Multiomic analysis of malignant pleural mesothelioma identifies molecular axes and specialized tumor profiles driving intertumor heterogeneity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Through a large series of whole-genome sequencing data, integrated with transcriptomic and epigenomic data using multiomics factor analysis, we demonstrate that the current World Health Organization classification only accounts for up to 10% of interpatient molecular differences. Instead, the MESOMICS project paves the way for a morphomolecular classification of MPM based on four dimensions: ploidy, tumor cell morphology, adaptive immune response and CpG island methylator profile. We show that these four dimensions are complementary, capture major interpatient molecular differences and are delimited by extreme phenotypes that-in the case of the interdependent tumor cell morphology and adapted immune response-reflect tumor specialization. These findings unearth the interplay between MPM functional biology and its genomic history, and provide insights into the variations observed in the clinical behavior of patients with MPM.

Comprehensive Molecular and Pathologic Evaluation of Transitional Mesothelioma Assisted by Deep Learning Approach: A Multi-Institutional Study of the International Mesothelioma Panel from the MESOPATH Reference Center.

INTRODUCTION: Histologic subtypes of malignant pleural mesothelioma are a major prognostic indicator and decision denominator for all therapeutic strategies. In an ambiguous case, a rare transitional mesothelioma (TM) pattern may be diagnosed by pathologists either as epithelioid mesothelioma (EM), biphasic mesothelioma (BM), or sarcomatoid mesothelioma (SM). This study aimed to better characterize the TM subtype from a histological, immunohistochemical, and molecular standpoint. Deep learning of pathologic slides was applied to this cohort. METHODS: A random selection of 49 representative digitalized sections from surgical biopsies of TM was reviewed by 16 panelists. We evaluated BAP1 expression and CDKN2A (p16) homozygous deletion. We conducted a comprehensive, integrated, transcriptomic analysis. An unsupervised deep learning algorithm was trained to classify tumors. RESULTS: The 16 panelists recorded 784 diagnoses on the 49 cases. Even though a Kappa value of 0.42 is moderate, the presence of a TM component was diagnosed in 51%. In 49% of the histological evaluation, the reviewers classified the lesion as EM in 53%, SM in 33%, or BM in 14%. Median survival was 6.7 months. Loss of BAP1 observed in 44% was less frequent in TM than in EM and BM. p16 homozygous deletion was higher in TM (73%), followed by BM (63%) and SM (46%). RNA sequencing unsupervised clustering analysis revealed that TM grouped together and were closer to SM than to EM. Deep learning analysis achieved 94% accuracy for TM identification. CONCLUSION: These results revealed that the TM pattern should be classified as non-EM or at minimum as a subgroup of the SM type.

Sarcoidosis, inorganic dust exposure and content of bronchoalveolar lavage fluid: the MINASARC pilot study.

Inhalation of mineral dust was suggested to contribute to sarcoidosis. We compared the mineral exposome of 20 sarcoidosis and 20 matched healthy subjects. Bronchoalveolar lavage (BAL) samples were treated by digestion-filtration and analyzed by transmission electron microscopy. The chemical composition of inorganic particles was determined by energy-dispersive X-ray (EDX) spectroscopy. Dust exposure was also assessed by a specific questionnaire. Eight sarcoidosis patients and five healthy volunteers had a high dust load in their BAL. No significant difference was observed between the overall inorganic particle load of each group while a significant higher load for steel was observed in sarcoidosis patients (p=0.029). Moreover, the building activity sub-score was significantly higher in sarcoidosis patients (p=0.018). These results suggest that building work could be a risk factor for sarcoidosis which could be considered at least in some cases as a granulomatosis caused by airborne inorganic dust. The questionnaire should be validated in larger studies. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 327-332).

[Technical recommendations and best practice guidelines for May-Grünwald-Giemsa staining: literature review and insights from the quality assurance]. / Recommandations techniques et règles de bonne pratique pour la coloration de May-Grünwald-Giemsa : revue de la littérature et apport de l'assurance qualité.

May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples.

French multicentric validation of ALK rearrangement diagnostic in 547 lung adenocarcinomas.

Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening. Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded. 140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (<15%). FISH was not interpretable for seven cases. ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p<0.0001). 13 cases were IHC negative but FISH ≥15%, including six cases with FISH between 15% and 20%; eight were IHC positive with FISH between 10% and 14%. Sensitivity and specificity for 5A4 and D5F3 were 87% and 92%, and 89% and 76%, respectively. False-negative IHC, observed in 2.4% of cases, dropped to 1.3% for FISH >20%. Variants were undetected in 36% of ALK tumours. Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.

Usual interstitial pneumonia end-stage features from explants with radiologic and pathological correlations.

Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe idiopathic interstitial pneumonia, with typical high-resolution computed tomography (HRCT) features and histologic pattern of usual interstitial pneumonia (UIP); its main differential diagnosis is fibrotic nonspecific interstitial pneumonia (F-NSIP). Usual interstitial pneumonia was mainly described from lung biopsies, and little is known on explants. Twenty-two UIP/IPF explants were analyzed histologically and compared with previous open lung biopsies (OLBs; n = 11) and HRCT (n = 19), when available. Temporospatial heterogeneity and subpleural and paraseptal fibrosis were similarly found in UIP/IPF explants and OLB (91%-95%). Fibroblastic foci were found in 82% of OLBs and 100% of explants, with a higher mean score in explants (P = .023). Honeycombing was present in 64% of OLBs and 95% of explants, with a higher mean score in explants (P = .005). Almost 60% of UIP/IPF explants showed NSIP areas and 41% peribronchiolar fibrosis; inflammation, bronchiolar metaplasia, and vascular changes were more frequent in UIP/IPF explants; and Desquamative Interstitial Pneumonia (DIP)-like areas were not common (18%-27%). Numerous large airspace enlargements with fibrosis were frequent in UIP/IPF explants (59%). On HRCT, honeycombing was observed in 95% of the cases and ground-glass opacities in 53%, correlating with NSIP areas or acute exacerbation at histology. Six patients had combined IPF and emphysema. Lesions were more severe in UIP/IPF explants, reflecting the worsening of the disease. Usual interstitial pneumonia/IPF explants more frequently presented with confounding lesions such as NSIP areas, peribronchiolar fibrosis, and airspace enlargements with fibrosis sometimes associated with emphysema.

Intimal sarcoma is the most frequent primary cardiac sarcoma: clinicopathologic and molecular retrospective analysis of 100 primary cardiac sarcomas.

We report novel molecular and pathologic features of sarcomas involving the heart. Intimal sarcoma appears as the most frequent primary cardiac sarcoma within the largest described series of 100 primary cardiac sarcomas. Immunohistochemical analysis, fluorescence in situ hybridization, real-time polymerase chain reaction, and array-comparative genomic hybridization were performed on materials from 65 women and 35 men, aged 18 to 82 years (mean 50 y), retrieved from the French Departments of Pathology, between 1977 and early 2013. Right and left heart was involved in 44 and 56 cases, respectively. There were 42 intimal sarcomas, 26 angiosarcomas, 22 undifferentiated sarcomas, 7 synovial sarcomas, 2 leiomyosarcomas, and 1 peripheral neuroectodermal tumor. All but 1 angiosarcomas originated from the right heart, whereas 83% of the intimal sarcomas and 72% of the undifferentiated sarcomas were from the left heart. MDM2 overexpression was immunohistochemically observed in all intimal sarcomas, as well as in 10 of the 22 undifferentiated sarcomas and in 5 of the 26 angiosarcomas. MDM2 amplification was only demonstrated in intimal sarcomas. Genomic analysis showed a complex profile, with recurrent 12q13-14 amplicon involving MDM2, 4q12 amplicon involving KIT and PDGFRA, 7p12 gain involving EGFR, and 9p21 deletion targeting CDKN2A. Immunohistochemical detection of MDM2 overexpression can easily detect intimal sarcoma, provided that molecular aberration is proved. As resections are limited to the left atrium, this histologic subtype could benefit from therapies targeting PDGFRA or MDM2.

