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Mediastinal germ cell tumors share similar histopathological, immunohistochemical, and molecular features with their counterparts in the gonads. Therefore, proper clinical and radiological evaluation of patients with an anterior mediastinal mass becomes essential in the final interpretation of these tumors. The gold standard for the diagnosis of these tumors remains histopathological evaluation. However, immunohistochemical stains and molecular studies also provide an aid in cases in which the histology is not typical. It is also important to keep in mind that a small mediastinoscopic biopsy may not be representative of the entire neoplasm. In this review, we will provide our perspective regarding histopathological diagnosis, staging, immunohistochemical and molecular profile, and briefly family of tumors address pertinent epidemiological, clinical and treatment options. However, the main emphasis is to review the process of pathological assessment in pre and post-treated tumors. Knowledge of the different growth patterns and histological associations is important, mainly when confronted with mediastinoscopic biopsies, which ultimately will determine treatment options.

Two cases with the co-existence of micronodular thymoma and lymphoma are presented. The patients are two female patients 54 and 60 years of age who presented with symptoms of chest pain, general malaise, cough, and dyspnea. Diagnostic imaging demonstrated the presence of an anterior mediastinal mass. Initial biopsy was interpreted as an epithelial neoplasm compatible with thymoma. Surgical resection took place demonstrating the presence of micronodular thymoma in both cases. In addition, in both cases the lymphoid proliferation infiltrated surrounding adipose tissue. However, in one case the lymphoid proliferation was composed of atypical lymphocytes with irregular nuclei and increase mitotic activity positive for CD3 and TDT, while in the second case, the lymphoid proliferation was composed of more mature lymphocytes with a monocytoid appearance and positive for CD20. Both of these cases represent an unusual association and one that must be kept in the differential diagnosis especially in cases of micronodular thymoma.

Over the years the nomenclature of thymomas has been debated regarding the best manner in which these tumors should be grouped. In every schema presented thus far, the main issue has been the presence or lack of lymphocytes and accordingly, the tumors have been place into a specific category. However, even though this concept applies for most of the cases, there are numerous tumors that do not necessarily fit into those categories as either the thymomas show another cellular proliferation associated with the epithelial cells or the epithelial cell themselves are arranged in a pattern that departs from the conventional features of the classic thymoma. Herein we will emphasize those features, which in some circumstances, mainly with small mediastinoscopic biopsies may pose a considerable problem in interpretation. We do consider that the most important issue is to be familiar with the different growth pattern that these tumors may show in order to avoid misdiagnosis. In addition, we consider that regardless of the growth pattern or cellular composition of the tumor, it is highly recommended that these tumors just like any other be carefully sampled and properly stage. Although we are fully aware of the different growth pattern and specific cellular details that thymoma may show, the discussion of each one of those tumors is beyond the scope of this review. Therefore, we have placed more emphasis in those, which in our judgment are more commonly encountered in the daily practice.

PDF file 87K, Summary of multivariate analysis of outcome in lung adenocarcinoma patients by combined expression of EZH2 and TTF-1

Purpose: To understand the role of nuclear factor erythroid-2–related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in non–small cell lung cancer (NSCLC), we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy.Experimental Design: We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients' clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n = 26) or adjuvant platinum-based chemotherapy (n = 63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens.Results: We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P < 0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P = 0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival [P = 0.0139; hazard ratio (HR), 1.75] in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P = 0.0181; HR, 2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P = 0.0410; HR, 3.37).Conclusions: Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma. Clin Cancer Res; 16(14); 3743–53. ©2010 AACR.

Purpose: Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non–small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes.Experimental Design: EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCC) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status.Results: EZH2 expression was significantly (P < 0.0001) higher in SCCs compared with adenocarcinomas and in brain metastasis relative to matched primary tumors (P = 0.0013). EZH2 expression was significantly (P < 0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking, and higher TNM stage (P = 0.02 to P < 0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P = 0.025; HR = 1.54) and overall survival (OS; P = 0.0002; HR = 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P = 0.009; HR = 0.51) and OS (P = 0.0011; HR = 0.45), which was confirmed in the independent set of 91 adenocarcinomas.Conclusion: In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas. Clin Cancer Res; 19(23); 6556–65. ©2013 AACR.

