Marco Chilosi

Aberrant Wnt/β-Catenin Pathway Activation in Idiopathic Pulmonary Fibrosis

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of β-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear β-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (ΔN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear β-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the β-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/β-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue.

Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death.

The anaplastic lymphoma kinase (ALK) gene is characteristically translocated in Anaplastic Large Cell Lymphomas (ALCL) and the juxtaposion of the ALK gene to multiple partners results in its constitutive protein tyrosine kinase activity. We show here that expression of activated ALK induces the constitutive phosphorylation of Stat3 in transfected cells as well as in primary human ALCLs. Furthermore, immunohistochemical studies demonstrate that among distinct human B and T cell lymphomas, activation of Stat3 nuclear translocation is uniquely associated with ALK expression. NPM-ALK also binds and activates Jak3; however, Jak3 is not required for Stat3 activation or for cell transformation in vitro. Moreover, src family kinases are not necessary for NPM-ALK-mediated Stat3 activation or transformation, suggesting that Stat3 may be phosphorylated directly by ALK. To evaluate relevant targets of ALK-activated Stat3, we investigated the regulation of the anti-apoptotic protein Bcl-xL and its role in cell survival in NPM-ALK positive cells. NPM-ALK expression caused enhanced Bcl-xL transcription, largely mediated by Stat3. Increased expression of Bcl-xL provided sufficient anti-apoptotic signals to protect cells from treatment with specific inhibitors of the Jaks/Stat pathway or the Brc-Abl kinase. These studies support a pathogenic mechanism whereby stimulation of anti-apoptotic signals through activation of Stat3 contributes to the successful outgrowth of ALK positive tumor cells.

Clonal mast cell disorders in patients with systemic reactions to Hymenoptera stings and increased serum tryptase levels

BACKGROUND Anaphylaxis after Hymenoptera stings has been reported in subjects with mastocytosis, but few data exist regarding disease prevalence in populations allergic to these insects. OBJECTIVE The incidence of clonal mast cell (MC) disorders in subjects with both systemic reactions to Hymenoptera stings and increased serum baseline tryptase (sBT) levels was assessed by using bone marrow (BM) aspirates and biopsy specimens. METHODS Subjects with a history of a systemic reaction caused by a Hymenoptera sting underwent the standard diagnostic work-up for Hymenoptera allergy, and sBT levels were measured. Subjects with an increased sBT level had BM evaluation that included histology/cytology, flow cytometry, and detection of KIT mutations. RESULTS Forty-four (11.6%) of 379 subjects with systemic reactions had increased sBT levels (>11.4 ng/mL), and 31 (70.5%) of these had a history of anaphylaxis. Thirty-four subjects with increased sBT levels underwent a BM analysis. Histology detected diagnostic or subdiagnostic MC infiltrates in 22 (65%) of 34 patients. Abnormal MCs were identified by means of flow cytometry and cytology in 26 (78.8%) of 33 and 20 (58.8%) of 34 subjects, respectively. A KIT mutation was detected in 17 (54.8%) of 31 subjects. The diagnosis was indolent systemic mastocytosis in 21 (61.7%) of 34 subjects and monoclonal MC activation syndrome in 9 (26.5%) of 34 subjects. All subjects with anaphylaxis had one of those 2 disorders. CONCLUSION The concomitant presence of systemic reactions (especially anaphylaxis) after Hymenoptera stings and increased sBT levels strongly suggests that a BM examination is indicated for the diagnosis of clonal MC disease.

Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ T cells by producing CCL22.