Extensive expression of collapsin response mediator protein 5 (CRMP5) is a specific marker of high-grade lung neuroendocrine carcinoma.

The diagnosis of high-grade neuroendocrine tumors has strong clinical relevance because it identifies patients at higher risk of an unfavorable outcome who should receive multimodal treatment. However, these tumors can be mistaken for poorly differentiated nonsmall cell carcinoma or carcinoid lung tumors. In fact, no immunohistochemical marker can currently distinguish between histologic lung subtypes. Because the collapsin response mediator protein (CRMP) family is involved in an autoimmune disease associated with small cell lung carcinoma, we explored the relationship between CRMP5 expression and lung tumor behavior. Using World Health Organization morphologic criteria, 123 lung neuroendocrine tumors and 41 randomly selected non-neuroendocrine tumors were classified. CRMP5 protein expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining method. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade neuroendocrine lung tumors, including small cell lung carcinoma and large cell lung neuroendocrine carcinoma, but not in any of the squamous cell carcinomas or lung adenocarcinomas in our series. In contrast, the majority of low-grade neuroendocrine lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with 2 different staining patterns of either scattered positive cells or small foci of positive cells. Our findings point at CRMP5 as a novel marker for routine pathologic evaluation of lung tumors surgical samples in distinguishing between highly aggressive neuroendocrine carcinoma and the other lung cancers.

Influence of residual stress/strain on the biomechanical stability of vulnerable coronary plaques: potential impact for evaluating the risk of plaque rupture.

In a vulnerable plaque (VP), rupture often occurs at a site of high stress within the cap. It is also known that vessels do not become free of stress when all external loads are removed. Previous studies have shown that such residual stress/strain (RS/S) tends to make the stress distribution more uniform throughout the media of a normal artery. However, the influence of RS/S on the wall stress distribution in pathological coronaries remains unclear. The aim of this study was to investigate the effects of RS/S on the biomechanical stability of VPs. RS/S patterns were studied ex vivo in six human vulnerable coronary plaque samples. Because the existence of RS/S can only be assessed by releasing it, the opening angle technique was the experimental approach used to study the geometrical opening configurations of the diseased arteries, producing an arterial wall in a near-zero stress state. Reciprocally, these opening geometries were used in finite element simulations to reconstruct the RS/S distributions in closed arteries. It was found that the RS/S 1) is not negligible, 2) dramatically affects the physiological peak stress amplitude in the thin fibrous cap, 3) spotlights some new high stress areas, and 4) could be a landmark of the lipid core's developmental process within a VP. This study demonstrates that plaque rupture is not to be viewed as a consequence of intravascular pressure alone, but rather of a subtle combination of external loading and intraplaque RS/S.

Valvular heart disease associated with fenfluramine detected 7 years after discontinuation of treatment.

We report the case of a patient referred to us for mitral and aortic valvular disease with a rheumatic appearance. The unusual macroscopic appearance on valve resection was not compatible with a rheumatic cause. A detailed review of this patient's clinical history (ie, a history of treatment with fenfluramine) suggested an iatrogenic cause, which was confirmed by histology. For the first time, a case of valvular heart disease that deteriorated was discovered 7 years after treatment with fenfluramine, whereas this iatrogenic disease classically resolves after discontinuation of treatment. This case illustrates the need for continuing heart valve surveillance of patients who have used these anorectics.

Comparison between 3 aortic clamps for video-assisted cardiac surgery: a histological study in a pig model.

