Novel insights into the GCN2 pathway and its targeting. Therapeutic value in cancer and lessons from lung fibrosis development.

Defining the mechanisms that allow cells to adapt to environmental stress is critical for understanding the progression of chronic diseases and identifying relevant drug targets. Among these, activation of the pathway controlled by the eIF2-alpha kinase GCN2 is critical for translational and metabolic reprogramming of the cell in response to various metabolic, proteotoxic, and ribosomal stressors. However, its role has frequently been investigated through the lens of a stress pathway signaling via the eIF2α-activating transcription factor 4 (ATF4) downstream axis, while recent advances in the field have revealed that the GCN2 pathway is more complex than previously thought. Indeed, this kinase can be activated through a variety of mechanisms, phosphorylate substrates other than eIF2α, and regulate cell proliferation in a steady state. This review presents recent findings regarding the fundamental mechanisms underlying GCN2 signaling and function, as well as the development of drugs that modulate its activity. Furthermore, by comparing the literature on GCN2's antagonistic roles in two challenging pathologies, cancer and pulmonary diseases, the benefits, and drawbacks of GCN2 targeting, particularly inhibition, are discussed.

Spread Through Air Spaces in Stage I to III Resected Lung Adenocarcinomas: Should the Presence of Spread Through Air Spaces Lead to an Upstaging?

In recent years, the concept of spread through air spaces (STAS) has been discussed as an adverse prognostic factor for lung cancer. The aim of our study is to clarify the prognostic role of STAS in relation to the main recognized prognostic factors in a retrospective cohort of 330 European patients who underwent stages I to III lung adenocarcinoma resection. On univariate analysis, the presence of STAS was related to progression-free survival (PFS; hazard ratio [HR]: 1.48; 95% CI: 1.02-2.19; P = 0.038) and overall survival (OS; HR: 1.61; 95% CI: 1.03-2.52; P = 0.50). On multivariate analysis, STAS was related to PFS (HR: 1.51; 95% CI: 1.00-2.17; P = 0.050) and to OS (HR: 1.67; 95% CI: 1.00-2.81; P = 0.050). We showed that the presence of STAS was associated with lower PFS, equivalent to the next pathologic T stage, especially the median PFS of T3 stages without STAS was at 62.8 months while the median PFS of T3 stages with STAS was at 15.7 months, closer to the median PFS of 17.4 months in T4 stages. To conclude, STAS is an independent prognostic factor of PFS in this European cohort and is close to significance for OS. We suggest that the presence of STAS might lead to an upstaging of lung adenocarcinoma.

Value of immunohistochemistry in crushed areas of pulmonary neuroendocrine carcinoma.

Immunohistochemical demonstration of neuroendocrine differentiation is often performed in routine diagnostic practice for lung neuroendocrine carcinoma. However, these carcinomas are often crushed, especially on small specimens. The value of immunohistochemistry on crushed areas is not known. We aimed to assess the value of immunohistochemical markers in crushed areas. We performed a retrospective study of 299 patients with a diagnosis of pulmonary neuroendocrine carcinoma. We showed that the markers TTF-1, synaptophysin, chromogranin A, CD56, and INSM1 were more often negative in crushed areas compared with well-preserved areas. The proliferation index with anti-Ki67 was decreased but remained on average around 90%. For all markers, the percentage of labeled cells was lower than in the preserved areas. Finally, we show that cases without labeling in the crushed areas and maintained labeling in the non-crushed areas have a lower percentage of labeling than cases without this labeling mismatch. Finally, there were no false positives of these stains. Neuroendocrine markers are valid in crushed areas when positive. However, the percentage of labeled cells may be lower than on preserved areas and lead to false negatives. Finally, the proliferation index, although decreased, remains close to that on preserved areas.

Anti-CK7/CK20 Immunohistochemistry Did Not Associate with the Metastatic Site in TTF-1-Negative Lung Cancer.

