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Background The efficacy of pre-operative systemic treatment (PST) combined with immune checkpoint inhibition (ICI) for triple-negative breast cancer (TNBC) has been recognized recently as being independent of the degree of programmed death ligand-1 (PD-L1) positivity of infiltrating immune cells, especially for patients with axillary lymph node metastasis (ALNM). Methods TNBC patients with ALNM were treated surgically between 2002 and 2016 in our facility (n = 109), of whom 38 received PST before resection. The presence of tumor-infiltrating lymphocytes (TILs) expressing CD3, CD8, CD68, PD-L1 (detected by antibody SP142) and FOXP3 at primary and metastatic LN sites was quantified. Results The size of invasive tumor and the number of metastatic axillary LN were confirmed as prognostic markers. The numbers of both CD8+ and FOXP3+ TILs at primary sites were also recognized as prognostic markers, especially for overall survival (OS) (CD8, p = 0.026; FOXP3, p Conclusions PD-L1+ , CD8+ or FOXP3+ immune cells in the tumor microenvironment (TME) at both primary and metastatic sites are significant on prognosis, which could be a clue to expect the potential for better responses to the combination of chemotherapy and ICI, especially for patients with ALNM.

Background A subset of patients with estrogen receptor (ER)-positive, HER2-negative, and node-negative breast cancer experience recurrences. Predicting patients who will have recurrences within 5 years of surgery is essential so that patients can be selected to receive adjuvant chemotherapy. The 95-gene classifier (95-GC) has been validated as a method to differentiate patients into high and low-risk groups for early recurrence. Methods In this study, we performed 95-GC analysis on 56 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients who underwent surgery for ER-positive, HER2-negative, and node-negative breast cancer and did not receive adjuvant chemotherapy. We associated the obtained high- and low-risk groups with clinicopathological characteristics and recurrence-free survival (RFS).Results We classified 12 out of 56 patients into the high-risk recurrence group. We found significantly higher KI67 scores in patients in the high-risk group. Other clinicopathological characteristics were not associated with the 95-GC risk groups. Patients in the 95-GC low-risk group had a significantly better prognosis than those in the high-risk group (p = 0.0387). The 5-year RFS rate was 97.6% in the low-risk group and 74.1% in the high-risk group, while the 10-year RFS rates were 90.1% and 74.1%, respectively.Conclusions Our study shows that the 95-GC score can accurately predict RFS within 5 years of surgery for ER-positive, HER2-negative, and node-negative breast cancer using FFPE tissue samples. These prediction models could help assign patients to the most effective treatment regimen.

Takashi Mitamura,1 Tianyue Zhai,1 Kanako C Hatanaka,2 Yutaka Hatanaka,2,3 Toraji Amano,4 Lei Wang,5,6 Shinya Tanaka,5,6 Hidemichi Watari1 1Department of Obstetrics and Gynecology, Hokkaido University Faculty of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan; 2Center for Development of Advanced Diagnostics, Hokkaido University Hospital, Sapporo, Hokkaido, Japan; 3Research Division of Genome Companion Diagnostics, Hokkaido University Hospital, Sapporo, Hokkaido, Japan; 4Clinical Research and Medical Innovation Center, Hokkaido University Hospital, Sapporo, Japan; 5Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Hokkaido, Japan; 6Department of Cancer Pathology, Faculty of Medicine, Hokkaido University, Sapporo, Hokkaido, JapanCorrespondence: Takashi Mitamura, Department of Obstetrics and Gynecology, Hokkaido University Faculty of Medicine, Hokkaido University, North15, West 7, Kita-Ku, Sapporo, 0608638, Hokkaido, Japan, Tel +81 11 706 5941, Fax +81 11 706 7711, Email takami@huhp.hokudai.ac.jpPurpose: To identify the germline genetic characteristics of long-term recurrence-free survivors that can be applied to establishing a new strategy for curing advanced cancer, we investigated the whole-genome single nucleotide variants of ovarian cancer patients.Patients and Methods: DNA specimens were obtained from rare long-term recurrence-free survivors with FIGO stage III–IV ovarian cancer with no recurrence for 8– 23 years after primary treatments for a whole-genome analysis of approximately 660,000 single nucleotide variants. We then established a mouse model with a notable gene alteration by CRISPR/Cas9 to confirm the biological role.Results: The long-term recurrence-free survivors more frequently had germline heterozygous variant rs2185379 of the PRDM1 gene exon than patients with early recurrence (6.8-fold, P=0.013) and the general population. In the mouse model, primary intraperitoneal disseminated tumors of allograft ID8 were significantly smaller in the germline heterozygous rs2185379 group than in the wild-type group (57.4% decrease, P=0.008). Immunohistochemistry showed that the area of distribution of infiltrating T lymphocytes with positive CD8 staining was significantly increased in the germline heterozygous rs2185379 group in comparison to the wild-type group.Conclusion: Germline heterozygous rs2185379 in PRDM1 is correlated with an excellent prognosis and can be used to establish a new strategy for treating advanced ovarian cancer.Keywords: ovarian cancer, long-term survivors, rs2185379, PRDM1