OBJECTIVES: To assess histological traumatic effects of aortic clamps used in video-assisted surgery, an experimental study was undertaken in a pig model, comparing the Portaclamp, Endoclamp, and a metallic clamp. MATERIAL AND METHODS: In 3 groups of 5 pigs each, the descending aorta was exposed through a posterolateral left thoracotomy. External clamps (Portaclamp and metallic clamp) were positioned at the middle of the aorta. Endoclamps were inserted at the top of the descending aorta through a small purse and inflated lower. After 60 minutes of clamping, the clamp was removed and the animal reperfused for 60 minutes. It was then sacrificed and the descending aorta was harvested for blind histological study using hemotoxylin-eosin staining of 4 samples per animal: A, before the clamping spot; B, at the clamping spot; C, after the clamping spot; D, a remote sample as control. RESULTS: In the Portaclamp and metallic clamp groups, there were no lesions of the intima in all aortic samples. In the Endoclamp group, severe lesions of the intima were observed on the clamping spot: endothelium crushing with flattening of cell nucleus (3/5) or endothelium stripping with vanishing of cell nucleus all gathered in 1 point (2/5). Only spongy lesions (clearance between fibers) located on the external third of the media and moderate inflammatory lesions of the adventice were observed with a random distribution in aortic samples without difference between groups. CONCLUSIONS: This study reveals the impressive lesions of the aortic intima due to the Endoclamp. The nonspecific lesions observed in media or adventice may be related to the surgical trauma of the procedure.

Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Quantitative study of nerves of the human left atrium.

OBJECTIVES: To quantify and study the distribution of innervation of the left atrium and the pulmonary veins in humans. BACKGROUND: Damage to cardiac nerves has been hypothesized as the explanation for successful radiofrequency ablation of atrial fibrillation. METHODS: From January 2003 to September 2003, histologic and quantitative studies of innervation of the left atrium and the pulmonary veins was performed in 43 consecutive necropsied adult hearts (30 men and 3 women; mean age 45.5 +/- 12.4 years). The left atrium was sectioned in 1-cm slices from left to right, with the plane of section perpendicular to the long axis of the heart. Sections of the pulmonary veins at their ostia and sections 1 cm away of this structure also were obtained. Nerve fiber density was counted manually for each case and expressed as the mean number per slice. RESULTS: Numerous epicardial nerve fibers and ganglia having distinct patterns of distribution in the left atrium were found. Nerve density was significantly higher at the ostia of the four pulmonary veins than in their distal part (7.1 +/- 2.1 vs 5.2 +/- 1.3 for left upper pulmonary vein; 6.3 +/- 1.5 vs 5.2 +/- 1.7 for right upper pulmonary vein; 7.4 +/- 2 vs 5.9 +/- 2 for left lower pulmonary vein; 6.7 +/- 1.8 vs 3.9 +/- 1.3 for right lower pulmonary vein). The left superior vein was significantly more innervated than the right inferior vein (12.3 +/- 3 vs 10.6 +/- 1.4). Gradients of innervation were found from right to left (9.8 +/- 4.6 vs 18.5 +/- 6.6, P < .05) and from the front to the rear of the atrium (17.2 +/- 6.4 vs 20.7 +/- 6.5, P < .05). The same heterogeneous distribution was observed at the myocardial level but with thinner nerve fibers, making quantification difficult. Only very thin nerve fibers were present in the endocardium. CONCLUSIONS: The human left atrium exhibits several gradients of innervation at discrete sites. These findings may have clinical implications for radiofrequency ablation of atrial fibrillation.

Expression of class III beta tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy.

This study determined the prevalence and the prognostic value of the expression of microtubule components in tumors of 19 patients with non small cell lung cancer receiving taxane-based regimens. Patient samples were stained with antibodies directed against total beta tubulin, classes I, II and III beta tubulin isotypes, delta2 alpha tubulin, tau protein, and the P-gp protein involved in the classical multidrug resistance phenotype. All tumors were stained with pan-beta tubulin antibody and class I tubulin isotype. A majority of the tumor samples expressed class II and class III, although the percentage of positive cells varied significantly between tumors. delta2 alpha tubulin, tau protein and Pgp protein were found in only one tumor sample each. Progression-free survival was shorter (41 days) in patients whose tumors expressed high levels of class III tubulin isotype in comparison to patients with low levels (288 days, p = 0.02). There were 2 responses to chemotherapy among 9 patients (22%) with high levels of class III tubulin vs. 6 among 10 patients (60%) with low levels of expression (Fisher exact test: p = 0.11). These data suggest that high expression of class III tubulin by tumor cells is associated with poor prognosis in patients with NSCLC receiving a taxane-based regimen.