Purpose: New prognostic markers to guide treatment decisions in early stage non–small cell lung cancer are necessary to improve patient outcomes. In this report, we assess the utility of a predefined mRNA expression signature of cell-cycle progression genes (CCP score) to define 5-year risk of lung cancer–related death in patients with early stage lung adenocarcinoma.Experimental Design: A CCP score was calculated from the mRNA expression levels of 31 proliferation genes in stage I and stage II tumor samples from two public microarray datasets [Director's Consortium (DC) and GSE31210]. The same gene set was tested by quantitative PCR in 381 formalin-fixed paraffin-embedded (FFPE) primary tumors. Association of the CCP score with outcome was assessed by Cox proportional hazards analysis.Results: In univariate analysis, the CCP score was a strong predictor of cancer-specific survival in both the Director's Consortium cohort (P = 0.00014; HR = 2.08; 95% CI, 1.43–3.02) and GSE31210 (P = 0.0010; HR = 2.25; 95% CI, 1.42–3.56). In multivariate analysis, the CCP score remained the dominant prognostic marker in the presence of clinical variables (P = 0.0022; HR = 2.02; 95% CI, 1.29–3.17 in Director's Consortium, P = 0.0026; HR = 2.16; 95% CI, 1.32–3.53 in GSE31210). On a quantitative PCR platform, the CCP score maintained highly significant prognostic value in FFPE-derived mRNA from clinical samples in both univariate (P = 0.00033; HR = 2.10; 95% CI, 1.39–3.17) and multivariate analyses (P = 0.0071; HR = 1.92; 95% CI, 1.18–3.10).Conclusions: The CCP score is a significant predictor of lung cancer death in early stage lung adenocarcinoma treated with surgery and may be a valuable tool in selecting patients for adjuvant treatment. Clin Cancer Res; 19(22); 6261–71. ©2013 AACR.

Supplementary Data from Nrf2 and Keap1 Abnormalities in Non–Small Cell Lung Carcinoma and Association with Clinicopathologic Features

PDF file - 212K, Final list of 31 cell cycle genes in the CCP score and their correlation with the CCP mean in the MDACC-IEO cohort. Supplementary Table 2. Set of 15 housekeeper genes in the CCP score and the correlation of their Ct mean with the housekeeper gene mean in the MDACC-IEO cohort. Supplementary FigureS1. Patient sets and exclusion criteria for the microarray cohorts (A) and the FFPE cohort (B). Supplementary FigureS2. Association between CCP score and lung cancer death in stage I patients: MDACC/IEO combined (A), MDACC subset (B) and IEO subset (C). Each patient set was divided by the median CCP score into two equally sized groups. The patient group with lower CCP scores defines a subpopulation with improved survival.

PDF file 68K, C-index of Cox proportional hazard models of outcome of lung adenocarcinoma patients

PDF file 55K, Legend Supplementary Figure 1

PDF file 106K, Kaplan-Meir curves depicting recurrence-free survival rates in the two sets of lung adenocarcinoma patients by EZH2 and combined EZH2/TTF-1 expressions

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.

Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.

Supplemental Fig. S5. Representative CP110 immunohistochemistry staining of lung normal (N) versus malignant (T) tissues harvested independently from KRAS wild-type transgenic mice and KRAS mutant transgenic mice.The table displays KRAS mutation information, CP110 scoring and intensity of CP110 immunostaining in 6 lung cancer specimens from cyclin-E transgenic mice and Kras LSL-G12D driven engineered mice respectively.

Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Three cases of primary neuroblastomas presenting as anterior mediastinal tumors are presented. The patients are two women and one man between the ages of 57 and 63 year. Clinically, the patients presented with symptoms of chest pain, cough, and shortness of breath. Diagnostic imaging revealed the presence of an anterior mediastina mass. Initial biopsy was non-diagnostic in two patients, while in one patient no biopsy was obtained. Surgical resection via thoracotomy was performed in all three patients. Grossly, the tumors vary in size from 3 to 4.5 cm in greatest dimension, and they were described as well circumscribed but not encapsulated, light brown in color. Areas of hemorrhage and/or necrosis were not described. Histologically, at low power the tumors were surrounded by a rim of adipose tissue containing remnants of thymic tissue with Hassall's corpuscles. At higher magnification, the tumors show the characteristic small round cell proliferation with varying amounts of neurophil. Mitotic activity was present but not in large number. Areas of necrosis and/or hemorrhage were not identified. Immunohistochemically, the tumors show positive staining for NSE, while synaptophysin highlighted neurophil. Other markers epithelial and neuroendocrine were negative. Clinical follow-up information shows that two patients have remained alive 8 and 12 months after initial surgical resection. One patient was lost to follow-up.

The interpretation of biopsy specimens in the diagnosis of thymoma is a subject that is generally not addressed in the literature. Even though the diagnosis of thymoma may seem to be an easy step in the assessment of these tumors, in reality, it is the biopsy specimen interpretation that will be use to determine course of action in any particular patient. It may determine whether a patient is a surgical candidate or on the contrary whether a patient may be benefited the most by medical therapy. In addition, there may be conditions in which all that is required is surgical resection without any further treatment, and that the evaluation of those conditions does not necessarily required the careful pathologic staging that thymomas need. In addition, it is important to highlight that in small biopsies, there are limitations not only in terms of the cellularity and other features that may not be present in such biopsy but also the limitation in term of immunohistochemical interpretation. Herein we have attempted to highlight numerous tumoral conditions that are frequently encountered in the daily practice of mediastinal pathology, some of them pose significant problems in separating them from thymomas. Needles to say, the entire spectrum of mediastinal pathology that may at any given time mimic thymoma is well beyond the scope of this review. Furthermore, we also herein emphasize the need for proper clinical and radiologic information and correlation in order to lead to a better interpretation of the biopsy specimen. The emphasis in this review is on thymoma and their possible pitfall and shortcomings while evaluating small biopsy specimens.