The natural history of B‐chronic lymphocytic leukemia (CLL) is not entirely explained by intrinsic defects of the neoplastic cell, but is also favored by microenvironmental signals. As CLL cells retain the capacity to respond to CD40 ligand (CD40L) and as CD4+ T cells are always present in involved tissues, we asked whether malignant CLL cells might produce T cell‐attracting chemokines. We studied the chemokine expression of CD19+/CD5+ malignant B cells from peripheral blood (PB), lymph nodes (LN) or bone marrow (BM) of 32 patients and found a major difference. LN‐ and BM‐, but not PB‐derived cells, expressed a readily detectable reverse transcription‐PCR band for CCL22 and one for CCL17 of variable intensity. CD40 ligation of PB cells induced the mRNA expression of both CCL22 and CCL17. CCL22 was also released in the culture supernatants. These supernatants induced the migration of activated CD4+, CD40L+ T cells expressing the CCL22 receptor, CCR4. T cell migration was abrogated by anti‐CCL22 antibodies. Immunohistochemistry and cytofluorography studies revealed that a proportion of CD4+ T cells in CLL LN and BM expressed CD40L. Our data demonstrate that malignant CLL cells chemo‐attract CD4+ T cells that in turn induce a strong chemokine production by the leukemic clone, suggesting a vicious circle, leading to the progressive accumulation of the neoplastic cells.

Differential Activity of Nivolumab, Pembrolizumab and MPDL3280A according to the Tumor Expression of Programmed Death-Ligand-1 (PD-L1): Sensitivity Analysis of Trials in Melanoma, Lung and Genitourinary Cancers

Background The potential predictive role of programmed death-ligand-1 (PD-L1) expression on tumor cells in the context of solid tumor treated with checkpoint inhibitors targeting the PD-1 pathway represents an issue for clinical research. Methods Overall response rate (ORR) was extracted from phase I-III trials investigating nivolumab, pembrolizumab and MPDL3280A for advanced melanoma, non-small cell lung cancer (NSCLC) and genitourinary cancer, and cumulated by adopting a fixed and random-effect model with 95% confidence interval (CI). Interaction test according to tumor PD-L1 was accomplished. A sensitivity analysis according to adopted drug, tumor type, PD-L1 cut-off and treatment line was performed. Results Twenty trials (1,475 patients) were identified. A significant interaction (p<0.0001) according to tumor PD-L1 expression was found in the overall sample with an ORR of 34.1% (95% CI 27.6-41.3%) in the PD-L1 positive and 19.9% (95% CI 15.4-25.3%) in the PD-L1 negative population. ORR was significantly higher in PD-L1 positive in comparison to PD-L1 negative patients for nivolumab and pembrolizumab, with an absolute difference of 16.4% and 19.5%, respectively. A significant difference in activity of 22.8% and 8.7% according to PD-L1 was found for melanoma and NSCLC, respectively, with no significant difference for genitourinary cancer. Conclusion Overall, the three antibodies provide a significant differential effect in terms of activity according to PD-L1 expression on tumor cells. The predictive value of PD-L1 on tumor cells seems to be more robust for anti-PD-1 antibody (nivolumab and pembrolizumab), and in the context of advanced melanoma and NSCLC.

CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs

Lung metastases from colorectal carcinomas (CRC) can be resected with improved survival. The distinction between primary lung adenocarcinomas and metastases from CRC may sometimes be difficult, especially on cytologic specimens or small bronchoscopic biopsies. Immunohistochemistry may be of help in this setting: available markers include TTF-1 and SP-A, which are markers of lung origin, whereas there are no good markers of intestinal origin, besides cytokeratin 7 and 20 coexpression pattern, which is not very specific. The nuclear CDX-2 transcription factor, which is the product of a homeobox gene necessary for intestinal organogenesis, is expressed in normal colonic epithelia and most colorectal adenocarcinomas, and could potentially be of diagnostic usefulness. Our aim was to investigate CDX-2 immunohistochemical expression using a new monoclonal antibody and to verify if CDX-2 can be a reliable marker to identify the colorectal origin of lung metastases. CDX-2 expression was evaluated in formalin-fixed, paraffin-embedded samples of normal adult human tissues (50 samples) and in 299 surgically resected carcinomas of different origins, including 125 non-lung adenocarcinomas, 117 primary lung tumors, 5 mesotheliomas, and 52 adenocarcinomas metastatic to the lung. CDX-2 was also evaluated on a series of 20 bioptic and 10 cytologic specimens (5 cases of colorectal metastases to the lung, 5 cases of metastases from other organs, and 10 primary lung adenocarcinomas). In normal tissues CDX-2 immunoreactivity was observed only in ileal and colorectal epithelia. CDX-2 was expressed in almost all primary and metastatic CRC (88 of 90) and was never observed in primary lung tumors. CDX-2 was also expressed in a limited group of adenocarcinomas of other sites (gastric, biliopancreatic, and mucinous ovarian adenocarcinomas). CDX-2 could be easily detected in all bioptic and cytologic samples of CRC metastases. CDX-2 is a reliable, specific, and sensitive immunohistochemical marker of normal and neoplastic intestinal epithelium. CDX-2 can be easily applied to routine histologic and cytologic material and is therefore a useful marker in the differential diagnosis of primary versus metastatic adenocarcinomas in the lung, and among metastases from an unknown primary, supports intestinal origin.

