Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non‐small‐cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune‐related proteins in fine‐needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD‐L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody‐based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore^® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD‐L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD‐L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

Brain metastases (BMs) are a key challenge in the management of anaplastic lymphoma kinase-rearranged non-small-cell lung cancer (ALK+ NSCLC), but prognostic scores are complicated or rely on data before the era of tyrosine kinase inhibitors (TKIs). This study aimed to validate the novel ALK-Brain Prognostic Index (ALK-BPI), which was originally proposed based on 44 TKI-treated ALK+ NSCLC patients from Karolinska University Hospital, using an external clinical cohort. TKI-treated ALK+ NSCLC patients with BM from Heidelberg (n = 82, cohort 1) were retrospectively analyzed alone and together with the original Karolinska cohort (n = 126, cohort 2). Cox regression models were used to determine the association of clinical variables and scores with overall survival (OS) after BM diagnosis (BM-related OS). Both cohorts showed a similar median age (58 years), roughly balanced sex distributions (52%-56% females), and Eastern Cooperative Oncology Group performance status (PS) 0-2 for most patients (87%-92%) at the time of BM development, which were present already at initial diagnosis in 36%-38% of the patients. Most patients had received next-generation ALK inhibitors (54%-63%), while 55%-56% of patients did not receive any radiotherapy. The ALK-BPI identified poor-risk patients (i.e. featuring ≥ 2/3 risk factors: PS > 2, male sex, development of BM after initial diagnosis) with a significantly shorter BM-related OS than other patients in both cohorts: 32/82 in cohort 1 with 21.3 versus 62.2 months in median [hazard ratio (HR) = 2.5, P < 0.001]; 59/126 in cohort 2 with 23.1 versus 67.2 months in median (HR = 2.6, P < 0.001). The five-parameter Lung-molGPA score did not achieve statistical significance and/or clear prognostic separation in all four groups, while the Disease-Specific Graded Prognostic Assessment score did not show consistent results. The ALK-BPI is a reliable tool for easy prognostic dichotomization of TKI-treated ALK+ NSCLC patients with BM in daily clinical practice, without the complexity of previous models.

Background: Disease-specific Graded Prognostic Assessment (DS-GPA) is the most validated prognostic tool for patients with brain metastasized lung cancer. The Lung-molGPA scoring system was recently introduced for oncogenic-driven brain metastasized lung cancer, but has not yet been validated in cohorts including only ALK-translocated tumors. Methods: We designed a retrospective cohort study consisting of 44 patients with brain metastasized ALK-positive, non-small cell lung cancer (NSCLC) who were treated between January 2009 and November 2019 at Karolinska University Hospital in Stockholm, Sweden. Information about demographics and clinicopathological parameters were collected. Predictors of overall survival (OS) were identified by Cox regression analyses. A bootstrap validation with 1000 samples was performed in order to compare the different prognostic scores. Results: The variables found to independently influence OS in the multivariate analysis, i.e., PS, sex and brain metastases at diagnosis, were used as prognostic variables in our new prognostic index (ALK-BPI). Patients were divided into two prognostic groups. The median OS was 65.7 months for the good prognostic group and 22.7 months for the poor prognostic group (p = 0.0068). In the univariate analysis of the different prognostic scores, ALK-BPI performed better than the others (HR = 3.6; 95% CI: 1.3–9.9). The mean C-statistics of the different prognostic scores were compared to each other, and no significant difference was observed. Conclusion: We propose the ALK-BPI score as a new prognostic tool that can easily be applied for ALK-positive lung cancer patients with brain metastases in daily clinical practice, as it has at least the same prognostic value as Lung-molGPA.

Stereotactic radiosurgery (SRS) is a standard treatment of brain metastases (BM), but it may lead to radionecrosis (RN) or local recurrence (LR). This study evaluates irradiated tumor volume as a predictor of LR and RN in SRS-treated lung cancer patients with BM. We retrospectively analyzed 431 lung cancer patients with BM who underwent SRS at Karolinska University Hospital (all-comers, 2009-2020). Associations among irradiated tumor volume and risks of RN, symptomatic RN, and LR at 6 and 12 months were assessed using Cox regression models. Furthermore, we evaluated the diagnostic performance of methionine positron emission tomography-computed tomography (PET-CT) in differentiating RN from LR. Forty patients (9.3%) developed asymptomatic RN, 36 (8.4%) symptomatic RN, and 67 (15.5%) LR. Larger tumor volumes significantly increased RN and LR risks. At 6 months, a volume of 4.75 cm³ corresponded to an RN risk reaching up to 20%. By 12 months, substantially smaller volumes were correlated with the same risk levels. Symptomatic RN followed a similar trend, at 6 months and 12 months. A 20% risk of LR was observed with volumes of 5.66 cm³ and 3.28 cm³ at 6 months and 12 months, respectively. Methionine PET-CT had a sensitivity of 0.909 and specificity of 0.600 in detecting RN or LR, with no significant diagnostic advantage compared with magnetic resonance imaging (MRI). Larger irradiated tumor volumes were correlated with increased RN or LR risk. Smaller volumes can lead to RN and LR, not evident at 6 months, but emerging by 12 months after SRS. Methionine PET-CT showed high sensitivity but modest specificity and offered no significant benefit over MRI in differentiating RN from LR.

Patients with NSCLC have an increased risk of developing venous thromboembolism (VTE), a condition associated with a poorer prognosis. Those with ALK rearrangements have a three- to five-fold higher risk of VTE than those with other oncogenic drivers. This study aimed to investigate the incidence, risk factors, and impact of VTE on overall survival. We retrospectively analyzed patients with advanced ALK-positive NSCLC treated at Karolinska University Hospital from 2009 to 2021. VTE events were recorded from 90 days before histologic cancer diagnosis to death or end of follow-up. The impact of VTE on survival was assessed using a time-dependent Cox model, and risk factors were evaluated using Fine-Gray competing risk regression. A total of 97 patients were included, of whom 35 (36.1%) developed VTE. Pulmonary embolism accounted for 24 cases (68.6%), with 16 events (45.7%) occurring more than 6 months after treatment initiation. The median time from diagnosis to the first clot was 167 days. Patients with VTE had a 3.43-fold increased risk of death. Competing risk regression analysis revealed that the presence of baseline adrenal metastasis (hazard ratio [HR] = 2.92, 95% confidence interval [CI]: 1.19–7.16), leukocyte count more than 11 × 10⁹/L (HR = 3.74, 95% CI: 1.91–7.31), and hemoglobin less than 10 g/dL (HR = 5.18, 95% CI: 2.07–12.94) significantly increased VTE risk, whereas albumin more than or equal to 3.5 g/dL reduced the risk (HR = 0.24, 95% CI: 0.10–0.56). In advanced ALK-positive NSCLC, baseline adrenal metastases, leukocytosis, and anemia were associated with increased VTE risk and may warrant heightened clinical vigilance.