Sixty-four cases of sarcomatoid pleural mesothelioma represent the basis of this study. The patients are 51 men and 13 women between the ages of 42 and 79 years, who presented with symptoms of chest pain, cough, and weight loss. Diagnostic imaging showed the presence of diffuse pleural thickening with encasement of the lung parenchyma in all the cases. All patients had surgical resection via extrapleural pneumonectomy. By immunohistochemistry, all cases were positive for cytokeratin AE1/AE3; however, reactivity with other markers including keratin 5/6, calretinin, and D2-40 was seen in different proportions, whereas a few cases showed positive staining for GATA3, WT1, and p40. All tumors were negative for carcinomatous epitopes (carcinoembryonic antigen, CD15, and TTF1). Our findings show that even though the use of immunohistochemical stains plays an important role in the final interpretation, the best results are accomplished by a global interpretation of clinical, radiographical, and immunohistochemical findings. It is also important to highlight that it does not seem to be a single immunohistochemical stain that is pathognomonic of sarcomatoid mesothelioma and that some other stains that are commonly used for other tumors may also show positive staining in a small percentage of sarcomatoid mesotheliomas.

Introduction Immune infiltration in lung adenocarcinomas (LUADs) has been associated with response to immune checkpoint inhibitors. Clinical features underlying differential responses of patients with LUADs to immunotherapy are not well understood. Here, we analyzed the association between LUAD immune infiltration and clinicopathologic variables. Materials and Methods Intratumoral CD3, CD8, and CD68 cell densities (tumor-associated immune cells [TAICs]) were immunohistochemically assessed in 146 surgically resected LUADs. LUADs were classified into 2 groups, low and high TAICs, based on the median values of cell densities for CD3, CD8, and CD68. Somatic mutation burden and driver gene mutation status were analyzed in a subset of the cases (n = 92). We statistically analyzed the association between the TAIC groups and various clinicopathologic and molecular variables by using the χ2/Fisher and Wilcoxon sum tests and multivariable logistic regression models. Results Patient gender, tumor size, and STK11 mutations were significantly associated with TAIC levels in LUAD. Female patients exhibited significantly elevated TAIC levels (P = .005) compared with male patients. Tumor size was inversely associated with TAIC levels (P = .012). STK11 mutated tumors were associated with lower TAICs (P = .008). Higher TAICs were consistently observed in female patients with LUADs after adjusting for stage, tumor size, and age. Multivariable regression models confirmed female gender as an independent variable associated with TAIC levels in LUAD (P = .0141). Conclusion Immune infiltration in LUADs was significantly higher in female patients, warranting further exploration into the association between this clinical variable and immunotherapeutic response in LUAD.

Three cases of primary giant cell tumors of the lung akin to those described in the soft tissues are presented. The patients are 3 men between the ages of 43 and 54 years who presented with nonspecific symptoms of cough, chest pain, and shortness of breath. None of the patients had any prior history of malignancy anywhere else. Diagnostic imaging disclosed the presence of an intrapulmonary mass. All the patients underwent lobectomy. Grossly, the tumors were described as solid, slightly hemorrhagic, and measuring between 1.8 and 2.4 cm in greatest diameter. Histologically, the tumors were characterized by a dual population of multinucleated giant cells admixed with a mononuclear proliferation. Nuclear atypia was mild to moderate, and mitotic activity varied but was under 5 mitotic figures per 10 high power fields. Immunohistochemical stains showed positive staining for vimentin, CD68, and cathepsin K, whereas the tumors were negative for keratin, TTF-1, p40, S-100 protein, and SABT-2. Clinical follow-up was obtained in 2 patients who have remained alive and without evidence of recurrence or metastasis up to 12 months after surgery. One patient was lost to follow-up. The current neoplasms represent a tumor that to the best of our knowledge has not been reported as a primary neoplasm of the lung. The cases herein described represent an unusual occurrence and should be maintained in the differential diagnosis of primary pulmonary tumors rich in multinucleated giant cells.

Introduction CD73 is a membrane-bound enzyme crucial in adenosine generation. The adenosinergic pathway plays a critical role in immunosuppression and in anti-tumor effects of immune checkpoint inhibitors (ICI). Here, we interrogated CD73 expression in a richly annotated cohort of human lung adenocarcinoma (LUAD) and its association with clinicopathological, immune, and molecular features to better understand the role of this immune marker in LUAD pathobiology. Materials and methods Protein expression of CD73 was evaluated by immunohistochemistry in 106 archived LUADs from patients that underwent surgical treatment without neoadjuvant therapy. Total CD73 (T +) was calculated as the average of luminal (L +) and basolateral (BL +) percentage membrane expression scores for each LUAD and was used to classify tumors into three groups based on the extent of T CD73 expression (high, low, and negative). Results CD73 expression was significantly and progressively increased across normal-appearing lung tissue, adenomatous atypical hyperplasia, adenocarcinoma in situ, minimally invasive adenocarcinoma, and LUAD. In LUAD, BL CD73 expression was associated with an increase in PD-L1 expression in tumor cells and increase of tumor-associated immune cells. Stratification of LUADs based on T CD73 extent also revealed that tumors with high expression of this enzyme overall exhibited significantly elevated immune infiltration and PD-L1 protein expression. Immune profiling demonstrated that T-cell inflammation and adenosine signatures were significantly higher in CD73-expressing lung adenocarcinomas relative to those lacking CD73. Conclusion Our study suggests that higher CD73 expression is associated with an overall augmented host immune response, suggesting potential implications in the immune pathobiology of early stage lung adenocarcinoma. Our findings warrant further studies to explore the role of CD73 in immunotherapeutic response of LUAD.

