Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. Significance: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer. See related commentary by Letai, p. 290. This article is highlighted in the In This Issue feature, p. 275

Thymoma and thymic carcinoma are thymic epithelial tumors (TETs). We performed a molecular profiling to investigate the pathogenesis of TETs and identify novel targets for therapy. We analyzed 37 thymomas (18 type A, 19 type B3) and 35 thymic carcinomas. The sequencing of 50 genes detected nonsynonymous mutations in 16 carcinomas affecting ALK, ATM, CDKN2A, ERBB4, FGFR3, KIT, NRAS and TP53. Only two B3 thymomas had a mutation in noncoding regions of the SMARCB1 and STK11 gene respectively. Three type A thymomas harbored a nonsynonymous HRAS mutation. Fluorescence in situ hybridization detected in 38 % of carcinomas a CDKN2A, in 32 % a TP53 and in 8 % an ATM gene deletion, whereas only one B3 thymoma exhibited a CDKNA deletion, and none of the type A thymomas showed a gene loss. Sequencing of the total miRNA pool of 5 type A thymomas and 5 thymic carcinomas identified the C19MC miRNA cluster as highly expressed in type A thymomas, but completely silenced in thymic carcinomas. Furthermore, the miRNA cluster C14MC was downregulated in thymic carcinomas. Among non-clustered miRNAs, the upregulation of miR-21, miR-9-3 and miR-375 and the downregulation of miR-34b, miR-34c, miR-130a and miR-195 in thymic carcinomas were most significant. The expression of ALK, HER2, HER3, MET, phospho-mTOR, p16^INK4A, PDGFRA, PDGFRB, PD-L1, PTEN and ROS1 was investigated by immunohistochemistry. PDGFRA was increased in thymic carcinomas and PD-L1 in B3 thymomas and thymic carcinomas. In summary, our results reveal genetic differences between thymomas and thymic carcinomas and suggest potential novel targets for therapy.

Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) is associated with poor outcome. The clinical behavior and genetic landscape of DLBCL is heterogeneous and still not fully understood. TP53 mutations in DLBCL have been identified as markers of poor prognosis and are often associated with therapeutic resistance. Chimeric antigen receptor T-cell therapy is an innovative therapeutic concept and represents a game-changing therapeutic option by supporting the patient’s own immune system to kill the tumor cells. We investigated the impact of TP53 mutations on the overall survival of refractory/relapsed DLBCL patients treated with comparable numbers of therapy lines. The minimum number of therapy lines was 2 (median 4), including either anti-CD19 CAR T-cell therapy or conventional salvage therapy. A total of 170 patients with DLBCL and high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements (DHL/THL), diagnosed and treated in our hospital between 2000 and 2021, were included. Twenty-nine of them received CAR T-cell therapy. TP53 mutations were found in 10/29 (35%) and 31/141 (22%) of patients in the CAR T-cell and conventional groups, respectively. Among the 141 patients not treated with CAR T cells, TP53 mutation was an independent prognostic factor for overall survival (OS) (median 12 months with TP53 vs. not reached without TP53 mutation, p < 0.005), but in the CAR T cell treated group, this significance could not be shown (median OS 30 vs. 120 months, p = 0.263). The findings from this monocentric retrospective study indicate that TP53 mutation status does not seem to affect outcomes in DLBCL patients treated with CAR T-cell therapy. Detailed evaluation in large cohorts is warranted.