Transbronchial lung cryobiopsy in the diagnosis of fibrotic interstitial lung diseases

Background Histology is a key element for the multidisciplinary diagnosis of fibrotic diffuse parenchymal lung diseases (f-DPLD) when the clinical-radiological picture is nondiagnostic. Transbronchial lung cryobiopsy (TBLC) have been shown to be useful for obtaining large and well-preserved biopsies of lung parenchyma, but experience with TBLC in f-DPLD is limited. Objectives To evaluate safety, feasibility and diagnostic yield of TBLC in f-DPLD. Method Prospective study of 69 cases of TBLC using flexible cryoprobe in the clinical-radiological setting of f-DPLD with nondiagnostic high resolution computed tomography (HRCT) features. Results Safety: pneumothorax occurred in 19 patients (28%). One patient (1.4%) died of acute exacerbation. Feasibility: adequate cryobiopsies were obtained in 68 cases (99%). The median size of cryobiopsies was 43.11 mm2 (range, 11.94–76.25). Diagnostic yield: among adequate TBLC the pathologists were confident (“high confidence”) that histopathologic criteria sufficient to define a specific pattern in 52 patients (76%), including 36 of 47 with UIP (77%) and 9 nonspecific interstitial pneumonia (6 fibrosing and 3 cellular), 2 desquamative interstitial pneumonia/respiratory bronchiolitis–interstitial lung disease, 1 organizing pneumonia, 1 eosinophilic pneumonia, 1 diffuse alveolar damage, 1 hypersensitivity pneumonitis and 1 follicular bronchiolitis. In 11 diagnoses of UIP the pathologists were less confident (“low confidence”). Agreement between pathologists in the detection of UIP was very good with a Kappa coefficient of 0.83 (95% CI, 0.69–0.97). Using the current consensus guidelines for clinical-radiologic-pathologic correlation 32% (20/63) of cases were classified as Idiopathic Pulmonary Fibrosis (IPF), 30% (19/63) as possible IPF, 25% (16/63) as other f-DPLDs and 13% (8/63) were unclassifiable. Conclusions TBLC in the diagnosis of f-DPLD appears safe and feasible. TBLC has a good diagnostic yield in the clinical-radiological setting of f-DPLD without diagnostic HRCT features of usual interstitial pneumonia. Future studies should consider TBLC as a potential alternative to SLBx in f-DPLD.