Thymic typical and atypical carcinoids are rare and appear to be more aggressive than similar tumors in other sites. We retrospectively analyzed a group of biomarkers that hold therapeutic and prognostic utility, in 27 of these tumors. All cases were immunohistochemically stained with PAX5, MET, CRMP5, paxillin, p21, p27, EZH2, PDL-1, and Ki-67, and then H-scored. Clinicopathologic and survival data were statistically analyzed against staining (χ2 test). Five- and 10-year-survival rates were 53% and 18%, respectively. Mitotic counts ≥4 per 2 mm2 and tumor size ≥5 cm, associated with death of disease (DoD; P = .010 and .016). Ki-67 expression ≥1% associated with DoD ( P = .003) and death within 5 years ( P = .031). Biomarkers stained tumor cases as follows: PDL-1 = 0%, PAX-5 = 0%, MET = 7.4%, paxillin = 41%, CRMP5 = 78%, p21 = 63%, p27 = 63%, EZH2 = 37%, and MASH1 = 59%. Overall ± staining did not associate with survival or grade. Cases with low CRMP5 H-scores (80 associated with 10-year survival ( P = .022). Cases with high MASH1 H-score (>100) associated with DoD ( P = .021). Accurate grading and staging remain paramount in predicting clinical outcome. Biomarkers may have significance in subsets of patients and the use of these studies likely should be focused on a more personalize type of approach.

Twenty-five cases of micronodular thymomas with prominent cystic changes are presented. The patients are 13 men and 12 women between the ages of 38 and 69 years. Clinically, the majority of patients presented with nonspecific symptoms of cough, chest pain, and dyspnea. Four patients were asymptomatic. Diagnostic imaging showed the presence of an anterior mediastinal mass and surgical resection of the tumor mass was performed in all the patients. Histologically, all the tumors were characterized by the presence of cystic structures of varying sizes lined by different types of epithelium. In addition, the tumors were characterized by nodules of epithelial cells embedded in a lymphocyte-rich stroma with germinal centers. Twenty-one tumors were encapsulated while 4 tumors were minimally invasive. Immunohistochemical stains were positive for keratin in the nodular epithelial component while CD45 and CD20 were positive in the lymphoid component. Clinical follow-up ranging from 12 to 24 months was obtained in 19 patients. All the patients were alive and well without disease. No clinical follow-up was available in 6 patients. The cases presented in this article highlight the existence of cystic micronodular thymomas, which can be easily misdiagnosed as a multilocular thymic cyst.

Background Small cell lung cancer (SCLC) has the strongest association with smoking among lung cancers. The characteristics of never smokers with SCLC is not known. Research Question Are the clinical characteristics, prognostic factors, survival, genomic alterations, and tumor mutational burdens of SCLC in patients who have never smoked different from those who have smoked? Study Design and Methods A retrospective multicenter cohort study of patients with clinician-confirmed SCLC was performed with the use of a longitudinal and nationally representative electronic medical records database. Smoking history was assessed through technology-enabled abstraction and confirmed for never smokers via chart review. Genomic characteristics of never smoker patients with SCLC were examined with the use of a next-generation sequencing-based gene panel and whole exome sequencing. Results One hundred of 5,632 patients (1.8%) with SCLC were never smokers. Relative to smokers, never smokers were more likely to be female (66.0% vs 52.4%; P = .009) and present with extensive stage (70.0% vs 62.2%; P = .028). Never smokers had a higher proportion of patients in age groups 35 to 49 years (7.0% vs 3.0%; P = .006) and ≥80 years (17.0% vs 8.2%; P = .006). Known risk factors for lung cancer were found in Interpretation The sex- and age-specific distribution of SCLC among never smokers, along with differences that were identified by genomic analyses, suggests a distinct biology of SCLC in never smokers compared with smokers.