Background: Superficial temporal artery (direct) and encephalomyosynangiosis (indirect) revascularization may develop after combined bypass surgery in pediatric patients with moyamoya disease (MMD). However, arterial development varies widely among patients, and the underlying mechanisms remain unknown. Objectives: We evaluated the relationship between the development of donor arteries after bypass surgery in pediatric patients with MMD and the MMD-susceptibility gene variant c.14576G>A of ring finger protein (RNF) 213. Methods: The data of pediatric patients with MMD (age RNF213 c.14576G>A status was examined. Results: Forty-eight hemispheres from 28 pediatric patients with MMD were examined. Three hemispheres belonged to patients with the AA genotype; 33 to patients with the AG genotype (AA/AG group); and 12 to patients with the GG genotype (GG group; wild type). The CCRs for the DTA were significantly higher in patients with RNF213 variant (AA/AG group; 2.5 ± 0.1) than in the GG group (2.0 ± 0.2) (p = 0.03), whereas the CCRs for the STA were significantly higher in the GG (1.6 ± 0.1) than in the AA/AG group (1.3 ± 0.6) (p = 0.02). There was no significant difference in the CCRs for the MMA and basilar artery between the groups. Other factors, including sex, age, and MRA grading, were not associated with the development of specific bypass development. Conclusions: The extent of collateral development associated with direct or indirect bypass was found to differ between the genotypes of the RNF213 c.14576G>A associated with pediatric MMD. This genetic variant correlates with the development of the disease and affects revascularization after bypass surgery in pediatric patients with MMD.

This study aimed to validate the Proactive Molecular Risk Classifier for Endometrial Cancer, a modified version of The Cancer Genome Atlas, using data from 184 patients with endometrial cancer (median age: 57.5 years; median follow-up period: 109 months) who had undergone radical surgery (including systemic lymphadenectomy) and subsequent adjuvant chemotherapy (patients with intermediate or high recurrence risk) from 2003 to 2015. Tissue microarrays were prepared from surgical specimens and classified using the conventional clinical risk classifier. Immunohistochemistry was used to detect mismatch repair proteins, L1 cell adhesion molecule, and p53. Direct sequencing was used to identify hotspot mutations in the polymerase-epsilon gene. Forty-five patients were identified as having high L1 cell adhesion molecule expression, 41 as low risk, 34 as mismatch repair-deficient, 13 as polymerase-epsilon gene-mutated, five as having abnormal p53, and 46 as other. Patients were stratified into significantly different prognostic groups (p < 0.0001): favorable (low risk and polymerase-epsilon gene-mutated), intermediate (mismatch repair-deficient and other), and unfavorable (high L1 cell adhesion molecule expression and abnormal p53) with 5-year disease-specific survival rates of 100%, 93.8%, and 75.1%, respectively (Kaplan-Meier method). The combination of conventional recurrent risk classification, sequencing for polymerase-epsilon gene mutations and immunohistochemistry for L1 cell adhesion molecule, p53, and mismatch repair proteins can be used to determine the prognoses of patients with endometrial cancer.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

Simple Summary Extrahepatic cholangiocarcinoma (EHCC) is an aggressive tumor. The five-year survival rate for patients who undergo surgical resection is only 20-40% due to recurrences. Therefore, elucidating the molecular mechanisms underlying invasion and metastasis in EHCC is crucial for developing adjuvant therapy. The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in two EHCC cell lines. CCR7 mediates cell invasion and migration in EHCC by inducing EMT, which was abrogated by a CCR7 antagonist. CCR7 may be a potential target for adjuvant therapy in EHCC. The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Background Because a number of years may be required for normal cells to develop into carcinoma, genes involved in tumorigenesis and progression might differ among breast cancers in young women and those in older women. The present study sought to analyze subclonality during breast cancer evolution as well as diversity within each individual in our young patients’ cohort. Methods A total of 13 women aged, Highlights • Subclonality during breast cancer progression in young patients were analyzed. • No two patients had the same germline and/or somatic gene alterations. • Somatic gene mutations were present in normal breast tissue.

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be >= 30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

Background Combination therapy with the B-Raf inhibitor, dabrafenib, and the MEK inhibitor, trametinib (DT) is commonly used to treat patients with B-Raf proto-oncogene, serine/threonine kinase V600E (BRAF V600E)-mutated non-small cell lung cancer (NSCLC). However, the mechanisms through which cancer develops DT resistance are unclear. Here, we investigated new mechanisms underlying acquired DT-resistant NSCLC with the BRAF V600E mutation. Methods We compared genomic signatures before and after DT treatment in patients with NSCLC. Results Two of four patients treated with DT developed carcinomatous pleuritis within 3 months. Target DNA sequencing and quantitative polymerase chain reaction (PCR) analyses revealed the increased expression level of cyclin-dependent kinase 4 (CDK4). We also found prominent protein expression of CDK4 after DT treatment. Induction of CDK4 expression in a cell line derived from a patient with the BRAF V600E mutation resulted in partial resistance to dabrafenib. Conclusions Our findings suggest a possible relationship between CDK4 upregulation and acquired resistance to DT therapy.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that has shown marked antitumor activity in patients with EGFR-mutated non-small-cell lung cancer (NSCLC). However, these effects are transient and most patients develop resistance. Reversible drug-tolerant persister (DTP) cells are defined as a small subpopulation of cells with markedly reduced sensitivity and non-genetic acquired resistance to EGFR-TKIs. Notch is a transmembrane receptor that plays an important role in tumorigenesis. We previously reported that there is significant crosstalk between the Notch and EGFR pathways in NSCLC. Moreover, the Notch pathway is associated with resistance to previous-generation EGFR-TKIs. However, the role of Notch in osimertinib resistance is not fully understood. In this study, we evaluated whether Notch is involved in osimertinib resistance. We show that NOTCH1 and Notch target genes are upregulated in osimertinib DTP cells, and that the addition of a gamma-secretase inhibitor (GSI), a Notch inhibitor, impairs drug-tolerant persistence in vitro and in vivo. Compared with osimertinib, combined GSI and osimertinib suppress phospho-ERK partly by enhancing DUSP1 expression. Furthermore, Notch1 and HES1 were upregulated after EGFR-TKI treatment in half of human EGFR-mutated NSCLC tumor tissues. These results suggest that the combination of GSI and osimertinib may be a potential therapy for EGFR-mutated NSCLC.