Anti-CK7 and anti-CK20 immunohistochemistry is sometimes used to establish a diagnosis of primary lung cancer. We performed a retrospective study on the value of anti-CK7 and anti-CK20 immunohistochemistry in 359 biopsies of patients with suspected lung carcinoma in order to assess the usefulness of these antibodies in the evaluation of lung tumors in biopsies. Our results showed TTF-1 positivity in 73.3% of patients. EGFR mutations and ALK rearrangements were significantly different between TTF-1 positive and TTF-1 negative tumors (p < 0.001 and p = 0.023, respectively). Our results show a significant difference (p < 0.001) between TTF-1 positive and TTF-1 negative carcinomas with a median survival of 21.97 months (CI95% = 17.48−30.9 months) and 6.52 months (CI95% = 3.34−10.3 months), respectively. In the group of TTF-1 negative patients, anti-CK7 and CK20 immunohistochemistry was performed in 70 patients and showed CK7+/CK20- staining in 61 patients (87.1%), CK7-/CK20- in 4 patients (5.7%), CK7+/CK20+ in 3 patients (4.3%), and CK7-/CK20- in 2 patients (2.8%). No specific or molecular pattern was found in these groups of CK7/CK20 combinations. In total, this work brings arguments concerning the uselessness of anti-CK7/CK20 immunohistochemistry in the case of suspicion of primary lung cancer in biopsies.

Spread Through Air Spaces (STAS) Is an Independent Prognostic Factor in Resected Lung Squamous Cell Carcinoma.

Objective: There is no histoprognostic grading for lung squamous cell carcinoma (LUSC). Different prognostic factors have been described in the recent literature and are not always studied in parallel. Our objective was to search for morphological histopathological prognostic factors in LUSC. Materials and Methods: In this single-center retrospective study of 241 patients, all patients with LUSC who underwent surgical excision over a 12-year period were included. The primary endpoint was 5-year overall survival. Results: STAS was present in 86 (35.7%) patients. The presence of Spread Through Air Spaces (STAS) was correlated with tumor location (p < 0.001), pathological stage (p = 0.039), tumor differentiation (p = 0.029), percentage of necrosis (p = 0.004), presence of vascular and/or lymphatic emboli, budding (p = 0.02), single cell invasion (p = 0.002) and tumor nest size (p = 0.005). The percentage of tumor necrosis was correlated with the overall survival at 5 years: 44.6% of patients were alive when the percentage of necrosis was ≥50%, whereas 68.5% were alive at 5 years when the necrosis was <30% (p < 0.001). When vasculolymphatic emboli were present, the percentage of survival at 5 years was 42.5% compared to 65.5% when they were absent (p = 0.002). The presence of isolated cell invasion was correlated with a lower 5-year survival rate: 51.1% in the case of presence, versus 66% in the case of absence (p = 0.02). In univariate analysis, performance status, pathological stage pT or pN, pleural invasion, histopathological subtype, percentage of tumor necrosis, vasculolymphatic invasion, single-cell invasion, budding and tumor nest size correlated with the percentage of survival at 5 years. On multivariate analysis, only STAS > 3 alveoli (HR, 2.74; 95% CI, 1.18−6.33) was related to overall survival. Conclusion: In conclusion, extensive STAS is an independent factor of poor prognosis in LUSC. STAS is correlated with the presence of other poor prognostic factors such as emboli and pleural invasion and would reflect greater tumor aggressiveness.

WHO grading system for invasive pulmonary lung adenocarcinoma reveals distinct molecular signature: An analysis from the cancer genome atlas database.

Lung adenocarcinoma grading has gained interest in the past years. Recently a three-tier tumor grading was proposed showing that it is related to patients' prognosis. Nevertheless, the underlying molecular basis of this morphological grading remains partly unknown. The aim of our work is to take advantage of The Cancer Genome Atlas lung adenocarcinoma (TCGA_LUAD) cohort to describe the molecular data associated to tumor grading. We performed a study on publicly available data of the TCGA database first by assessing a tumor grade on downloadable tumor slides. Secondly we analyzed the molecular features of each tumor grade group. Our work was performed on a study group of 449 patients. We show that aneuploidy score was significantly different between grade 2 and grade 3 groups with different chromosomal imbalance (p < 0.001). SCGB1A1 mRNA expression was higher in grade 2 (p = 0.0179) whereas NUP155, CHFR, POLQ and CDC7 have a higher expression in grade 3 (p = 0.0189, 0.0427, 0.0427 and 0.427 respectively). GZMB and KRT80 have a higher methylation of DNA in grade 2 (p = 0.0201 and 0.0359 respectively). MT1G, CLEC12B and NDUFA7 have a higher methylation of DNA in grade 3 (p < 0.001, 0.0246 and 0.0359 respectively). We showed that the number of activated pathways is different between grade 2 and grade 3 patients (p = 0.004). We showed that differentially expressed genes by mRNA analysis and DNA methylation analysis involve several genes implied in chemoresistance. This could suggest that grade 3 lung adenocarcinoma might be more resistant to chemotherapy.