BACKGROUND: Mutations in the serine-threonine kinase LKB1/STK11 have been implicated in mediating resistance to checkpoint blockade among patients with advanced lung adenocarcinoma. We sought to examine the associations between clinicopathologic characteristics, tumor LKB1 expression, features of the immune microenvironment, and postoperative prognosis among patients with early-stage lung adenocarcinoma undergoing surgical therapy. METHODS: Formalin-fixed, paraffin-embedded specimens of patients undergoing resection of stage I-III, chemotherapy-naïve adenocarcinomas (1997–2008) were analyzed using tissue microarray sectioning. Sublobar resections were excluded. Intratumoral LKB1/STK11 expression was quantified as H-score. In a subset, tumor associated immune cell populations were quantified using whole tumor sections in peritumoral and intratumoral compartments. RESULTS: 104 patients met inclusion criteria. LKB1/STK11 expression (median H-score 102.9) was higher in women (median 123.3) than men (100.0, p=0.004) and in never-smokers (median 145.0) than former/current smokers (100.0, p=0.002). LKB1/STK11 expression was positively correlated with intratumoral infiltration of CD3(+) (r=0.351, P=0.005), CD4(+) (r=0.436, P

Mucous gland adenomas represent a small percentage of primary lung neoplasms. The accurate diagnosis of these benign tumors can be challenging not only on resected specimens but also more challenging in small bronchoscopic biopsies. If to that problem we add the issue that these tumors may also exist in the periphery of the lung, then it is easy to conclude that there is much difficulty in properly diagnosing these tumors with a core needle biopsy. Furthermore, there is little knowledge on the immunohistochemical properties and radiologic features of these tumors. Therefore, pathologists need to be aware of the spectrum of histopathologic features in these tumors and place them in perspective regarding the proper radiologic and immunohistochemical correlations. Needless to say, mucous gland adenomas exhibit a gamut of histopathologic features that can be easily confused with other more common tumor of the lung. Therefore, awareness of such features become essential in a benign tumor that is essentially diagnosed on morphologic grounds.

Purpose: Nintedanib enhances the activity of chemotherapy in metastatic non–small cell lung cancer (NSCLC). In this phase I/II study, we assessed safety and efficacy of nintedanib plus neoadjuvant chemotherapy, using major pathologic response (MPR) as primary endpoint. Patients and Methods: Eligible patients had stage IB (≥4 cm)–IIIA resectable NSCLC. A safety run-in phase was followed by an expansion phase with nintedanib 200 mg orally twice a day (28 days), followed by three cycles of cisplatin (75 mg/m2), docetaxel (75 mg/m2) every 21 days plus nintedanib, followed by surgery. With 33 planned patients, the study had 90% power to detect an MPR increase from 15% to 35%. Results: Twenty-one patients (stages I/II/III, N = 1/8/12) were treated. One of 15 patients treated with nintedanib 200 mg achieved MPR [7%, 95% confidence interval (CI), 0.2%–32%]. Best ORR in 20 evaluable patients was 30% (6/20, 95% CI, 12%–54%). Twelve-month recurrence-free survival and overall survival were 66% (95% CI, 47%–93%) and 91% (95% CI, 79%–100%), respectively. Most frequent treatment-related grade 3–4 toxicities were transaminitis and electrolyte abnormalities. On the basis of an interim analysis the study was discontinued for futility. Higher levels of CD3+ and cytotoxic CD3+CD8+ T cells were found in treated tumors of patients who were alive than in those who died (652.8 vs. 213.4 cells/mm2, P = 0.048; 142.3 vs. 35.6 cells/mm2, P = 0.018). Conclusions: Although tolerated, neoadjuvant nintedanib plus chemotherapy did not increase MPR rate compared with chemotherapy historical controls. Additional studies of the combination in this setting are not recommended. Posttreatment levels of tumor-infiltrating T cells were associated with patient survival. Use of MPR facilitates the rapid evaluation of neoadjuvant therapies. See related commentary by Blakely and McCoach, p. 3499

Introduction We have suggested that major pathologic response (MPR) could serve as a surrogate endpoint for survival and provide rapid means of comparing different neoadjuvant treatment regimens. Here, we confirm that MPR is predictive of long-term overall survival (OS) in patients with non–small-cell lung cancer (NSCLC) who underwent neoadjuvant chemotherapy and surgical resection, to assess agreement on MPR between 2 observers, and to determine the minimum number of slides needed to obtain an accurate determination of MPR. Patients and Methods We identified 151 patients with NSCLC who had been treated with neoadjuvant chemotherapy followed by complete surgical resection from 2008 to 2012. Tissue specimens were retrospectively evaluated by 2 pathologists who had been blinded to patients’ treatment and outcome. We assessed the relationships between MPR and OS, the levels of agreement between the pathologists, and determined the number of slides needed to obtain an accurate determination of MPR. Results Our results reveal that MPR examined by either observer 1 (experienced) or by observer 2 (trained) was significantly predictive of long-term OS after neoadjuvant chemotherapy. MPR was associated with long-term OS in patients with NSCLC undergoing neoadjuvant chemotherapy on multivariable analysis (hazard ratio 2.68; P = .01). The levels of agreement between 2 pathologists were high after direct in-person training by one pathologist or the other (R2 = 0.994). Our data suggest that at least 3 slides should be read to accurately determine MPR. Conclusions MPR is significantly predictive of long-term OS in neoadjuvant chemotherapy–treated patients with NSCLC. MPR may serve as a surrogate endpoint for evaluating novel chemotherapies and immunotherapy response in biomarker-driven translational clinical trials.

