Assessment of associations between clinical and immune microenvironmental factors and tumor mutation burden in resected nonsmall cell lung cancer by applying machine learning to whole-slide images.

BACKGROUND: It is unclear whether clinical factors and immune microenvironment (IME) factors are associated with tumor mutation burden (TMB) in patients with nonsmall cell lung cancer (NSCLC). MATERIALS AND METHODS: We assessed TMB in surgical tumor specimens by performing whole exome sequencing. IME profiles, including PD-L1 tumor proportion score (TPS), stromal CD8 tumor-infiltrating lymphocyte (TIL) density, and stromal Foxp3 TIL density, were quantified by digital pathology using a machine learning algorithm. To detect factors associated with TMB, clinical data, and IME factors were assessed by means of a multiple regression model. RESULTS: We analyzed tumors from 200 of the 246 surgically resected NSCLC patients between September 2014 and September 2015. Patient background: median age (range) 70 years (39-87); male 37.5%; smoker 27.5%; pathological stage (p-stage) I/II/III, 63.5/22.5/14.0%; histological type Ad/Sq, 77.0/23.0%; primary tumor location upper/lower, 58.5/41.5%; median PET SUV 7.5 (0.86-29.8); median serum CEA (sCEA) level 3.4 ng/mL (0.5-144.3); median serum CYFRA 21-1 (sCYFRA) level 1.2 ng/mL (1.0-38.0); median TMB 2.19/ Mb (0.12-64.38); median PD-L1 TPS 15.1% (0.09-77.4); median stromal CD8 TIL density 582.1/mm2 (120.0-4967.6);, and median stromal Foxp3 TIL density 183.7/mm2 (6.3-544.0). The multiple regression analysis identified three factors associated with higher TMB: smoking status: smoker, increase PET SUV, and sCEA level: >5 ng/mL (P < .001, P < .001, and P = .006, respectively). CONCLUSIONS: The IME factors assessed were not associated with TMB, but our findings showed that, in addition to smoking, PET SUV and sCEA levels may be independent predictors of TMB. TMB and IME factors are independent factors in resected NSCLC.

Comparison of Clinically Relevant Mutation Profiles Between Preoperative Biopsy and Corresponding Surgically Resected Specimens in Japanese Patients With Non-Small-cell Lung Cancer by Amplicon-based Massively Parallel Sequencing.

BACKGROUND: Amplicon-based massively parallel sequencing (MPS) is an effective platform for identifying clinically actionable mutations across many genes in limited amounts of tissue. Most lung cancers are diagnosed and staged using small tissue samples obtained by transbronchial biopsy (TBB). To determine whether the mutations in TBB specimens detected by amplicon-based MPS reflect those present in the tumors, we compared the mutational profiles of preoperative TBB specimens and corresponding surgically resected specimens. PATIENTS AND METHODS: Fresh-frozen primary tumor specimens from non-small-cell lung cancer patients (n = 46) obtained preoperatively by TBB and during surgical resection were analyzed. The concordance of mutations detected by amplicon-based MPS in the 2 sample types was investigated, and the allele frequency of the mutations common to both specimens from the same patient was determined. RESULTS: An initial assessment of DNA quantity revealed that 46% of the TBB specimens (21 of 46) had less than the lower limit for amplicon-based MPS. These 21 TBB specimens were consequently omitted from the analysis. Of the 29 mutations detected in the TBB and/or surgically resected specimens from 25 patients, 23 were present in both samples, for a concordance rate of 79%. CONCLUSION: Amplicon-based MPS with TBB specimens approximately reflects clinically relevant tumor mutation profiles. However, the rate of TBB specimens with sufficient DNA quantity for amplicon-based MPS was only around 50%. Therefore, surgically resected specimens have a valuable role in exploratory and comprehensive genomic profiling.

Prospective genetic profiling of squamous cell lung cancer and adenosquamous carcinoma in Japanese patients by multitarget assays.