Florid Lymphoid Hyperplasia or Lymphoma-like Lesion of the Lower Genital Tract: A 35-Year Literature Review in View of the New WHO Classification.

Florid lymphoid hyperplasias of the lower female genital tract, also known as pseudolymphoma or lymphoma-like lesions, are benign lesions displaying histologic features which mimic aggressive B-cell lymphomas. Initially described by Young and colleagues in 1985, fewer than 100 cases have been published, making this lesion rather uncommon and subject to misdiagnoses. However, given that this entity has now been included in the World Health Organization's most recent classification, greater clarity would be beneficial for pathologists and physicians. Thus, our report aims to review these entities and provide all available data. We reviewed the available literature according to PRISMA guidelines. We found that lymphoma-like lesions, regardless of their localization, display numerous superficial lymphoid B cells admixed with a polymorphic small lymphocytic and plasmocytic background and, sometimes, superficial ulceration. Large lymphoid cells show prominent nucleoli and mitotic figures. Immunohistochemistry can usually exclude large cell lymphomas, such as high-grade follicular lymphoma and Burkitt lymphoma, when a starry sky pattern is found, as well as Hodgkin Lymphoma; however, the exclusion of diffuse large B-cell lymphoma or marginal zone lymphoma is more difficult. Explorations seeking infectious agents may show Epstein-Barr virus or, rarely, Borrelia burgdorferi involvement. Molecular study occasionally finds a monoclonal B-cell population, but without the subsequent follow-up which would otherwise be worrisome. Despite its somewhat aggressive histologic features, the benignity of this entity must be highlighted to avoid misdiagnosis and complications due to overtreating.

Histopathological and molecular profiling of lung adenocarcinoma skin metastases reveals specific features.

AIMS: Little is known regarding the histopathological and molecular features of lung adenocarcinoma skin metastases. Our study is the largest, to our knowledge, to comprehensively explore these to date. METHODS AND RESULTS: We performed a retrospective cohort study analysing 42 lung adenocarcinoma skin metastasis samples obtained from a database of 2659 lung adenocarcinomas collected between 2010 and 2020. EGFR exon 19 deletion was detected in one patient and KRAS mutations were detected in 12 (33.3%) patients. The programmed cell death ligand 1 (PD-L1) tumour proportion score was <1% in 27 patients, ≥1% and <50% in eight patients, ≥50% in six patients and not assessable in one patient. We showed that the predominant histopathological subtype is different from that at other metastatic sites (P = 0.024). Thyroid transcription factor I (TTF-1) was more often negative in skin metastases compared to other sites (P < 0.001). The EGFR mutation rate tended to be lower for skin metastases compared to other sites (P = 0.079). Skin metastases were associated with a high rate of PD-L1-negative cases (P = 0.022). CONCLUSION: Our work shows that the skin metastases of lung adenocarcinoma have a specific histopathological profile.

Histopathological typing in diffuse malignant epithelioid mesothelioma: implication for prognosis and molecular basis.

The prognostic impact of tumour grading, cytological and architectural patterns and stromal features in diffuse pleural malignant epithelioid mesothelioma (MEM) has been partly studied but not correlated to molecular features. We performed a retrospective study on 92 MEM in our department in order to assess the prognostic role of architectural and stromal patterns, especially tumour to stroma ratio. Secondly, based on The Cancer Genome Atlas (TCGA) database, we analysed the differentially expressed genes in prognostic groups of interest. Our results showed that tumour grading, tumour to stroma ratio and predominant pattern were related to overall survival, p≤0.001, p=0.01 and p=0.001, respectively. In univariate analysis, for high grade tumours hazard ratio (HR) was 4.75 (2.47-9.16), for stroma poor tumours HR=0.016, for predominant tubular or tubulopapillary pattern HR=0.044. In multivariate analysis, high grade tumours were related to overall survival [HR=3.09 (1.50-6.35), p=0.002] and predominant tubular or tubulopapillary pattern [HR=0.56 (0.32-0.99), p=0.045]. In TCGA analysis, after grading of diagnostic slides, we showed that KRTDAP and CXRCR1 expression was higher in low grade tumours, unlike PDZD7 and GPR176 expression which was higher in high grade tumours. FAM81B had a higher expression in stroma poor tumours. We did not find any differentially expressed genes in the architectural patterns group. Our work suggests that tumour grading is an important parameter in MEM with an underlying genomic basis. The role of tumour to stroma ratio needs to be investigated and might also have a genomic basis.