Enhanced tumor glycolytic activity is a mechanism by which tumors induce an immunosuppressive environment to resist adoptive T cell therapy; therefore, methods of assessing intratumoral glycolytic activity are of considerable clinical interest. In this study, we characterized the relationships among tumor (18)F-fluorodeoxyglucose (FDG) retention, tumor metabolic and immune phenotypes, and survival in patients with resected non-small cell lung cancer (NSCLC). We retrospectively analyzed tumor preoperative positron emission tomography (PET) (18)F-FDG uptake in 59 resected NSCLCs and investigated correlations between PET parameters (SUV(Max), SUV(Total), SUV(Mean), TLG), tumor expression of glycolysis- and immune-related genes, and tumor-associated immune cell densities that were quantified by immunohistochemistry. Tumor glycolysis-associated immune gene signatures were analyzed for associations with survival outcomes. We found that each (18)F-FDG PET parameter was positively correlated with tumor expression of glycolysis-related genes. Elevated (18)F-FDG SUV(Max) was more discriminatory of glycolysis-associated changes in tumor immune phenotypes than other (18)F-FDG PET parameters. Increased SUV(Max) was associated with multiple immune factors characteristic of an immunosuppressive and poorly immune infiltrated tumor microenvironment, including elevated PD-L1 expression, reduced CD57(+) cell density, and increased T cell exhaustion gene signature. Elevated SUV(Max) identified immune-related transcriptomic signatures that were associated with enhanced tumor glycolytic gene expression and poor clinical outcomes. Our results suggest that (18)F-FDG SUV(Max) has potential value as a noninvasive, clinical indicator of tumor immunometabolic phenotypes in patients with resectable NSCLC and warrants investigation as a potential predictor of therapeutic response to immune-based treatment strategies.

Three cases of primary Rosai-Dorfman disease of the thymus and lung are presented. The patients are 3 men between the ages of 42 and 47 years who presented with non-specific symptoms including cough, chest pain, and shortness of breath. Clinically, the patients did not have any other pertinent clinical history. Diagnostic imaging revealed in one patient a cystic anterior mediastinal mass, while in two other patients the imaging was that of an intrapulmonary mass, one in the right upper lobe and the other in the left lower lobe. The three patients undergo surgical resection of the mass. In the cases in which the tumor mass was in the lung, both patients had a lobectomy while in the patient with anterior mediastinal mass, surgical resection via thoracotomy was performed. The intrapulmonary tumors were described as soft and yellowish measuring 2.5 and 3.0 cm in greatest diameter, while the mediastinal mass was described as cystic measuring 4.0 cm in diameter. Histologically, all tumors show similar features in terms of a proliferation of large histiocytes admixed with an inflammatory component composed predominantly of plasma cells. Immunohistochemical stains show positive staining for CD68 and S-100 protein, while negative for keratin, CD1a, and langerin. The cases herein presented highlight the ubiquitous distribution of Rosai-Dorfman disease and the importance of keeping this entity in the differential diagnosis of histiocytic proliferation in the thymus or lung.

Five cases of an unusual primary benign stromal tumor designated as hemangioblastoma-like clear cell stromal tumor of the lung are presented. The patients are 4 women and 1 man between the ages of 39 and 52 years of age (average: 45.5 y). The patients presented with nonspecific symptoms of cough, chest pain, or dyspnea. None of the patients had any prior history of malignancy or tumor elsewhere. Diagnostic imaging showed the presence of an intrapulmonary tumor. Lobectomy was performed in all 5 patients. Grossly, the tumors were well-demarcated but not encapsulated with focal areas of hemorrhage without necrosis. Histologically, low power examination showed a cellular proliferation alternating with discrete dilated vessels reminiscent of a vascular neoplasm. Higher magnification showed medium-sized cells with clear cytoplasm arranged in sheets and cords. Mitotic activity and marked cellular atypia were not present. A wide panel of immunohistochemical studies was performed including epithelial, neural, muscle, vascular, and neuroendocrine markers, all of which showed negative staining. Tumor cells showed positive staining for vimentin. In 2 cases, fluorescence in situ hybridization for the solitary fibrous tumor was performed and was negative. Clinical follow-up in 3 patients showed no evidence of recurrence. The cases herein presented highlight an unusual benign stromal tumor of the lung, which needs to be considered in the differential diagnosis of tumors with a clear cell and vascular appearance.