BACKGROUND: Despite considerable recent progress in the treatment of lung adenocarcinoma, there has been little progress in the development of efficacious molecular targeted therapies for squamous cell lung cancer. In addition to the recent comprehensive genome-wide characterization of squamous cell lung cancer, it is also important to genotype this form of cancer. We therefore conducted the Shizuoka Lung Cancer Mutation Study to analyze driver mutations in patients with thoracic malignancies. Here we report the results of genotyping in patients with squamous cell lung cancer. METHODS: Based on the biobanking system, in conjunction with the clinic and pathology lab, we developed a genotyping panel designed to assess 24 mutations in 10 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, HER2 and DDR2), EGFR, MET, PIK3CA, FGFR1 and FGFR2 copy numbers, and EML4-ALK and ROS1 translocations, using pyrosequencing plus capillary electrophoresis, quantitative polymerase chain reaction (PCR) and reverse-transcription PCR, respectively. RESULTS: A total of 129 patients with squamous cell lung cancer and adenosquamous carcinoma were enrolled in this study between July 2011 and November 2012. We detected genetic alterations in 40% of all cases. Gene alterations included: EGFR mutations, 6%; KRAS mutations, 4%; PIK3CA mutations, 13%; NRAS mutations, 1%; KIF5b-RET fusion gene, 1%; EGFR copy number gain, 5%; PIK3CA copy number gain, 15%; and FGFR1 copy number gain, 5%. Twelve patients (9%) harbored simultaneous genetic alterations. Genetic alterations were detected more frequently in surgically-resected, snap-frozen samples than in formalin-fixed, paraffin-embedded samples (50% vs. 29%). In addition, patients aged ≤70 years old and never-smokers showed high frequencies of genetic alterations. CONCLUSIONS: This study represents one of the largest prospective tumor-genotyping studies to be performed in Asian patients with squamous cell lung cancer. These results suggest that incorporation of genetic profiling into lung cancer clinical practice may facilitate the administration of personalized cancer treatments in patients with squamous cell lung cancer.

Assessment of mutational profile of Japanese lung adenocarcinoma patients by multitarget assays: a prospective, single-institute study.

BACKGROUND: Integration of mutational profiling to identify driver genetic alterations in a clinical setting is necessary to facilitate personalized lung cancer medicine. A tumor genotyping panel was developed and the Shizuoka Lung Cancer Mutation Study was initiated as a prospective tumor genotyping study. This study reports the frequency of driver genetic alterations in Japanese lung adenocarcinoma patients, and clinicopathologic correlations with each genotype. METHODS: Between July 2011 and January 2013, 411 lung adenocarcinoma patients admitted to the Shizuoka Cancer Center were included in this study with their written informed consent. Surgically resected tissues, tumor biopsies, and/or body cavity fluids were collected and tested for 23 hotspot sites of driver mutations in 9 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, and HER2), gene amplifications in 5 genes (EGFR, MET, PIK3CA, FGFR1, and FGFR2), and ALK, ROS1, and RET fusions. RESULTS: Genetic alterations were detected in 54.3% (223 of 411) of all patients. The most common genetic alterations detected in this study were EGFR mutations (35.0%) followed by KRAS mutations (8.5%) and ALK fusions (5.0%). Concurrent genetic alterations were detected in 22 patients (5.4%), and EGFR mutations were observed in 16 patients as the most common partner for concurrent genetic alteration. Significantly more concurrent genetic alterations were observed in older patients. CONCLUSIONS: This is one of the largest reports of a prospective tumor genotyping study on Japanese patients with adenocarcinoma. These data suggest that mutational profiling data using a multimutational testing platform would be valuable for expanding the range of molecular-targeted therapeutics in lung cancer.

Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections.

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR.

18F-FDG uptake on PET in primary mediastinal non-thymic neoplasm: a clinicopathological study.

BACKGROUND: The usefulness of 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) positron emission tomography (PET) has been investigated in thymic epithelial tumors. However, little is known about PET imaging of (18)F-FDG in primary non-thymic mediastinal neoplasms. The aim of this study is to explore the clinicopathological significance of (18)F-FDG PET in primary mediastinal (non-thymic) neoplasms. METHODS: Twenty-one patients with mediastinal neoplasms who underwent (18)F-FDG PET before treatment were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (Glut1); glucose transporter 3 (Glut3); hypoxia-inducible factor-1 alpha (HIF-1α); hexokinase I; vascular endothelial growth factor (VEGF); microvessels (CD34); epidermal growth factor receptor (EGFR); Akt/mTOR signaling pathway (p-Akt and p-mTOR); cell cycle control (p53). RESULTS: Seventeen of 21 patients were imaged on PET system using (18)F-FDG, but 4 patients with a histology of cyst showed nothing abnormal in PET scans. The histology of the resected tumors was as follows: 6 schwannoma, 3 teratoma, 4 cyst, 3 sarcoma, 1 undifferentiated carcinoma, 1 seminoma, 1 mediastinal goiter, 1 ganglioneuroma, and 1 Hodgkin lymphoma. (18)F-FDG uptake was significantly correlated with Glut1, HIF-1α, EGFR, p-Akt and p-S6K. These biomarkers were highly expressed in schwannoma, teratoma and high grade malignancies, whereas all patients with cyst and ganglioneuroma had no positive expression of these biomarkers. High uptake of (18)F-FDG was significant associated with Glut1, VEGF, EGFR, p-Akt, p-S6K and tumor maximal size. CONCLUSION: The amount of (18)F-FDG uptake in primary mediastinal non-thymic neoplasms is determined by the presence of glucose metabolism (Glut1), hypoxia (HIF-1α) and upstream components of HIF-1α (EGFR, p-Akt and p-S6K).