The objective of this study is to clarify clinical significance of the H3F3A K27M mutation (H3K27M) and analyze the correlation between H3K27M, H3K27me3 status, and EZH2 expression and prognosis in spinal cord gliomas. Patients with spinal cord diffuse glioma regardless of World Health Organization (WHO) grade underwent genetic analysis for H3F3A, HIST1H3B, TERT promoter, IDH1/2, and BRAF. H3K27me3 status and EZH2 expression were analyzed through immunohistochemistry. Thereafter, the association between H3K27M, H3K27me3 status, and EZH2 expression and prognosis was retrospectively analyzed using the log-rank test. A total of 26 cases, 5 with WHO grade 4, 9 with grade 3, and 12 with grade 2 glioma, were analyzed. Although WHO grade 2 cases tended to present favorable overall survival, the difference was not statistically significant. H3K27M, which was detected in four grade 4 cases (80%) and three grade 3 cases (33%), was not associated with prognosis among grade 3 and 4 cases. Among WHO grade 2-4 cases, the combination of retained H3K27me3 and negative EZH2 expression was correlated with favorable overall survival (p = 0.03). The combination of H3K27me3 status and EZH2 expression was considered as a potential prognostic marker in WHO grade 2-4 diffuse spinal cord gliomas.

Background/aim This study aimed to develop a new pathological finding, namely, invasion front grade and verify its clinical usefulness. Materials and methods We re-examined haematoxylin-eosin-stained specimens in 162 stage II-III colorectal cancer patients who underwent radical resection. We assessed the desmoplastic reaction, Klintrup grade, and poorly differentiated cluster. These three findings were combined to form the invasion front grade (good prognosis group; Grade A, poor prognosis group; Grade B), and its reproducibility and prognostic stratification ability were statistically analysed. Results Invasion front grade was Grade A in 116 cases and Grade B in 46 cases, and its kappa coefficient was 0.81 for interobserver and 0.74 for intraobserver variability. The 3-year recurrence-free survival rates of Grade A and Grade B were 90.4% and 55.9%. Multivariate analysis showed that invasion front grade was an independent prognostic factor. Conclusion Invasion front grade is useful as a prognostic stratification factor for stage II-III colorectal cancer.

To identify the germline genetic characteristics of long-term recurrence-free survivors that can be applied to establishing a new strategy for curing advanced cancer, we investigated the whole-genome single nucleotide variants of ovarian cancer patients.DNA specimens were obtained from rare long-term recurrence-free survivors with FIGO stage III-IV ovarian cancer with no recurrence for 8-23 years after primary treatments for a whole-genome analysis of approximately 660,000 single nucleotide variants. We then established a mouse model with a notable gene alteration by CRISPR/Cas9 to confirm the biological role.The long-term recurrence-free survivors more frequently had germline heterozygous variant rs2185379 of theGermline heterozygous rs2185379 in

Background: Estrogen signals play an important role in the phenotype of estrogen receptor positive breast cancer (BC). However, comprehensive analyses of the effect of estrogen signals on the tumor microenvironment (TME) and treatment response in large cohorts of primary BC patients have been lacking. We aimed to test the hypothesis that estrogen reactivity effects gene expression and immune cell infiltration profiles in the TME and relates to response to chemotherapy (CT) and endocrine therapy (ET). Methods: A total of 3091 BC cases were analyzed; 1075 from TCGA cohort, 1904 from METABRIC cohort, and 112 from Hokkaido University hospital cohort. We divided the group into estrogen reactivity-high and estrogen reactivity-low groups utilizing estrogen response genes.Results: BC with high estrogen reactivity was related to Myc targets, Metabolism-related signaling, cell stress response, TGF-beta signaling, androgen response, and MTORC1 signaling gene sets in the TME. Low estrogen reactivity was related to immune-related proteins, IL2-STAT5 signaling, IL6-JAK-STAT3 signaling, KRAS signaling, cell cycle related gene sets, and EMT. In addition, BC with high levels of estrogen reactivity had low immune cytolytic activity, low levels of immunostimulatory cells, and high levels of immunosuppressive cells. It also had low levels of stimulatory and inhibitory factors of the cancer immunity cycle (CIC). Patients with high estrogen reactivity were also associated with a better prognosis. Regarding the effect of estrogen reactivity on treatment, patients who were treated with ET and CT but relapsed (BC with CT rec) were related with higher levels of E2F targets and G2M checkpoint, but lower levels of immunosuppressive M2 macrophages or Tregs cells. In addition, the TME in CT rec had higher levels of CIC regulators.Conclusions: We demonstrated the relationship between estrogen reactivity and the profiles of immune cells and gene expression within the TME, as well as the treatment effect of CT given in addition to ET.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared genomic DNA from the cancer tissue and peripheral blood of 5 cancer patients and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants and 25 bp delins in EGFR. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. By conducting this pilot study using patient samples, we were able to obtain a glimpse of the current status of cancer genome testing at participating laboratories. To enhance domestic cancer genome testing, it is important to conduct local PT and to involve the parties concerned as organizers and participants.