Non-small cell lung cancer remains a highly lethal malignancy. Using the tamoxifen inducible Hnf1b:CreERT2 (H) transgenic mouse crossed to the LsL-KrasG12D (K) transgenic mouse, we recently discovered that an Hnf1b positive cell type in the lung is sensitive to adenoma formation when expressing a mutant KrasG12D allele. In these mice, we observe adenoma formation over a time frame of three to six months. To study specificity of the inducible Hnf1b:CreERT2 in the lung, we employed lineage tracing using an mTmG (G) reporter allele. This technique revealed recombined, GFP+ cells were predominantly SPC+. We further employed this technique in HKG mice to determine Hnf1b+ cells give rise to adenomas that express SPC and TTF1. Review of murine lung tissue confirmed a diagnosis of adenoma and early adenocarcinoma, a pathologic subtype of non-small cell lung cancer. Our expanded mouse model revealed loss of Mst1/2 promotes aggressive lung adenocarcinoma and large-scale proteomic analysis revealed upregulation of PKM2 in the lungs of mice with genetic deletion of Mst1/2. PKM2 is a known metabolic regulator in proliferating cells and cancer. Using a human lung adenocarcinoma cell line, we show pharmacologic inhibition of Mst1/2 increases the abundance of PKM2, indicating genetic loss or pharmacologic inhibition of Mst1/2 directly modulates the abundance of PKM2. In conclusion, here we report a novel model of non-small cell lung cancer driven by a mutation in Kras and deletion of Mst1/2 kinases. Tumor development is restricted to a subset of alveolar type II cells expressing Hnf1b. Our data show loss of Mst1/2 regulates levels of a potent metabolic regulator, PKM2.

Primary mediastinal non-Hodgkin lymphomas (PM-NHLs) represent ~5% of all NHLs and comprise lymphomas of B-cell and T-cell origin. PM-NHLs are defined as involvement of mediastinal lymph nodes, thymus, and/or mediastinal organs (heart, lung, pleura, pericardium) by NHL without evidence of systemic disease at presentation. The clinical scenario is variable and depends on the lymphoma subtype. The radiologic presentation is also variable ranging from a mediastinal mass with or without superior vena cava syndrome, a pleural or a cardiac mass associated with an effusion, or as an effusion only. The diagnosis of PM-NHLs can only be established by microscopic evaluation, and therefore, general pathologists should be aware of these tumors and familiar with their diagnostic approach. The most common anterior mediastinal NHLs (90% to 95%) are primary mediastinal large B-cell lymphoma and T lymphoblastic lymphoma. Thymic marginal zone lymphoma and mediastinal gray zone lymphoma are very rare. The remainder PM-NHLs involving middle or posterior mediastinum include diffuse large B-cell lymphoma (DLBCL) and rare cases of T-cell lymphoma, including anaplastic large cell lymphoma and breast implant-associated anaplastic large cell lymphoma extending to the anterior mediastinum. Primary pleural and cardiac NHLs are mostly DLBCLs. Other rare subtypes of PM-NHLs include DLBCL associated with chronic inflammation/pyothorax-associated lymphoma, fibrin-associated DLBCL (both EBV), and pleural and/or pericardial primary effusion lymphoma (HHV-8/EBV). We review the historical aspects, epidemiology, clinico-radiologic features, histopathology, immunohistochemistry, differential diagnosis, and relevant cytogenetic and molecular features of PM (thymic) LBCL, PM "nonthymic" DLBCL, BCL, unclassifiable, with features intermediate between DLBCL and classic Hodgkin lymphoma (mediastinal gray zone lymphoma), DLBCL associated with chronic inflammation (pyothorax-associated lymphoma), fibrin-associated DLBCL, and primary effusion lymphoma. This review represents the first part of 2 manuscripts covering PM-NHLs.

Rationale: Uninvolved normal-appearing airway epithelium has been shown to exhibit specific mutations characteristic of nearby non–small cell lung cancers (NSCLCs). Yet, its somatic mutational landscape in patients with early-stage NSCLC is unknown. Objectives: To comprehensively survey the somatic mutational architecture of the normal airway epithelium in patients with early-stage NSCLC. Methods: Multiregion normal airways, comprising tumor-adjacent small airways, tumor-distant large airways, nasal epithelium and uninvolved normal lung (collectively airway field), matched NSCLCs, and blood cells (n = 498) from 48 patients were interrogated for somatic single-nucleotide variants by deep-targeted DNA sequencing and for chromosomal allelic imbalance events by genome-wide genotype array profiling. Spatiotemporal relationships between the airway field and NSCLCs were assessed by phylogenetic analysis. Measurements and Main Results: Genomic airway field carcinogenesis was observed in 25 cases (52%). The airway field epithelium exhibited a total of 269 somatic mutations in most patients (n = 36) including key drivers that were shared with the NSCLCs. Allele frequencies of these acquired variants were overall higher in NSCLCs. Integrative analysis of single-nucleotide variants and allelic imbalance events revealed driver genes with shared “two-hit” alterations in the airway field (e.g., TP53, KRAS, KEAP1, STK11, and CDKN2A) and those with single hits progressing to two in the NSCLCs (e.g., PIK3CA and NOTCH1). Conclusions: Tumor-adjacent and tumor-distant normal-appearing airway epithelia exhibit somatic driver alterations that undergo selection-driven clonal expansion in NSCLC. These events offer spatiotemporal insights into the development of NSCLC and, thus, potential targets for early treatment.