Biologic correlates of ¹8F-FDG uptake on PET in pulmonary pleomorphic carcinoma.

BACKGROUND: Pulmonary pleomorphic carcinoma is a rare epithelial tumor, and little is also known about the information on the usefulness of 2-[¹8F]-fluoro-2-deoxy-d-glucose (¹8F-FDG) positron emission tomography (PET). Therefore, we conducted the study including the underlying biologic analysis of ¹8F-FDG uptake. METHODS: Fifteen patients with pulmonary pleomorphic carcinoma who underwent ¹8F-FDG PET before treatment were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (Glut1); glucose transporter 3 (Glut3); hypoxia-inducible factor-1 alpha (HIF-1α); cell proliferation (Ki-67 labeling index); vascular endothelial growth factor (VEGF); microvessels (CD34); cell cycle control marker (p53); and apoptosis marker (bcl-2). These parameters were correlated with a control group of patients with other non-small cell lung cancer (NSCLC) (n=33). RESULTS: The maximal standardized uptake value (SUV(max)) of the primary tumors in 15 patients ranged from 6.1 to 26.8 (median 19.3). There were positive correlation between ¹8F-FDG uptake and Glut1 (p=0.0016), Glut3 (p=0.0080), VEGF (p=0.0048), and microvessel density (MVD) (p=0.0005). HIF-1α, p53 and bcl-2 showed no positive correlation with ¹8F-FDG uptake. ¹8F-FDG uptake, Glut1, Glut3, HIF-1α, VEGF and Ki-67 were significantly higher in patients with pulmonary pleomorphic carcinoma than those with other NSCLC. CONCLUSION: ¹8F-FDG uptake in pulmonary pleomorphic carcinoma is closely associated with the presence of glucose metabolism (Glut1 and Glut3) and angiogenesis (VEGF and MVD). The relationship between ¹8F-FDG uptake and these biomarkers may lead to a more rational use of PET scan in pulmonary pleomorphic carcinoma.

Biologic correlation of 2-[18F]-fluoro-2-deoxy-D-glucose uptake on positron emission tomography in thymic epithelial tumors.

PURPOSE: The usefulness of 2-[(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]FDG) positron emission tomography (PET) can help predict the grade of malignancy and staging in thymic epithelial tumors. However, no satisfactory biologic explanation exists for this phenomenon. The aim of this study was to investigate the underlying biologic mechanisms of [(18)F]FDG uptake. PATIENTS AND METHODS: Forty-nine patients with thymic epithelial tumors who underwent [(18)F]FDG PET were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), hypoxia-inducible factor-1 alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), microvessels (CD31 and CD34), cell cycle control marker (p53), and apoptosis marker (bcl-2). We also conducted an in vitro study of [(18)F]FDG uptake in a thymic tumor cell line. RESULTS: There was a positive correlation between [(18)F]FDG uptake and GLUT1 (P < .0001), HIF-1alpha (P = .0036), VEGF (P < .0001), microvessel density (MVD; P < .0001), and p53 (P = .0002). GLUT3 and bcl-2 showed no positive correlation with [(18)F]FDG uptake. The expression of Glut1, HIF-1alpha, VEGF, CD31, CD34, and p53 was also significantly associated with the grade of malignancy and poor outcome in thymic epithelial tumors, whereas this relationship was not observed in GLUT3 and bcl-2. Our in vitro study demonstrated that upregulation of GLUT1 and HIF-1alpha was closely associated with [(18)F]FDG uptake into thymic tumor cell. CONCLUSION: [(18)F]FDG uptake in thymic epithelial tumors is determined by the presence of glucose metabolism (GLUT1), hypoxia (HIF-1alpha), angiogenesis (VEGF and MVD), and cell cycle regulator (p53).

Optimal extent of lymph node dissection for T1 gastric cancer, with special reference to the distribution of micrometastasis, and accuracy of preoperative diagnosis for wall invasion.