The aim of this study was to analyze the clinical and radiological characteristics of glioblastomas (GBMs) harboring a BRAF mutation. Sequencing analysis of BRAF, IDH1/2, and TERT promoters was performed on GBM samples of patients older than 15 years. The clinical, pathological, and radiological data of patients were retrospectively reviewed. Patients were classified into three groups according to their BRAF and IDH1/2 status: BRAF group, IDH group, and BRAF/IDH-wild-type (WT) group. Among 179 GBM cases, we identified nine cases with a BRAF mutation and nine with IDH mutation. The WT group had 161 cases. Age at onset in the BRAF group was significantly lower compared to the WT group and was similar to the IDH group. In cases with negative IDH1-R132H staining and age < 55 years, 15.2% were BRAF-mutant cases. Similar to the IDH group, overall survival of the BRAF group was significantly longer compared with the WT group. Among nine cases in the BRAF group, three cases had hemorrhagic onset and prior lesions were observed in two cases. In conclusion, age < 55 years, being IDH1-R132H negative, with hemorrhagic onset or the presence of prior lesions are factors that signal recommendation of BRAF analysis for adult GBM patients.

Although the routine use of immunohistochemistry has improved the accuracy of histopathologic diagnosis in clinical practice, new methods for discovering novel diagnostic markers are still needed. We sought new diagnostic markers for malignant pleural mesothelioma (MPM) using a reverse translational approach with limited archival tissues from a very rare case. Total RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues of a synchronous collision tumor consisting of MPM and pulmonary adenocarcinoma (PAC) was employed for gene expression profiling (GEP) analysis. Among the 54 genes selected by GEP analysis, we finally identified the following two candidate MPM marker genes: PHGDH and TRIM29. Immunohistochemical analysis of 48 MM and 20 PAC cases showed that both PHGDH and TRIM29 had sensitivity and specificity almost equivalent to those of calretinin (sensitivity 50% and 46% vs. 63%, and specificity 95% and 100% vs. 100%, respectively). Importantly, of the 23 epithelioid MMs, all 3 calretinin-negative cases were positive for TRIM29. These two markers may be diagnostically useful for immunohistochemical distinction between MPMs and PACs. This successful reverse translational approach based on FFPE samples from one very rare case encourages the further use of such samples for the development of novel diagnostic markers.

Simple Summary Pancreatic ductal adenocarcinoma is one of the most lethal malignancies, and there are vast unmet medical needs. In this study, we hypothesized that chimeric antigen receptor engineered T cell (CAR-T) targeting carcinoembryonic antigen (CEA) would be effective in the treatment of pancreatic ductal adenocarcinoma. In vivo experiments in a more clinically similar environment were considered necessary; we examined the antitumor effects of adoptive anti-CEA-CAR-T, using orthotopic xenograft mouse models of pancreatic ductal adenocarcinoma. As result, the therapeutic effect of anti-CEA-CAR-T therapy was related to the CEA expression level. Furthermore, the retrospective analysis of pathological findings from pancreatic ductal adenocarcinoma patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. These findings show that anti-CEA-CAR-T therapy can be useful for pancreatic ductal adenocarcinoma; furthermore, the pathological findings of CEA can be clinically used as biomarkers to select cases for anti-CEA-CAR-T therapy. Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared 5 samples from patients with lung or colorectal cancer, extracted genomic DNA from the cancer tissue and peripheral blood, and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants, and 25-bp delins in the EGFR exon 19, not identified by the in vitro diagnostics testing. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. All NGS platforms and bioinformatics pipelines have advantages and disadvantages. We propose the use of a PT program using patient samples to ascertain the quality status of cancer gene testing in laboratories and to ensure that laboratories have sufficient information to develop advancements in precision medicine for cancer.

OBJECTIVEThe authors aimed to investigate genetic alterations in low-grade gliomas (LGGs) in pediatric and young adult patients presenting with spontaneous hemorrhage.METHODSPatients younger than 30 years of age with a pathological diagnosis of World Health Organization (WHO) grade I or II glioma and who had undergone treatment at the authors’ institution were retrospectively examined. BRAF V600E, FGFR1 N546/K656, IDH1 R132, IDH2 R172, and KIAA1549-BRAF (K-B) fusion genetic alterations were identified, and the presence of spontaneous tumoral hemorrhage was recorded.RESULTSAmong 66 patients (39 with WHO grade I and 27 with grade II tumors), genetic analysis revealed K-B fusion in 18 (27.3%), BRAF V600E mutation in 14 (21.2%), IDH1/2 mutation in 8 (12.1%), and FGFR1 mutation in 4 (6.1%). Spontaneous hemorrhage was observed in 5 patients (7.6%); 4 of them had an FGFR1 mutation and 1 had K-B fusion. Univariate analysis revealed a statistically significant association of an FGFR1 mutation and a diencephalic location with spontaneous hemorrhage. Among 19 diencephalic cases including the optic pathway, hypothalamus, and thalamus, an FGFR1 mutation was significantly associated with spontaneous hemorrhage (p < 0.001). Four FGFR1 mutation cases illustrated the following results: 1) a 2-year-old female with pilomyxoid astrocytoma (PMA) harboring the FGFR1 K656E mutation presented with intraventricular hemorrhage (IVH); 2) a 6-year-old male with PMA harboring FGFR1 K656E and D652G mutations presented with intratumoral hemorrhage (ITH); 3) a 4-year-old female with diffuse astrocytoma harboring FGFR1 K656M and D652G mutations presented with IVH; and 4) a young adult patient with pilocytic astrocytoma with the FGFR1 N546K mutation presented with delayed ITH and IVH after 7 years of observation.CONCLUSIONSAlthough the mechanism remains unclear, the FGFR1 mutation is associated with spontaneous hemorrhage in pediatric and young adult LGG.