Purpose: HER3 belongs to a family of receptor tyrosine kinases with oncogenic properties and is targeted by a variety of novel anticancer agents. There is a huge unmet medical need for systemic treatment options in patients with brain metastases (BM). Therefore, we aimed to investigate HER3 expression in BM of breast (BCa) and non–small cell lung cancer (NSCLC) as the basis for future clinical trial design. Experimental Design: We analyzed 180 BM samples of breast cancer or NSCLC and 47 corresponding NSCLC extracranial tissue. IHC was performed to evaluate protein expression of HER3, and immune cells based on CD3, CD8, and CD68. To identify dysregulated pathways based on differential DNA methylation patterns, we used Infinium MethylationEPIC microarrays. Results: A total of 99/132 (75.0%) of BCa-BM and 35/48 (72.9%) of NSCLC-BM presented with HER3 expression. Among breast cancer, HER2-positive and HER2-low BM showed significantly higher rates of HER3 coexpression than HER2-negative BM (87.1%/85.7% vs. 61.0%, P = 0.004). Among NSCLC, HER3 was more abundantly expressed in BM than in matched extracranial samples (72.9% vs. 41.3%, P = 0.003). No correlation of HER3 expression and intratumoral immune cell density was observed. HER3 expression did not correlate with overall survival from BM diagnosis. Methylation signatures differed according to HER3 status in BCa-BM samples. Pathway analysis revealed subtype-specific differences, such as TrkB and Wnt signaling pathways dysregulated in HER2-positive and triple-negative breast cancer BM, respectively. Conclusions: HER3 is highly abundant in BM of breast cancer and NSCLC. Given the promising results of antibody–drug conjugates in extracranial disease, BM-specific trials that target HER3 are warranted. See related commentary by Kabraji and Lin, p. 2961

Primary diffuse leptomeningeal melanomatosis (PDLMM) is an extremely rare and aggressive cancer type for which best treatment strategies remain to be elucidated. Herein, we present current and prospective diagnostic strategies and treatment management of PDLMM. Against the background of an extensive literature review of published PDLMM cases and currently employed therapeutic strategies, we present an illustrative case of a pediatric patient suffering from PDLMM. We report the first case of a pediatric patient with PDLMM who received combination treatment including trametinib and everolimus, followed by intravenous nivolumab and ipilimumab with concomitant intensive intraventricular chemotherapy, resulting in temporary significant clinical improvement and overall survival of 7 months. Following this clinical experience, we performed a comprehensive literature review, identifying 26 additional cases. By these means, we provide insight into current knowledge on clinical and molecular characteristics of PDLMM. Analysis of these cases revealed that the unspecific clinical presentation, such as unrecognized increased intracranial pressure (present in 67%), is a frequent reason for the delay in diagnosis. Mortality remains substantial despite diverse therapeutic approaches with a median overall survival of 4 months from diagnosis. On the molecular level, to date, the only oncogenic driver reported so far is mutation of NRAS (n = 3), underlining a close biological relation to malignant melanoma and neurocutaneous melanosis. We further show, for the first time, that this somatic mutation can be exploited for cerebrospinal fluid liquid biopsy detection, revealing a novel potential biomarker for diagnosis and monitoring of PDLMM. Last, we use a unique patient derived PDLMM cell model to provide first insights into in vitro drug sensitivities. In summary, we provide future diagnostic and therapeutic guidance for PDLMM and first insights into the use of liquid biopsy and in vitro models for this orphan cancer type.

Targeting oncogenic fusion-genes in pediatric high-grade gliomas (pHGG) with entrectinib has emerged as a highly promising therapeutic approach. Despite ongoing clinical studies, to date, no reports on the treatment of cerebrospinal fluid (CSF) disseminated fusion-positive pHGG exist. Moreover, clinically important information of combination with other treatment modalities such as intrathecal therapy, radiotherapy and other targeted agents is missing. We report on our clinical experience of entrectinib therapy in two CSF disseminated ROS1/NTRK-fusion-positive pHGG cases. Combination of entrectinib with radiotherapy or intrathecal chemotherapy appears to be safe and has the potential to act synergistically with entrectinib treatment. In addition, we demonstrate CSF penetrance of entrectinib for the first time in patient samples suggesting target engagement even upon CSF dissemination. Moreover, in vitro analyses of two novel cell models derived from one case with NTRK-fusion revealed that combination therapy with either a MEK (trametinib) or a CDK4/6 (abemaciclib) inhibitor synergistically enhances entrectinib anticancer effects. In summary, our comprehensive study, including clinical experience, CSF penetrance and in vitro data on entrectinib therapy of NTRK/ROS1-fusion-positive pHGG, provides essential clinical and preclinical insights into the multimodal treatment of these highly aggressive tumors. Our data suggest that combined inhibition of NTRK/ROS1 and other therapeutic vulnerabilities enhances the antitumor effect, which should be followed-up in further preclinical and clinical studies.