Cyclin D1 expression in ganglioglioma, pleomorphic xanthoastrocytoma and pilocytic astrocytoma.

Ganglioglioma, pleomorphic xanthoastrocytoma (PXA) and pilocytic astrocytoma are rare brain neoplasms with frequent activation of mitogen-activated protein (MAP) kinase pathway. A downstream marker of MAP-kinase pathway activation is cyclin D1. However, the expression of cyclin D1 has not been studied in the differential diagnosis between these brain tumors. The aim of this work is to compare the expression of cyclin D1 in ganglioglioma, PXA, pilocytic astrocytoma. We also compared cyclin D1 expression in giant cell glioblastoma and in IDH wild type glioblastoma. Our work shows that roughly half of gangliogliomas have ganglion cells stained by cyclin D1 while two third of PXA have pleormophic cells stained by cyclin D1 and 15% of giant cell glioblastoma have pleomorphic cells stained by cyclin D1 (p < 0.001). Cyclin D1 never stains normal neurons either in the adjacent cortex of circumscribed tumor, or in entrapped neurons in IDH wild type glioblastomas. The expression of cyclin D1 is correlated to the presence of BRAF V600E mutation in ganglioglioma and PXA (p = 0.002). To conclude, cyclin D1 positivity might be used to confirm the neoplastic nature of ganglion cells. Cyclin D1 is expressed in most cases of BRAF V600E mutated gangliogliomas but also in cases without BRAF mutations suggesting an activation of MAP-kinase pathway through another way. Cyclin D1 immunohistochemistry has currently no or little role in the differential diagnosis of pilocytic astrocytoma. Its role in the differential diagnosis between PXA and giant cell glioblastoma needs to be further investigated on external series.

Immune Escape Is an Early Event in Pre-Invasive Lesions of Lung Squamous Cell Carcinoma.

Bronchial dysplasia is the pre-neoplastic lesion recognized for invasive squamous cell carcinoma. The mechanisms leading to invasive squamous cell carcinoma for this lesion are not fully known. Programmed Death-Ligand 1 (PD-L1) expression by the bronchial dysplasia neoplastic epithelium might suggest a response to immunotherapy. The objective of this work is to further characterize PD-L1 and CD8 expression in bronchial dysplasia and bronchial metaplasia compared to normal bronchial epithelium. Immunohistochemical analysis of PD-L1 and CD8 staining were characterized in bronchial dysplasia of 24 patients and correlated with clinical data. We also compared PD-L1 expression in dysplasia samples to 30 normal epithelium and 20 samples with squamous bronchial metaplasia. PD-L1 was never expressed in normal epithelium and in metaplastic epithelium whereas 37.5% of patients with bronchial dysplasia were stained by PD-L1 (p < 0.001). PD-L1 expression was not related to the degree of dysplasia or a medical history of invasive squamous cell carcinoma, while CD8 expression and its localization were related to medical history of squamous cell carcinoma (p = 0.044). Our results show that PD-L1 is expressed in roughly one third of patients with bronchial dysplasia and is not expressed in normal and metaplastic epithelium. This suggests that PD-L1 is expressed in preneoplastic lesions of squamous cell carcinoma.

Histologic findings in hysterectomies after endometrial ablation.

BACKGROUND: Endometrial ablation for abnormal uterine bleeding is used as a less invasive alternative to hysterectomy, however, in cases of treatment failure hysterectomy may be finally performed. The histologic changes in these post-treatment uteri are not well-described. OBJECTIVE: To describe the histological findings in post-endometrial ablation uteri. STUDY DESIGN: During a ten-year period, 321 patients were treated with endometrial ablation. Twenty-five patients (7.8%), 10 treated with NovaSure® and 15 treated with ThermaChoice® endometrial ablation were finally subjected to hysterectomy mostly due to persistent uterine bleeding. Histologic features of these hysterectomies are described. RESULTS: The patients' age ranged from 33 to 73 years (mean 44.5) and 34-53 (mean 42) for the NovaSure® and ThermaChoice® group, respectively. The time from endometrial ablation to hysterectomy was 2-24 months (mean 8.8) and 2-60 months (mean 23.2) for the two groups, respectively (p = 0.01). Hysterectomies performed later (mean 22 months) showed no fibrosis (p = 0.04) compared with those performed earlier (mean 5 months). Endometrial lining was found more frequently in hysterectomies performed later (mean 13 months) than those performed earlier (mean 2 months, p = 0.0004). Abundant necrotic tissue of myometrial origin was found in 28% of the cases, but it was not associated with the time of hysterectomy (p = 0.2). A zonation effect and vascular changes also seen. Granulomatous reaction was not found. Ten patients (40%) harbored adenomyosis and another three (12%) extensive leiomyomas/diffuse leiomyomatosis. CONCLUSION: Necrosis, fibrosis and vascular changes are found during the first year of post-thermal uterine effect. Hysterectomies performed later show less prominent changes and almost normal endometrial lining. Adenomyosis is found in an important part of post-endometrial ablation hysterectomies.