Chronic allograft rejection is the most common cause of long-term allograft failure. One reason is that current diagnostics and therapeutics for chronic allograft rejection are very limited. We here show that enhanced NFκB signaling in kidney grafts contributes to chronic active antibody-mediated rejection (CAAMR), which is a major pathology of chronic kidney allograft rejections. Moreover, we found that urinary orosomucoid 1 (ORM1) is a candidate marker molecule and therapeutic target for CAAMR. Indeed, urinary ORM1 concentration was significantly higher in kidney transplant recipients pathologically diagnosed with CAAMR than in kidney transplant recipients with normal histology, calcineurin inhibitor toxicity, or interstitial fibrosis and tubular atrophy. Additionally, we found that kidney biopsy samples with CAAMR expressed more ORM1 and had higher NFκB and STAT3 activation in tubular cells than samples from non-CAAMR samples. Consistently, ORM1 production was induced after cytokine-mediated NFκB and STAT3 activation in primary kidney tubular cells. The loss- and gain-of-function of ORM1 suppressed and promoted NFκB activation, respectively. Finally, ORM1-enhanced NFκB-mediated inflammation development in vivo. These results suggest that an enhanced NFκB-dependent pathway following NFκB and STAT3 activation in the grafts is involved in the development of chronic allograft rejection after kidney transplantation and that ORM1 is a non-invasive candidate biomarker and possible therapeutic target for chronic kidney allograft rejection.

Background/aim Orbital solitary fibrous tumor (SFT) is a rare lesion among orbital tumors, which can be misdiagnosed as another mesenchymal tumor. In this study we report two cases of orbital SFT, focusing on the imaging and pathological findings of the vascular structure inside the tumor. Case report A 26-year-old woman and 43-year-old man presented with orbital SFT. The pathological findings revealed a patternless growth pattern of the tumor cells and hemangiopericytoma-like vascularity as well as thickened, dilated blood vessels within the tumor tissue. Tumor cells revealed a diffuse strong positivity for cluster of differentiation 34 (CD34) and signal transducer and activator of transcription 6 (STAT6) in both cases, while B-cell lymphoma 2 (bcl-2) and CD99 were positive in one case. Characteristic findings within the tumor were the arterial components, where a variety of STAT6, CD99 and bcl-2-positive smooth muscle cells were intermingled. Conclusion Histologically, the tumor tissues might be characterized by not only conventional hemangiopericytoma-like vasculature but also dilated arterial vessels, which were shown to be part of the tumor components.

The efficacy of preoperative neoadjuvant chemoradiotherapy (NAC) in cases of pancreatic cancer with extremely poor prognoses has been reported. In this study, we aimed to identify novel biomarkers that reflect prognoses following chemoradiotherapy using tertiary lymphoid organs (TLO) expressed in the tumor microenvironment. Resected tumor specimens were obtained from 140 pancreatic cancer patients. We retrospectively investigated the clinical relevance of TLO by categorizing patients into those who underwent upfront surgery (surgery first [SF]) and those who received NAC. The immunological elements within TLO were analyzed by immunohistochemistry (IHC). In the IHC analysis, the proportions of CD8+ T lymphocytes, PNAd+ high endothelial venules, CD163+ macrophages and Ki‐67+ cells within the TLO were higher in the NAC group than in the SF group. In contrast, the proportion of programmed cell death‐1+ immunosuppressive lymphocytes within TLO was lower in the NAC group than in the SF group. The NAC group demonstrated favorable prognoses compared with the SF group. In the multivariate analysis, the TLO/tumor ratio was determined as an independent predictive prognostic factor. In conclusion, the administration of preoperative chemoradiotherapy may influence the immunological elements in the tumor microenvironment and result in favorable prognoses in pancreatic ductal adenocarcinoma patients.

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

BACKGROUND: Combination therapy with the B-Raf inhibitor, dabrafenib, and the MEK inhibitor, trametinib (DT) is commonly used to treat patients with B-Raf proto-oncogene, serine/threonine kinase V600E (BRAF V600E)-mutated non-small cell lung cancer (NSCLC). However, the mechanisms through which cancer develops DT resistance are unclear. Here, we investigated new mechanisms underlying acquired DT-resistant NSCLC with the BRAF V600E mutation. METHODS: We compared genomic signatures before and after DT treatment in patients with NSCLC. RESULTS: Two of four patients treated with DT developed carcinomatous pleuritis within 3 months. Target DNA sequencing and quantitative polymerase chain reaction (PCR) analyses revealed the increased expression level of cyclin-dependent kinase 4 (CDK4). We also found prominent protein expression of CDK4 after DT treatment. Induction of CDK4 expression in a cell line derived from a patient with the BRAF V600E mutation resulted in partial resistance to dabrafenib. CONCLUSIONS: Our findings suggest a possible relationship between CDK4 upregulation and acquired resistance to DT therapy.

We propose a method to accurately obtain the ratio of tumor cells over an entire histological slide. We use deep fully convolutional neural network models trained to detect and classify cells on images of H&E-stained tissue sections. Pathologists' labels consisting of exhaustive nuclei locations and tumor regions were used to trained the model in a supervised fashion. We show that combining two models, each working at a different magnification allows the system to capture both cell-level details and surrounding context to enable successful detection and classification of cells as either tumor-cell or normal-cell. Indeed, by conditioning the classification of a single cell on a multi-scale context information, our models mimic the process used by pathologists who assess cell neoplasticity and tumor extent at different microscope magnifications. The ratio of tumor cells can then be readily obtained by counting the number of cells in each class. To analyze an entire slide, we split it into multiple tiles that can be processed in parallel. The overall tumor cell ratio can then be aggregated. We perform experiments on a dataset of 100 slides with lung tumor specimens from both resection and tissue micro-array (TMA). We train fully-convolutional models using heavy data augmentation and batch normalization. On an unseen test set, we obtain an average mean absolute error on predicting the tumor cell ratio of less than 6%, which is significantly better than the human average of 20% and is key in properly selecting tissue samples for recent genetic panel tests geared at prescribing targeted cancer drugs. We perform ablation studies to show the importance of training two models at different magnifications and to justify the choice of some parameters, such as the size of the receptive field., To be published in SPIE Medical Imaging 2021 online proceedings