Context Invasive micropapillary adenocarcinoma (MPC) is an aggressive variant of lung adenocarcinoma, frequently manifesting with advanced stage lymph node metastasis and decreased survival. Objective Identification of this morphology is important, as it is strongly correlated with poor prognosis regardless of the amount of MPC component. To date, no study has investigated the morphological criteria used to objectively diagnose it. Design Herein, we selected 30 cases of potential MPC of lung, and distributed 2 digital images per case among 15 pulmonary pathology experts. Reviewers were requested to diagnostically interpret, assign the percentage of MPC component, and record the morphological features they identified. The noted features included: columnar cells, elongated slender cell nests, extensive stromal retraction, lumen formation with internal epithelial tufting, epithelial signet ring-like forms, intracytoplasmic vacuolization, multiple nests in the same alveolar space, back-to-back lacunar spaces, epithelial nest anastomosis, marked pleomorphism, peripherally oriented nuclei, randomly distributed nuclei, small/medium/large tumor nest size, fibrovascular cores, and spread through air-spaces (STAS). Results Cluster analysis revealed three subgroups with the following diagnoses: “MPC”, “combined papillary and MPC”, and “others”. The subgroups correlated with the reported median percentage of MPC. Intracytoplasmic vacuolization, epithelial nest anastomosis/confluence, multiple nests in the same alveolar space, and small/medium tumor nest size were the most common criteria identified in the cases diagnosed as MPC. Peripherally oriented nuclei and epithelial signet ring-like forms were frequently identified in both the “MPC” and “combined papillary and MPC” groups. Conclusions Our study provides objective diagnostic criteria to diagnose MPC of lung.

Fibrosing lesions of the mediastinum represent a small but challenging group of lesions that range in etiology from infectious to idiopathic to neoplastic. The diagnosis of such lesions becomes more challenging in the setting of mediastinoscopic biopsies. In addition, over the years, there has been further accumulation of knowledge of the clinical aspects of these lesions that needs to be incorporated into their evaluation. Therefore, it is essential that in the general evaluation of these fibrosing processes, one not only carefully examines the histopathologic features of the lesion, that of a fibroinflammatory process with the appropriate histochemical and immunohistochemical studies, but also carefully evaluates the clinical presentation and imaging findings. Needless to say, as will be illustrated in this review, determining a definitive unequivocal diagnosis on a small mediastinoscopic biopsy may be difficult, and often one needs to provide guidance on the perspective of the histologic features present. In some cases, mainly tumoral conditions with extensive fibrosis, a conclusive diagnosis can be made; however, it is those cases in which the extensive fibrosis is the only histopathologic feature where more appropriate guidance is required. While this review will focus more on the non-neoplastic fibroinflammatory lesions of the mediastinum, within the discussion of differential diagnoses, we will discuss some neoplastic conditions that commonly show extensive fibrosing features.

ObjectivesEight cases of encapsulated and cystic atypical thymomas (World Health Organization type B3) are presented.MethodsCases were identified after review of more than 500 thymomas. Histologic material from surgical resections was evaluated.ResultsThe patients are seven men and one woman aged 43 to 71 years. Seven patients presented with nonspecific symptoms while one mass was found on autopsy. The tumors were 4 to 6 cm in greatest dimension and showed prominent cystic changes with an epithelial proliferation arranged in sheets and cords. Increased mitotic activity and nuclear pleomorphism were not identified. All tumors were encapsulated. Immunohistochemical studies showed the tumor cells positive for keratin cocktail, keratin 5/6, and p63. Clinical follow-up was available in five patients. All were alive without recurrence.ConclusionsThese cases represent an unusual variant of atypical thymoma that can be misdiagnosed as thymic carcinoma or multilocular thymic cyst. Awareness of this histologic growth pattern is important for accurate diagnosis.

Summary Three cases of primary carcinomas of the lung each with an extensive osteoclast-like giant cell component are presented. The patients are 3 men between the ages of 58 and 67 years (average, 62.5 years) who presented with nonspecific symptoms. A history of malignancy, infectious, or granulomatous disease was negative in all the patients. Diagnostic imaging disclosed the presence of a large intrapulmonary mass; in 1 case in the right upper lobe and in 2 cases in the right lower lobe. Surgical resection via lobectomy was performed in the 3 patients. Grossly, the tumors were described as soft, friable intrapulmonary masses, reddish in color, and measuring from 6 to 13 cm in largest diameter. Histologically, the tumors were each characterized by the extensive presence of a multinucleated osteoclast-like giant cell component, which represented approximately 80% of the tumor mass. The osteoclast-like giant cell component was admixed with a sarcomatoid carcinoma in 2 cases and an adenocarcinoma in 1 case. Immunohistochemistry showed that the osteoclast-like giant cells were positive for CD-68, cathepsin K, and histone H3, whereas the carcinoma component was positive for keratin, thyroid transcription factor-1, and histone H3 (patchy). Molecular studies were performed in 2 patients with negative results. Clinical follow-up was obtained in 2 patients; 1 died 14 months after initial diagnosis, whereas 1 remains alive 6 months after initial diagnosis. One patient was lost to follow-up. The current neoplasms represent an unusual type of lung carcinoma that needs highlighting as a separate type from conventional giant cell carcinoma.