Impact of delayed fixation and decalcification on PD-L1 expression: a comparison of two clones.

The bone is a frequent localization for lung non-small cell cancer metastasis; decalcification is required to permit tissue section. Pre-analytical conditions can influence the detection of immunohistochemical markers. The aim of our work is to evaluate PD-L1 expression in samples with delayed fixation and in decalcified tissue with chelating agent or acid at different time. Tumor-expressing PD-L1 and placental tissue were fixed at different times or decalcified with an acid decalcifier or EDTA for different durations. For 22C3 antibody, when tissues were decalcified with DC3, there was a significant decrease in the percentage of tumor cells or placental villi stained which after 4 h (p = 0.035 at 4 h). When EDTA is used for 22C3 antibody, there was a slight decrease in the percentage of stained tumor cells or villi but although there was a trend (p = 0.058 at 20 h), this was never statistically significant. For E1L3N antibody, when tissues were decalcified either with DC3 or EDTA, there was no significant decrease for the proportion of stained tumor cells or placental villi, neither for staining intensity for the first 24 h. The proportion of placental villi and tumor stained or intensity of staining was not significantly lower for any sample after delayed fixation also at 24 h for both PD-L1 clones. Delayed fixation does not affect the proportion of stained cell and intensity with PD-L1 immunohistochemistry. Decalcification also performed with EDTA lower the proportion and intensity of stained cells with PD-L1 immunohistochemistry.

Nuclear grading, BAP1, mesothelin and PD-L1 expression in malignant pleural mesothelioma: prognostic implications.

For malignant pleural mesothelioma (MPM), histopathological subtype is one of the most important prognostic factors. Several immunohistochemical stains whose expressions have possible therapeutic implications have been identified in MPM such as BAP1, mesothelin and PD-L1. The aim of our work was to evaluate the clinical significance and prognostic implications of BAP1, mesothelin and PD-L1 expression in 117 patients with a diagnosis of MPM who were diagnosed in our institution between 2002 and 2017. We also correlated this immunohistochemical profile to a recently described nuclear grading and to histopathological subtype. Mesothelin expression, BAP1 loss and PD-L1 expression were associated with histopathological subtype (p < 0.0001), BAP1 loss was more frequent in epithelioid subtype whereas PD-L1 expression was more frequent in non-epithelioid subtype. For epithelioid MPM, BAP1 expression was associated with overall survival (p = 0.034), with a longer survival when BAP1 expression is lost. Necrosis and nuclear grading are associated with overall survival (p = 0.0048 and <0.0001, respectively), with longer survival when necrosis was absent and for grade I. For non-epithelioid MPM, overall survival was not related to clinical, histopathological or immunohistochemical expression of BAP1, mesothelin or PD-L1. In multivariate analysis, grade I for nuclear grading was an independent prognostic factor associated with overall survival (p < 0.0001). In epithelioid and non-epithelioid MPM, we analysed overall survival in subgroups with combined mesothelin, BAP1 and PD-L1 expression. In epithelioid MPM, BAP1 retained/mesothelin negativity/PD-L1 > 1%, and BAP1 retained/mesothelin positivity/PD-L1 > 1% profiles, are associated with shorter overall survival. In non-epithelioid MPM, BAP1 loss/mesothelin negativity/PD-L1 > 1% is associated with shorter overall survival. Our work confirms that nuclear grading in epithelioid MPM is a strong and independent prognosis factor. Moreover, this study on several promising immunohistochemical stains whose expressions have possible therapeutic implications identifies subgroups with a poor prognosis.