BACKGROUND/AIMS: Preoperative diagnosis for wall invasion and lymph node metastasis is sometimes difficult in T1 gastric cancer. Optimum dissection extent of lymph nodes for T1 gastric cancer was studied from the aspect of subclassification of wall invasion and lymph node metastasis including micrometastasis. METHODOLOGY: 184 patients with cT1 or pT1 gastric cancer were studied. The grade of clinical wall invasion (cT) and clinical lymph node status (cN) were diagnosed by endoscopy and computed tomography or intraoperative findings. Lymph node metastasis (pN) was studied by hematoxylin and eosin staining and immunohistochemistry (IHC). RESULTS: In 79 cM tumors, 60 (75.9%) were diagnosed as pM. In 88 cSM tumors, 42 (47.7%) were diagnosed as pSM. In 94 pM gastric cancers, micrometastases were found in two patients (2.1%) and in N1 stations. Two (1.9%) of 70 pSM cancers had micrometastasis in No. 7, 8a and 12a stations. Lymph node metastasis (pN) correlated significantly with the depth of tumor invasion, lymphatic invasion and venous invasion. Regarding the pN2 stations, one (1.1%) of 94 pM tumors had lymph node metastasis in No.7 station, and 9 (12.9%) of 70 pSM tumors had nodal involvement in No.7, 8a, 11p, 12a and 14v stations. All eight pN+/cM tumors were diagnosed as nN0 and four (1.4%) of 23 pN+/cSM tumors were correctly diagnosed as pN+. In contrast, 8 (9.9%) of 81 cN0/cM tumors and 19 (24.1%) of 79 cN0/cSM tumors had histological lymph node metastasis (pN+). CONCLUSIONS: Accuracy of the clinical diagnosis of lymph node metastasis is very low. Accordingly, prophylactic lymph node dissection is recommended even for cT1 and cN0 tumors. For cN0/cM cancer, D1+No.7 is recommended. D1+No.7, 8a, 9, 11p is recommended for cSM cancer, located in U or M region and additional dissection of No. 14v is recommended for cSM cancer located in L region.

Cartilaginous features in matrix-producing carcinoma of the breast: four cases report with histochemical and immunohistochemical analysis of matrix molecules.

Matrix-producing carcinoma of the breast is a well-established entity in the group of metaplastic carcinoma, which is histologically characterized by myxochondroid matrix formation and is extremely rare. We describe here four additional cases of matrix-producing carcinoma of the breast. All cases of matrix-producing carcinoma show nest-like, sheet-like, and cord-like growth of tumor cells with cellular atypia, in addition to scattered cancer cells within myxoid or myxohyalinous stroma. Three of four cases showed an acellular or oligocellular matrix-rich zone in the center of the tumor. Immunohistochemically, cancer cells of all cases were positive for cytokeratins and epithelial membrane antigens and partially positive for sox9 and p63. Aggrecan and type II collagen, which are cartilage-specific matrix molecules, were deposited in the stroma of all cases. Type I and type IV collagens were also deposited on the stroma of all cases. These findings suggest that, although cancer cells of matrix-producing carcinoma of the breast are epithelial, they transdifferentiate to chondrocyte-like cells and produce cartilage-specific matrix molecules, which are useful markers for diagnosing matrix-producing carcinoma.

Evaluation of lymphatic invasion in primary gastric cancer by a new monoclonal antibody, D2-40.

Lymphatic invasion is known as an independent predictor of lymph node metastasis in gastric cancer. However, the diagnosis of lymphatic invasion is sometimes difficult by hematoxylin-eosin (H&E) staining. Immunostaining using D2-40 was performed to study the distribution of lymphatic vessel and lymphatic invasion in a series of 78 primary gastric cancers. D2-40 showed specific staining for the lymphatic vessels, but not for blood vessels. The lymphatic invasion was most frequently found in the upper half of submucosal layer. Positive rate of lymphatic invasion by H&E staining was 27% (21/78), and that by D2-40 was 44% (34/78). Lymphatic invasion on H&E staining was diagnosed as false negative in 17 (21.8%) of 78 primary gastric cancers and false positive in 4 (5.1%) of 78 primary gastric cancers. Sensitivity for lymph node metastasis by the lymphatic invasion diagnosed by D2-40 was significantly higher (89%, 24/27) than by H&E staining (41%, 11/27). These results suggest that the diagnosis of lymphatic invasion by D2-40 is more sensitive than H&E staining. Sensitivity for the prediction of lymph node metastasis from the lymphatic invasion status in primary tumor by D2-40 was significantly higher than by H&E staining. Based on our results, we recommend the use of D2-40 immunoreactions for the routine evaluation of lymphatic invasion in gastric cancer.