SMARCA4-deficient thoracic sarcomatoid tumors were characterized by inactivating mutations of SMARCA4 and often found in the chest of young and middle-aged males with a smoking history. Recently, SMARCA4-deficient thoracic sarcomatoid tumors were reported to represent primarily smoking-associated undifferentiated/de-differentiated carcinomas rather than primary thoracic sarcomas. The main complication of this tumor is compression of the respiratory tract and/or blood vessels. A 39-year-old man presented with a 2-month history of fever and dyspnea. Computed tomography revealed a mediastinal tumor invading the right and left pulmonary arteries. Because of severe right heart failure, we considered him ineligible for bronchoscopy. We scheduled palliative irradiation with 40 Gy/20 Fr to improve hemodynamics and perform endobronchial ultrasound transbronchial needle aspiration later. However, irradiation was ineffective, and his general condition deteriorated quickly and he died after a 7-week hospitalization. An autopsy revealed that the diagnosis was SMARCA4-deficient thoracic undifferentiated carcinoma. It has been reported that this tumor is insensitive to radiotherapy and there were some cases which responded to an immune checkpoint inhibitor. Therefore, when caring for patients with mediastinal tumors that invade and compress the trachea and large vessels, it is important to consider this tumor as a differential diagnosis and try to make a pathological diagnosis as soon as possible.

Salivary duct carcinoma (SDC) is morphologically similar to breast cancer, with HER2-overexpression reported. With regard to the pattern of disease onset, SDC can arise from de novo or carcinoma ex-pleomorphic adenoma (Ca-ex-PA). Recently, multiple molecular profiles of SDC as well as breast cancer have been reported, with significant differences in HER2 expression between Ca-ex-PA and de novo. We assessed the differences in gene expression between onset classifications. We conducted immunohistochemical analysis and HER2-DISH for 23 patients and classified SDCs into three subtypes as follows: “HER2-positive” (HER2+/any AR), “Luminal-AR” (HER2-/AR+), and “Basal-like” (HER2-/AR-). We assessed the expression levels of 84 functional genes for 19 patients by using a qRT-PCR array. Ten cases were classified as HER2-positive, seven cases as Luminal-AR, and six cases as Basal-like. The gene expression pattern was generally consistent with the corresponding immunostaining classification. The expression levels of VEGFA, ERBB2(HER2), IGF1R, RB1, and XBP1 were higher, while those of SLIT2 and PTEN were lower in Ca-ex-PA than in de novo. The functions of those genes were concentrated in angiogenesis and AKT/PI3K signaling pathway (Fisher’s test: p-value = 0.025 and 0.004, respectively). Multiple machine learning methods, OPLS-DA, LASSO, and RandomForest, also show that VEGFA can be a candidate for the characteristic differences between Ca-ex-PA and de novo. In conclusion, the AKT/PI3K signaling pathway leading to angiogenesis was hyper-activated in all SDCs, particularly in those classified into the Ca-ex-PAs. VEGFA was over-expressed significantly in the Ca-ex-PA, which can be a crucial factor in the malignant conversion to SDC.

Hepatoblastoma (HB) is the most common malignant liver neoplasm in children. Despite progress in HB therapy, outcomes for patients with metastatic disease remain poor. Dysregulation of miRNA expression is one of the potential epigenetic mechanisms associated with pathogenesis of HB. However, miRNA profiles related to the different stages of HB tissues and cells, in particular of lung metastatic tumor cells, are unknown. In the present study, using array-based miRNA expression and DNA methylation analysis on formalin-fixed paraffin-embedded tissues, we aimed to identify miRNA changes that can discriminate between lung metastatic tumors, primary tumors (fetal and embryonal subtypes), and nontumorous surrounding livers. Our analysis demonstrated that a large cluster of microRNAs and snoRNAs located within the 14q32.2 DLK1-DIO3 region showed a strikingly upregulated expression pattern in HB tumors, especially metastatic tumors, compared to normal liver tissues. This revealed dysregulation of miRNAs similar to that seen in a malignant stem-like subtype of hepatocellular carcinoma associated with poor prognosis. These findings in HB mirror similar findings made in multiple other cancer types. With further analysis this may in future allow stratification of different stages and types of HB tumors based on their miRNA profiles, which could lead to new approaches to diagnosis and treatment in progressive HB patients.

Soft tissue sarcomas (STSs) are heterogeneous and aggressive malignancies with few effective therapies available. We have developed T cells expressing a vascular endothelial growth factor receptor 2 (VEGFR2)-specific chimeric antigen receptor (CAR) to establish a tumor angiogenesis-specific CAR-T cells impacting cancers (TACTICs) therapy. In this study, we optimized the manufacturing and transportation of mRNA-transfected anti-VEGFR2 CAR-T cells and collected information that allowed the extrapolation of the efficacy and safety potential of TACTICs therapy for STS patients. Although 5-methoxyuridines versus uridines did not improve CAR-mRNA stability in T cells, the utilization of CleanCap as a 5&prime, cap-structure extended the CAR expression level, increasing VEGFR2-specific cytotoxicity. Furthermore, 4 &deg, C preservation conditions did not affect the viability/cytotoxicity of CAR-T cells, contrarily to a freeze-thaw approach. Importantly, immunohistochemistry showed that most of the STS patients&rsquo, specimens expressed VEGFR2, suggesting a great potential of our TACTICs approach. However, VEGFR2 expression was also detected in normal tissues, stressing the importance of the application of a strict monitoring schedule to detect (and respond to) the occurrence of adverse effects in clinics. Overall, our results support the development of a &ldquo, first in humans&rdquo, study to evaluate the potential of our TACTICs therapy as a new treatment option for STSs.

