IMMUNOPHENOTYPICAL CHARACTERIZATION OF LUNG ADENOCARCINOMAS: MOLECULAR AND CLINICAL CORRELATIONS

Il carcinoma del polmone è la neoplasia maligna con incidenza maggiore nonchè la principale causa di morte per neoplasia al mondo. La classificazione dell'Organizzazione Mondiale della Sanità (WHO, 2004) utilizzata fino a poco tempo fa distingueva le neoplasie polmonari in due grandi categorie: i carcinomi a piccole cellule e i carcinomi non a piccole cellule. Tale classificazione risulta ad oggi inadeguata e superata poiché l’utilizzo di farmaci sempre più specifici e mirati a colpire la biologia del tumore impone una sempre più attenta e precisa classificazione diagnostica delle neoplasie cui far corrispondere una stratificazione prognostica dei pazienti corredata inoltre da dati indicativi di risposta alla terapia. Scopo di questo studio è quello di distinguere, sulla base dell’immunofenotipo, le differenti entità carcinomatose del polmone. Centocinquantanove casi (159) di carcinoma del polmone sono stati selezionati dagli archivi di cinque istituzioni (Anatomia Patologica di Verona, Negrar, Mestre, Trento, Forlì). Ogni caso è stato rivisto da 2 patologi per confermare la diagnosi e per selezionare l’incluso in paraffina significativo su cui effettuare le indagini immunoistochimiche. Abbiamo successivamente classificato i tumori sulla base delle diverse espressioni dei marcatori immunoistochimici ed, in sintesi, il 43% dei casi erano carcinomi "alveolari", il 24% erano carcinomi "bronchiolari", il 16% erano carcinomi a "cellule Clara", il 9% erano di tipo “metaplastico-enterico", il 4% erano carcinomi sarcomatoidi, il 3 % erano carcinomi neuroendocrini e l'1% erano di tipo “metaplastico-squamoso". I dati clinici hanno confermato la relazione tra fumo di sigaretta e l'incidenza di adenocarcinoma (82% dei casi), inoltre, il distretto più spesso coinvolto dalla recidiva tumorale è risultato essere quello dei linfonodi mediastinici (66% dei casi di recidiva), con una sopravvivenza media libera (DFS) da malattia di 9 mesi. In 18 casi la sopravvivenza globale media era di 19,8 mesi, senza significative correlazioni con l’immunofenotipo espresso. In particolare, l'entità metaplastico enterica mostrava morfologia prevalentemente mucinosa, K-RAS mutato e prognosi peggiore (DFS media di 5,5 mesi). Tali correlazioni, basate sui profili immunoistochimici delle neoplasie, devono essere verificate ed eventualmente validate su un maggior numero di casi, con un follow-up più lungo, in modo da consentire una eventuale e reale stratificazione prognostica dei pazienti.

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related mortality. The lung cancer classification of the World Health Organization (WHO, 2004) can efficiently distinguish small-cell (SCLC) and non-small-cell lung carcinomas (NSCLC) but it results inadequate when facing prognosis and therapeutic decisions, especially for the entity of adenocarcinoma. Knowledge concerning the molecular pathogenesis of this tumor type is far from complete and diagnostic problems are mainly related to the poor reproducibility of morphological and histological criteria. Lung adenocarcinoma represents indeed a heterogeneous group of tumours characterized by different morphological and immunophenotypical features and molecular pathogenesis. Since updated therapeutic approaches are available (e.g. molecular-targeted therapies using tyrosine kinase inhibitors), exact histological classification is required for proper therapy and prognostic estimates. Aim of this study is to distinguish different lung tumour entities outlining a classification on the basis of the neoplastic immunophenotype. One hundred and fifty-nine cases of lung neoplasm were retrieved from the archives of five institutions (Anatomia Patologica of Verona, Negrar, Mestre, Trento, Forlì). All the cases were diagnosed as NSCLC carcinomas according to the WHO classification of 2004 and reviewed by two pathologists (MC and AE) to confirm the diagnosis and to select a significative paraffin block containing both tumor and adjacent non-neoplastic lung tissue. For each case multiple (30) paraffine sections were obtained in order to perform immunohistochemistry. The immunohistochemical panel included the following antibodies: CD56 (123C3.D5, Neomarkers), CK20 (IT-KS20.8, Biogenex), CK7 (OV-TL12/30, Biogenex), TTF-1 (8G7G3/1, Dako), NAPSIN A (TMU-A d02, Arp), SP A-1 (PE-10, Dako), CD208 (I10-1112, Beckton Dickinson), CC10 (rabbit, Dako), MUC5 (CLH2, Novocastra), CK5 (XM26, Novocastra), P63 (4A4, Santacruz), VILLINA (CWWB1, Novocastra), CDX2 (CDX2-88, Biogenex), P53 (DO-7, Novocastra), P21 (SX-118, Dako), P16 (JC8, Neomarkers) and DELTA-N-P63 (rabbit, Oncogene). Of 159 patients, 69 underwent surgery in Verona in 2007-2008; of these, clinical data were available for 34 cases and included smoke, type of surgery, adjuvant therapy, relapse, disease free survival (DFS), type of chemotherapy and overall survival (OS). We classified the tumors on the basis of the immunophenotype. In summary, 43% of the cases were “alveolar” carcinomas, 24% were “bronchiolar” carcinomas, 16% were carcinomas with “Clara cells features”, 9% were “metaplastic-enteric type”, 4% were sarcomatoid carcinomas, 3% were neuroendocrine carcinomas and 1% were “metaplastic-squamous type”. The mean age was 64, 113 were males (70%) and 46 were females (30%). The mean tumor size was 3,3 cm. The cases with available clinical data were 34, of which 17 were “alveolar” carcinomas, 8 were “bronchiolar” carcinomas, 4 were carcinomas with “Clara cells features”, 3 were “metaplastic-enteric type” and 2 were sarcomatoid carcinomas. Six patients (18%) never smoked, 9 patients (26%) quit smoking and 19 patients (56%) are currently smokers. Pathological stage according to Cancer staging (6th edition) was IA in 12 cases (35%), IIA in 6 cases (18%), IIB in 6 cases (18%), IIIA in 6 cases (18%) and IV in 4 cases (11%). Seven patients (20%) underwent neoadjuvant radiotherapy. The disease recurred in 12 patients (35%) with a mean DFS of 9 months and a predilection (66%) of recurrency in mediastinal lymphnodes. In seven patients (20%) we did not observe any recurrency while in 15 patients (45%) the data were not available. Surgery type, regarding the resection margins was R0 in 26 cases (76%) and R1 in 8 cases (24%). In 18 cases OS was available with a mean period of 19,8 months. The goal of our classification is not only to provide the most accurate diagnosis but also to manage the tissue in a way that immunohistochemical and/or molecular studies can be performed to obtain predictive and prognostic data that will lead to improvement in patient outcomes. It is well known that immunohistochemistry is a practical and powerful method for diagnosis and research, since it is simple, not time consuming, less expensive than molecular investigations, feasible on paraffin sections, amenable on small biopsy and cytology samples and permits correlations with morphology at both the cellular and subcellular levels. Particularly, the immunohistochemical panel including CK7, CK20, CK5, TTF-1, NAPSIN-A, CC-10, SP-A, CD208, CDX-2, VILLIN, MUC-5 provides a useful tool in distinguishing different subtypes of adenocarcinomas. Available clinical data confirmed the well-known relationship between smoking and incidence of adenocarcinoma (82% of cases); moreover, the sites most often involved by tumor recurrence resulted to be mediastinal lymphnodes (66% of cases with recurrency) with a mean DFS of 9 months. In 18 cases OS was available with a mean period of 19,8 months without correlation to the histotype. The metaplastic enteric type entity of adenocarcinoma, related to “bronchiolar” derivation, shows mainly mucinous morphology, frequently harbour KRAS mutations and worse prognosis (mean DFS of 5,5 months with mediastinal lymphnodes recurrency). Among cases with available clinical data the follow-up period resulted probably not long enough in order to establish adequate and statistically significant relationships between different immunophenotypes and prognosis. Difficulties in clinical data collection have been determined mainly by patients’ compliance, different modalities and locations of post-operative care and by the fact that the cases were retrieved from the archives of different institutions. Although highly promising, these studies should be further validated in future investigation. Such correlations need in fact more cases with longer follow-up period to be reliably verified and to allow stratification of patients with different NSCLC entities on the basis of molecular-immunophenotypical features.