P6 cells and CT26-N5 cells identified interferon gamma response as the most significantly enriched pathway in CT26-cells. In addition, Immunoprecipitation coupled with LC-MS/MS identified potential interaction between Avil and a molecule X that regulates epithelial-mesenchymal transition in CRC. Consistent with the findings in mouse CT26 cells, AVIL knockdown in CRC patient-derived cells (PDCs) suppressed cell migration and invasion in vitro. While AVIL expression was induced by interferon gamma in CRC PDCs, JAK inhibitors, Momelotinib and Ruxolitinib, significantly reduced interferon gamma-induced AVIL expression, suggesting regulation of AVIL by interferon gamma. Conclusion: Molecular characterization of mouse colorectal cancer cell lines with different metastatic potential identified AVIL as a potential therapeutic target in CRC patients with peritoneal metastasis. Regulation of AVIL expression by interferon gamma may suggest functional relevance of tumor immune microenvironment in the development of peritoneal metastasis in CRC. Citation Format: Haruki Mori, Hisanori Isomura, Shuang Zhou, Taisuke Kajino, Yuichi Abe, Takashi Kinoshita, Seiji Natsume, Yusuke Sato, Akira Ouchi, Toru Miyake, Waki Hosoda, Koji Komori, Yasuhiro Shimizu, Masaji Tani, Ayumu Taguchi. Molecular characterization of mouse colorectal cancer cell lines with high potential of peritoneal metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3599.

Locoregional recurrence after curative resection remains an important issue in the treatment of colorectal cancer (CRC). The aim of the present study was to investigate the clinical significance of quantitative detection of intraperitoneal free cancer cells by a PCR-based method for predicting locoregional recurrence after CRC resection.A total of 114 patients with CRC were enrolled between March 2017 and December 2018, and 95 patients with Stage I-III CRC were analyzed. Peritoneal lavage fluid was collected before and after tumour resection and subjected to cytology and quantitative reverse transcription-PCR (qRT-PCR) with carcinoembryonic antigen (CEA) as a genetic marker.2.1% of patients had positive cytology after resection, whereas 9.5% had positive CEA qRT-PCR (PCR+) after resection. Eight of nine PCR+ patients after resection had tumours in the rectum. Fifteen (15.8%) patients developed recurrence during the follow-up period, including three with locoregional recurrence. One of 86 (1.2%) PCR- patients and 2 of 9 (22.2%) PCR+ patients after resection developed locoregional recurrence. Overall and in rectal cancer patients, the 3-year cumulative risk of locoregional recurrence was 25.0% and 28.6% for PCR+ patients, which is significantly higher than PCR- patients (1.3% and 0%, P 0.001 and P = 0.001, respectively).Intraperitoneal free cancer cells can serve as a sensitive predictor of locoregional recurrence after rectal cancer resection. qRT-PCR for CEA can be a suitable method for detecting intraperitoneal free cancer cells in peritoneal lavage fluid.

BRAF mutations are rare driver mutations in non-small cell lung cancer (NSCLC), accounting for 1%-2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non-V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non-V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation-specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non-V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non-V600E mutations, using BRAF mutation-specific IHC. None of the tumors with non-V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation-specific IHC is specific without any cross-reactions to non-V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.

Background/aim Pulmonary pleomorphic carcinoma (PPC) is rare, and few studies have reported its features. We assessed the clinicopathological features, surgical outcomes, oncogenic status and programmed death-ligand 1 (PD-L1) expression of PPC. Patients and methods We retrospectively reviewed data from 22 consecutive patients who underwent resection of PPC between 2007 and 2017. Results The predominant tissue type of the epithelial component was adenocarcinoma in 15 patients (68%) and the others in 7 patients (32%), and the 3-year disease-free survival rate tended to be better in patients with an adenocarcinoma component compared to patients with another component (40.0% vs. 17.1%, p=0.059). PD-L1 expression was observed in all eight tumors whose PD-L1 status could be examined and high PD-L1 expression (≥50%) was frequent (5/8, 63%). Conclusion A predominant adenocarcinoma epithelial component in PPC might be associated with better survival outcomes and high PD-L1 expression might be frequent in PPC.

Emerging findings suggest that Parkinson's disease (PD) pathology (α-synuclein accumulation) and neuronal dysfunction may occur first in peripheral neurons of the autonomic nervous system including the enteric branches of the vagus nerve. The risk of PD increases greatly in people over the age of 65, a period of life in which chronic inflammation is common in many organ systems including the gut. Here we report that chronic mild focal intestinal inflammation accelerates the age of disease onset in α-synuclein mutant PD mice. Wild-type and PD mice treated with 0.5% dextran sodium sulfate (DSS) in their drinking water for 12 weeks beginning at 3 months of age exhibited histological and biochemical features of mild gut inflammation. The age of onset of motor dysfunction, evaluated using a rotarod test, gait analysis, and grip strength measurements, was significantly earlier in DSS-treated PD mice compared to control PD mice. Levels of the dopaminergic neuron marker tyrosine hydroxylase in the striatum and numbers of dopaminergic neurons in the substantia nigra were reduced in PD mice with gut inflammation. Levels of total and phosphorylated α-synuclein were elevated in enteric and brain neurons in DSS-treated PD mice, suggesting that mild gut inflammation accelerates α-synuclein pathology. Markers of inflammation in the colon and brain, but not in the blood, were elevated in DSS-treated PD mice, consistent with retrograde transneuronal propagation of α-synuclein pathology and neuroinflammation from the gut to the brain. Our findings suggest that interventions that reduce gut inflammation may prove beneficial in the prevention and treatment of PD.

In patients with familial adenomatous polyposis (FAP), adenomas and even carcinomas may develop in the rectal remnant and the ileal pouch after surgical treatment. The aim of this study was to evaluate the outcome of endoscopic management in patients with FAP. The main outcome measurements were the appearance of secondary cancer, complications, and the need for additional surgery. Thirty-four FAP patients with Kock’s continent ileostomy (Kock) (n = 3), ileorectal anastomosis (IRA) (n = 12), and ileal pouch-anal anastomosis (IPAA) (n = 19) were identified. The median follow-up period of endoscopic surveillance was 11.5 years for pouch patients (Kock + IPAA) and 21.7 years for IRA. Metachronous adenomas appeared in 32 patients (94.1%). In pouch patients, a total of 120 treatments were given to 20 patients, and 12 sessions of delayed bleeding (10%) occurred, which was significantly higher compared to IRA patients, with 0 sessions (p < 0.001). In IRA patients, a total of 169 treatments were given to 11 patients, with one case of perforation. No adenocarcinoma has developed since the start of endoscopic surveillance. Regular endoscopic surveillance and treatment are feasible and safe. However, in pouch patients, one must be cautious about delayed bleeding in the treatment of adenomas.

Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention., Neoplastic pancreatic cysts are associated with invasive pancreatic cancer, but their origins and evolutionary relationships are unclear. Here, the authors present the evolutionary analysis of neoplastic cysts and report them as precursors of invasive pancreatic cancer, and that SMAD4/TGFBR2 alterations are likely drivers of invasion in a subset of cases.