bcl-2 EXPRESSION IN PLEURAL AND EXTRAPLEURAL SOLITARY FIBROUS TUMOURS

This study evaluated the immunoreactivity for bcl‐2, a molecule involved in the control of programmed cell death, in cases of pleural (14) and extrapleural (2) solitary fibrous tumour (SFT), malignant mesotheliomas of different histological types, and a variety of extrapleural CD34‐positive and CD34‐negative spindle‐cell tumours. In all SFTs, strong and diffuse immunostaining was demonstrated with anti‐bcl‐2 antibody, sharply contrasting with the complete lack of staining observed in all mesotheliomas. The specificity of immunodetection of bcl‐2 in SFT was confirmed by immunoblot analysis, showing a band consistent with the bcl‐2 protein. At extrapleural locations, strong bcl‐2 immunoreactivity was observed in Schwannoma (2/3 cases), synovial sarcoma (4/4 cases), and all cases of CD34‐positive gastrointestinal stromal tumour (GIST; 10/10 cases). Most sarcomas were bcl‐2‐negative. Lack of bcl‐2 expression was demonstrated in tumours which can pose problems in the differential diagnosis of SFT and can exhibit haemangiopericytoma‐like features, including haemangiopericytoma (3 cases), dermatofibrosarcoma protuberans (16 cases), and deep‐seated fibrous histiocytoma (3 cases). The constitutive expression of bcl‐2 in SFT widens the spectrum of available markers for these tumours, providing a useful adjunct to their differential diagnosis in difficult cases at pleural and extrapleural sites, and contributing to the understanding of their histogenesis and molecular pathogenesis.

CD30 and type 2 T helper (Th2) responses.

CD30 is one of the members of the tumor necrosis factor receptor superfamily, originally described as a marker of Reed‐Sternberg and Hodgkins cells in Hodgkins lymphoma. CD30 appears to be preferentially expressed on, and its soluble form (sCD30) released by, CD4+ and CD8+ T cell clones capable of producing T helper 2 (Th2)‐type cytokines. In nonneoplastic conditions, CD30+ T cells are barely detectable in vivo; however, a few allergen‐specific CD4+CD30+ T cells inducible to the production of Th2‐type cytokines could be sorted out from the circulation of allergic subjects after allergen exposure. Moreover, high numbers of CD30+ T cells were found in the lymph node of a patient suffering from Omenns syndrome, a rare congenital Th2‐mediated immunodeficiency disorder. More importantly, high serum levels of sCD30 were observed in some conditions in which a pathogenetic role for Th2 cells has been suggested, such as Omenns syndrome, atopy, systemic lupus erythematosus, and after infection with measles virus or human immunodeficiency virus. Thus, detection of CD30+ T cells and/or of increased levels of sCD30 may reflect the presence of immune responses or immune alterations characterized by the prevalent activation of Th2‐like cells. J. Leukoc. Biol. 57: 726‐730; 1995.

Abnormal Re-epithelialization and Lung Remodeling in Idiopathic Pulmonary Fibrosis: The Role of ΔN-p63

Products of the p63 gene, a recently described member of the p53 family, are constitutively expressed in the basal cells of human bronchi and bronchioli. The truncated isoforms of the p63 gene (ΔN-p63 proteins) counteract the apoptotic and cell cycle inhibitory functions of p53 after DNA damage, and this property is likely to be central in the cell renewal strategy of stratified epithelial tissues. To investigate the dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we immunohistochemically analyzed the expression of the transactivating and dominant-negative isoforms of the p63 gene on 16 tissue samples obtained from patients suffering from this disorder. In most IPF cases herein investigated, epithelial cells expressing ΔN-p63 were observed at sites of abnormal proliferation at the bronchiolo-alveolar junctions, characterized by epithelial hyperplasia, squamous metaplasia, bronchiolization, and abnormal p53 nuclear accumulation. Similar features were not observed in normal lung and in samples taken from other pulmonary diseases used as controls, including acute interstitial pneumonia, idiopathic bronchiolitis obliterans organizing pneumonia, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia. On the basis of these findings, we can hypothesize a new model for UIP pathogenesis, involving a deregulated development of mesenchymal-epithelial interactions and abnormal proliferation of epithelial cells at the bronchiolo-alveolar junction after cell injury. In our view, the progressive loss of alveolar tissue and lung remodeling after injury in IPF/UIP is concomitantly produced by pneumocyte loss and alveolar collapse on one hand and by progressive bronchiolar proliferation and architectural distortion on the other.