118 Chapter 10 The presence of loose tumor cells and other cells, in pulmonary arteries was scored in H&E and CK7 stained slides by two experienced pulmonary pathologists (HB, ET). Follow-up data were retrieved from patient files. Because the exploratory study contained a relatively small number of patients and consisted of a not uniformly defined set of patients, we expanded the study with two retrospective separate cohorts in search of validation of the initial findings: patients with resected pulmonary adenocarcinoma diagnosed between the 1st January 2011 and the 31th December 2016 in the (1) Onze Lieve Vrouwe Gasthuis (OLVG) Hospital, Amsterdam, The Netherlands and (2) San Raffaele Scientific Institute, Milan, Italy. The inclusion criteria for both of these cohorts were different from the exploratory cohort and included resection specimens with a primary pulmonary adenocarcinoma of a pathological tumor diameter 3 cm or less and available follow-up information. The exclusion criteria were the presence of nodal or hematogenous metastases at the time of resection, treatment with neoadjuvant chemotherapy, multiple nodules in the same or other lobes, synchronous previous lung carcinoma, and invasive mucinous adenocarcinoma or other special type patterns (intestinal and fetal adenocarcinoma). Freshly cut sections of the selected paraffin blocks from both institutes were stained for H&E, elastica von Gieson (EvG or elastin) staining, and CK7 immunohistochemistry at the department of Pathology of the VUmc. Both institutes used neutral buffered formaldehyde for the fixation of their resection specimens. It was estimated that a total of 70 to 80 cases would be sufficient to validate the initial finding. Therefore, after determining suitable cases in both institutes, a minimum of 40 cases in each were randomly selected. Age, sex, total tumor size, tumor location, pathological stage adjusted to the 8th edition of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) TNM classification system for non-small cell lung cancer (NSCLC)182, time to recurrence, death, and cause of death were determined by retrospective chart review after approval by the Institutional Review Board (OLVG: ACWO21-044; VUmc 2022.0269 [biobank 2017.023]; Milan CE 284/2022). In the OLVG in Amsterdam 218 cases of resected adenocarcinoma in the lung between January 1st 2011 and December 31st 2016 were retrieved from the pathology archives. One hundred fifty-eight cases were excluded, because they did not meet inclusion criteria (47 with nodal metastases, 26 metastases from other organ [mainly colorectal carcinoma], 20 with multiple nodules, 2 subjected to neoadjuvant therapy, 47 with a diameter > 3 cm, 4 with previous or synchronous lung cancer, 12 mucinous or other type). The remaining 60 cases were included for random selection. A total of 365 cases of resected primary lung adenocarcinoma between 1st January 2011 and 31th December 2016 were retrieved from San Raffaele archive in Milan. A total of 223 were firstly excluded because did not meet the inclusion criteria of the study (122 with nodal metastases, 12 without lymph nodes histologic examination, 6 subjected to neoadjuvant treatment, 8 with multiple lung nodules at the time of resection, 18 mucinous and 1 enteric adenocarcinoma, 18 mixed mucinous and non-mucinous
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