Mucin 1 (MUC1) expression is upregulated in multiple types of cancer, including lung cancer. However, the conventional anti‑MUC1 antibody is not useful for the differentiation of malignant lung tumors and benign lesions due to its limited specificity. Our previous study screened a novel epitope‑defined antibody against cancer‑associated sugar chain structures that specifically recognizes the MUC1 Tn antigen (MUC1‑Tn ED Ab). In the present study, its potential utility as a diagnostic marker and therapeutic tool for lung adenocarcinoma (ADC) was examined. Immunohistochemical analysis of a lung ADC tissue microarray was performed using the MUC1‑Tn ED Ab (clone SN‑102), and the results were compared with those of another clone and commercially available MUC1 antibodies. The association between positive immunoreactivity of SN‑102 and clinicopathologic factors was analyzed. Furthermore, the association between MUC1‑Tn expression and epithelial‑mesenchymal transition markers and radiological characteristics was analyzed. Moderate or high MUC1‑Tn expression (MUC1‑Tn‑H) was observed in 138 (78.9%) of the 175 lung ADC cases. MUC1‑Tn‑H was associated with male sex, cigarette smoking, tumor extension, pleural invasion, and higher preoperative serum carcinoembryonic antigen and cytokeratin 19 fragment levels. Tumors with MUC1‑Tn‑H had higher consolidation/tumor ratios according to computed tomography and greater uptakes of 18F‑fluorodeoxyglucose. A total of 46 (26.9%) of the tumors had mesenchymal features, and MUC1‑Tn positivity was higher in the mesenchymal group than in the epithelial and intermediate groups (P

This study was performed to determine the clinical significance of adenomatous polyposis coli (APC)-binding protein end-binding 1 (EB1) in hepatocellular carcinoma (HCC) and to characterize its biochemical role in comparison with previous reports. We performed immunohistochemical staining to detect EB1 expression in tissues from 235 patients with HCC and investigated its correlations with clinicopathological features and prognosis. We also investigated the roles of EB1 in cell proliferation, migration, and tumorigenesis in vitro and in vivo by siRNA- and CRISPR/Cas9-mediated modulation of EB1 expression in human HCC cell lines. The results showed that EB1 expression was significantly correlated with several important factors associated with tumor malignancy, including histological differentiation, portal vein invasion status, and intrahepatic metastasis. Patients with high EB1 expression in HCC tissue had poorer overall survival and higher recurrence rates than patients with low EB1 expression. EB1 knockdown and knockout in HCC cells reduced cell proliferation, migration, and invasion in vitro and inhibited tumor growth in vivo. Further, genes encoding Dlk1, HAMP, and SLCO1B3 that were differentially expressed in association with EB1 were identified using RNA microarray analysis. In conclusion, elevated expression of EB1 promotes tumor growth and metastasis of HCC. EB1 may serve as a new biomarker for HCC, and genes coexpressed with EB1 may represent potential targets for therapy.

// Naoko Ishida 1 , Motoi Baba 1 , Yutaka Hatanaka 2, 3 , Kanako Hagio 1 , Hiromi Okada 2 , Kanako C. Hatanaka 3 , Kenichi Togashi 4 , Yoshihiro Matsuno 2, 3 and Hiroko Yamashita 1 1 Department of Breast Surgery, Hokkaido University Hospital, Kita-ku, Sapporo 060-8648, Japan 2 Department of Surgical Pathology, Hokkaido University Hospital, Kita-ku, Sapporo 060-8648, Japan 3 Research Division of Companion Diagnostics, Hokkaido University Hospital, Kita-ku, Sapporo 060-8648, Japan 4 Roche Diagnostics K.K., Konan, Minato-ku, Tokyo 108-0075, Japan Correspondence to: Hiroko Yamashita, email: hirokoy@huhp.hokudai.ac.jp Keywords: breast cancer; estrogen receptor; PIK3CA; AKT Ser473; ERα Ser167 Received: June 27, 2017 Accepted: February 28, 2018 Published: April 03, 2018 ABSTRACT Although endocrine therapy is the most important treatment option in estrogen receptor (ER)-positive breast cancer, new strategies, such as molecular targeted agents together with endocrine therapy are required to improve survival. PIK3CA is the most frequent mutated gene in ER-positive early breast cancers, and PIK3CA mutation status is reported to affect activation of AKT and ERα. Moreover, recent studies demonstrate that patients had a better prognosis when tumors expressed ER, androgen receptor (AR), and vitamin D receptor (VDR). In this study, we examined expression of AR and VDR, phosphorylation of AKT serine (Ser) 473 (AKT phospho-Ser473) and ERα Ser167 (ERα phospho-Ser167) by immunohistochemistry in ER-positive, HER2-negative early breast cancer. PIK3CA gene mutations were also detected in genomic DNA extracted from tumor blocks. Correlations between these biological markers, clinicopathological factors and prognosis were analyzed. Levels of AKT phospho-Ser473 were significantly higher in premenopausal women than in postmenopausal women. In contrast, AR expression was significantly higher in postmenopausal women than in premenopausal women. PIK3CA mutations were detected in 47% in premenopausal women and 47% in postmenopausal women. Postmenopausal women with PIK3CA wild-type tumors had significantly worse disease-free survival than patients with PIK3CA mutant tumors. Low levels of AKT phospho-Ser473 and high levels of ERα phospho-Ser167 were strongly associated with increased disease-free survival in postmenopausal women. Evaluation of ERα activation, in addition to PIK3CA mutation status, might be helpful in identifying patients who are likely to benefit from endocrine therapy alone versus those who are not in postmenopausal ER-positive early breast cancer.