A < 1.7 cM interval is responsible for Dmo1 obesity phenotypes in OLETF rats.

1. Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes of male Otsuka Long Evans Tokushima Fatty (OLETF) rats. 2. Our congenic lines, produced by transferring Dmo1 chromosomal segments from the non-diabetic Brown Norway (BN) rat into the OLETF strain, have confirmed the strong, wide-range therapeutic effects of Dmo1 on dyslipidaemia, obesity and diabetes in the fourth (BC4) and fifth (BC5) generations of congenic animals. Analysis of a relatively small number of BC5 rats (n = 71) suggested that the critical Dmo1 interval lies within a < 4.9 cM region between D1Rat461 and D1Rat459. 3. To confirm the assignment of the Dmo1 critical interval, we intercrossed BC5 animals to produce a larger study population (BC5:F1 males; n = 406). For the present study, we used bodyweight at 18 weeks of age as an index of obesity; this phenotype is representative of the closely associated dyslipidaemia and hyperglycaemia phenotypes. 4. Interval mapping assigned logarithm of odds (LOD) peaks at the D1Rat90 marker (LOD = 9.11). One LOD support interval lies within the < 1.7 cM region between D1Rat461 and D1Rat459. 5. This large intercross study confirms that Dmo1 is likely localized within the interval.

Difference in cytoplasmic localization pattern of neutral mucin among lobular endocervical glandular hyperplasia, adenoma malignum, and common adenocarcinoma of the uterine cervix.

To investigate whether the cytoplasmic localization pattern of neutral mucin differs between lobular endocervical glandular hyperplasia (LEGH) and cervical adenocarcinoma (CxAd), including minimal-deviation adenocarcinoma (MDA), or adenoma malignum, alcian blue (pH 2.5)/periodic acid-Schiff (AB-PAS) staining was performed to formalin-fixed paraffin-embedded tissue sections of 13 lesions of LEGH and 53 tumors of CxAd, including 6 tumors of MDA. The cytoplasmic localization of neutral mucin was classified as a "whole cytoplasmic pattern," in which neutral mucin filled the cytoplasm entirely, or as an "apical pattern," in which neutral mucin was localized in the subsurface area only. Cytoplasmic neutral mucin patterns were detected in all 13 cases of LEGH and in 19 cases (36%) of CxAd, including five cases of MDA. The localization of neutral mucin was always the whole cytoplasmic pattern in 13 cases of LEGH, but was the apical pattern in these 19 cases of CxAd. The other 34 cases of CxAd, including 1 case of MDA, corresponded to the acid mucin pattern stained purple or blue or no staining pattern by AB-PAS. Among the 53 cases of CxAd, the apical neutral mucin pattern was an indicator of poorer patient prognosis by univariate and multivariate analyses. The examination of cytoplasmic localization of neutral mucin might be applicable, not only to differential diagnosis between LEGH and CxAd, including MDA, but also to estimate clinical aggressiveness of CxAd.

Interobserver variation in the diagnosis of adenoma malignum (minimal deviation adenocarcinoma) of the uterine cervix.

To examine the interobserver agreement level of the histological diagnosis of adenoma malignum (ADM), 52 proliferative endocervical glandular lesions were evaluated independently by four observers (A to D), each of whom is in charge of gynecological pathology at a different hospital. The correlation of diagnosis by each observer with patient outcome was also examined for 19 of these lesions. When the diagnoses were categorized into benign lesions including hyperplasias, ADM, and common types of adenocarcinoma, consistent diagnoses among all observers were achieved for only 12 lesions (23%), with a slight level of interobserver agreement (kappa=0.115). The points of disagreement were as follows: (i) whether proliferative endocervical glandular lesions preserving lobular structures were diagnosed as benign or as ADM; and (ii) whether proliferative endocervical glandular lesions with a discrete area of obvious adenocarcinoma were diagnosed as ADM or as common-type adenocarcinoma. The mortality rates of patients with ADM diagnosed by observers A, B, C, and D were 60% (3 of 5), 25% (3 of 12), 14% (1 of 7), and 13% (2 of 15), respectively. Therefore, ADM diagnosed by observers A and B was frequently lethal, whereas ADM diagnosed by observers C and D was mostly non-lethal and might contain benign lesions. The diagnosis of ADM covered various spectra of proliferative endocervical glandular lesions among the observers. Disagreement in the diagnosis was suggested to derive largely from the absence of consensus criteria for differential diagnosis among benign hyperplastic lesions, ADM, and common adenocarcinoma, and from differences in the observers' interpretations about cellular atypia and invasion.