A 5-year-old boy underwent biopsy of an intra-axial calcified tumor in the hypothalamus, which was incidentally found. Based on the presence of ganglion-like cells combined with glial cell element, the pathological diagnosis was ganglioglioma. Because the tumor grew gradually in size over the next 2 years, he underwent chemotherapy with temozolomide. However, at 8 years of age, the boy developed hydrocephalus and the cystic lesion had re-grown. Endoscopic cyst fenestration and tumor biopsy was performed, and pathological diagnosis was tentatively oligodendroglioma based on the presence of tumor cells with a perinuclear halo. At 10 years of age, hydrocephalus recurred and the cystic lesion had re-grown. A second round of endoscopic cyst fenestration and tumor biopsy led to a pathological diagnosis of pilocytic astrocytoma due to a biphasic appearance with areas of dense tumor cells and microcystic areas, tumor cells with eosinophilic processes, and the presence of an eosinophilic granular body. Genetic analysis of the first biopsy successfully identified the BRAF V600E mutation. Because pathological diagnosis of diencephalic low-grade glioma harboring BRAF V600E would be sometimes difficult due to pathological variations, pathological diagnosis should be performed under the consideration of molecular diagnosis of BRAF V600E for optimal diagnosis and treatment.

Intraocular lymphoma is a rare neoplasm that occurs only in the eyes and/or central nervous system. Diagnosis of intraocular lymphoma is difficult because its clinical manifestations mimic chronic uveitis. Pathological examination of the vitreous is one of the main diagnostic tools for intraocular lymphoma, but this is challenging due to the sparse cellularity and specimen degeneration. Here, we reviewed 33 cell block preparations from vitreous perfusion fluid in order to examine the significance of cytopathological findings for differential diagnosis using vitreous samples. The cases comprised 12 intraocular lymphomas and 21 non-lymphomatous diseases. Cytologically, vitreous samples from non-lymphoma cases showed lower cellularity than the lymphoma cases. Whereas vitreous material from cases with infectious endophthalmitis showed prominent neutrophilic infiltration, material from sarcoidosis cases showed infiltration of small lymphoid cells, especially CD4-positive T cells. On the other hand, lymphoma cases showed higher cellularity, with large, irregular and atypical lymphoid cells, frequent necrotic cells in the background, and less pronounced neutrophil infiltration. Immunocytochemically, 11 of the 12 lymphoma cases were of the B-cell phenotype and the remaining case was of the T/NK-cell phenotype. In conclusion, careful cytopathological examination or immunocytochemistry of vitreous material facilitates appropriate diagnosis of intraocular lymphoma.

Atypical adenoma of the thyroid is a rare form of tumor, and its accurate diagnosis prior to surgical resection is difficult as the histological and pathological morphologies are very similar to those of anaplastic thyroid carcinoma (ATC), and its anaplastic transformation remains to be elucidated. We reported a case of a 75-year-old female with a thyroid isthmus nodule diagnosed repeatedly by FNAC as anaplastic carcinoma. Both the first and second FNAC specimen slides showed a large number of scattered or aggregated atypical cells consisting of large, pleomorphic nuclei with irregular membranes, chromatin clumps and prominent nucleoli. The morphology of the surgical specimen was similar to that of an anaplastic carcinoma and although it showed signs of transition from a normal follicular epithelium, there was no invasive growth or mitosis. This lesion was diagnosed as an atypical adenoma, and a papillary carcinoma was also present in the right lobe of the thyroid. Here we evaluate the molecular features of atypical adenomas in comparison with 9 ATC samples, and discuss whether or not atypical adenomas represent a form of premalignant lesion. Ki-67 expression was found to be very low in atypical adenomas whereas all ATC samples showed high levels of Ki-67 expression. Epithelial-mesenchymal transition (EMT) marker expression suggested that atypical adenomas maintain their epithelial phenotype to a higher degree than do ATCs. Differential diagnosis between ATC and atypical adenoma is difficult by cytological and histological methods alone, and Ki-67 and EMT marker expression may support the diagnosis.

Background Muir–Torre syndrome (MTS) is a rare autosomal dominant genodermatosis caused by mutations in mismatch repair genes. It is characterized by the presence of at least one sebaceous skin tumor associated with internal malignancies. Whether positron emission tomography/computed tomography (PET/CT) is useful for the detection of malignancies in patients with MTS has not been determined. We herein report two cases in which PET/CT was useful for the diagnosis and follow-up of internal malignancies in patients with MTS. Case presentation In case 1, a 57-year-old woman underwent excision of a sebaceous carcinoma on the left upper eyelid. She underwent follow-up PET/CT once yearly thereafter. Forty-two months after the eyelid surgery, PET/CT showed intense tracer uptake in the right lower abdomen. An ascending colon tumor was identified, and examination of a biopsy specimen showed adenocarcinoma. In case 2, a 77-year-old man presented for evaluation of three continuous papules with telangiectasia on his right cheek. Examination of a skin biopsy specimen of the cheek papule revealed a sebaceous carcinoma. He underwent PET/CT to detect other malignancies. PET/CT showed intense tracer uptake in the sigmoid colon. A sigmoid colon tumor was identified, and examination of a biopsy specimen showed adenocarcinoma. Both patients underwent resection of their tumors, and both were still free of recurrence of the sebaceous and colon carcinomas at the time of this writing. Conclusion PET/CT is a reliable imaging modality for the detection of internal malignancies and is useful for the diagnosis and follow-up of MTS.