Abstract Background Congenital melanocytic nevus syndrome is a disorder characterized by postzygotic, mosaic NRAS Proto-Oncogene, GTPase mutations. Clinical manifestations include melanotic skin lesions and, optionally, central nervous system melanosis typically noted during early infancy. Affected individuals have an increased risk of developing malignant melanomas at an early age. Case We report a child with neurocutaneous melanosis due to this syndrome, who had innumerable nevi at birth and diffuse leptomeningeal thickening. He developed increased intracranial pressure at 4 weeks of age. The nucleoside analogue azacitidine and the Mitogen-Activated Protein Kinase, Kinase inhibitor trametinib were started at 6 weeks of age resulting in rapid reduction of leptomeningeal thickening. At 53 months of age, the patient still takes trametinib and has met all developmental milestones. There has been no evidence of melanoma, and he exhibits minimal residual leptomeningeal changes. Conclusion To our best knowledge, this is the first child with this syndrome who has undergone successful therapy to reduce leptomeningeal thickening.

3041 Background: Profiling of targetable genetic alterations within molecular tumor boards (MTB) guides for personalized treatment selection in patients with advanced cancers. During therapy, response is typically assessed by CT scans or MRI, which often have suboptimal sensitivity and specificity. Circulating tumor DNA (ctDNA) from blood plasma has emerged as a promising biomarker for noninvasive profiling of tumor mutational landscapes and disease monitoring. Here, we applied a pan-cancer next-generation sequencing (NGS) technology to assess the role of ctDNA for comprehensive tumor genotyping, early response prediction, and characterization clonal heterogeneity in patients receiving MTB recommended therapies. Methods: We developed and applied a custom targeted NGS approach (ExTARGET), which covers 266 genes across a 540 kb genomic region, to 157 plasma samples obtained at distinct milestones from 57 patients with diverse solid cancers. Plasma samples from healthy individuals ( n = 24) were used to determine the specificity of our technology. Results: We identified variants in 96% of baseline plasma samples by ctDNA profiling, with a median of 7 mutations per patient (range: 1-41). Most frequently mutated genes included KRAS (35%), BRAF (24%), ERBB2 (22%) and TP53 (22%). Targetable tumor variants that led to treatment recommendations within the MTB were found non-invasively in 69% of patients. Longitudinal monitoring of baseline ctDNA variants in on-treatment samples, obtained early during therapy ( n = 21), revealed that ctDNA dynamics were predictive of disease progression and preceded radiological/clinical progression in 8/19 (42%) patients. All patients with increasing ctDNA levels early during treatment showed radiologic disease progression in subsequent CT scans. On the other hand, an early decrease of ctDNA levels was associated with durable disease control in most patients and significantly favorable progression-free survival ( p = 0.008; HR = 0.1, 95%CI: 0.02-0.6). Next, we explored temporal clonal heterogeneity in plasma samples collected from 16 patients with disease progression following MTB-recommended therapies. We observed substantial clonal evolution over time, with all samples harboring at least one emerging variant. Among these emerging alterations, 19% were classified as ‘oncogenic’ and 5% were identified as potentially targetable. Conclusions: We here developed an NGS-based technology for ctDNA profiling in heavily pretreated patients receiving MTB-recommended therapies. Non-invasive genotyping from plasma robustly identifies targetable aberrations and allows comprehensive tumor genotyping. Monitoring of ctDNA during treatment and at disease progression facilitates early prediction of treatment response and profiling of temporal clonal heterogeneity that could enable subsequent treatment selection.

INTRODUCTION:Neuroendocrine neoplasms (NENs) are rare and biologically heterogeneous tumors with limited evidence-based systemic treatment options, particularly in advanced disease stages. Molecular Tumor Boards (MTBs) offer an interdisciplinary framework for interpreting genomic alterations and identifying personalized treatment strategies. METHODS:We conducted a retrospective two-center analysis of patients with NENs discussed at the MTBs of the University Medical Center Freiburg and the Technical University of Munich between 2019 and 2024. Clinical characteristics, molecular profiling results, MTB-based therapy recommendations, treatment implementation, and outcomes were evaluated. Overall survival (OS) was analyzed using a predefined 6-month landmark approach to account for immortal time bias. Treatment efficacy was further assessed using progression-free survival (PFS) and PFS2/PFS1 ratio. RESULTS:A total of 87 patients were discussed at both MTBs, 74 of whom underwent molecular profiling. Actionable therapy recommendations were issued for 58 patients, and 20 patients ultimately received MTB-guided therapy. Molecular alterations frequently involved DNA damage repair pathways, immune-related biomarkers, and signaling cascades relevant to targeted therapy. 6-month landmark OS analysis demonstrated significantly improved OS for patients receiving MTB-guided therapy. At the individual level, 8 of 20 treated patients achieved a PFS2/PFS1 ratio >1.3. CONCLUSION:Molecular-guided precision oncology can provide clinically meaningful benefit for selected patients with NENs, despite limited overall implementation rates. Earlier integration of molecular profiling may increase the feasibility and impact of personalized treatment approaches in these rare tumor entities.

Abstract Circulating tumor DNA (ctDNA) from blood plasma has emerged as a promising biomarker for noninvasive profiling of tumor mutational landscapes and disease monitoring across cancers. In this study, we developed a targeted next-generation sequencing approach to explore the role of ctDNA for comprehensive tumor genotyping, early response prediction, and characterization of clonal heterogeneity in patients with advanced and rare cancers treated within molecular tumor boards. We applied our technology to 157 plasma specimens from 57 patients at distinct disease milestones and detected tumor variants in 96% of baseline samples, with 65% of them harboring actionable aberrations. Longitudinal monitoring of baseline mutations in on-treatment plasma revealed that ctDNA dynamics were significantly associated with clinical outcomes and enabled early prediction of disease progression. Finally, we observed substantial clonal heterogeneity over time, identifying emerging mutations in all analyzed plasma samples obtained at progression, including potentially targetable variants for subsequent personalized therapies.

Despite therapeutic advancements, approximately 50% of advanced melanoma patients succumb to metastatic disease. Molecular tumor boards (MTB) aim to identify targetable molecular alterations to guide individualized treatment strategies. Yet, real-world data on patient selection, referral timing, recommendation rates, implementation, and clinical impact remain limited. In this exploratory retrospective bicenter analysis, we evaluated 80 patients with advanced melanoma who presented at institutional MTBs of two comprehensive cancer centers. Clinical and molecular tumor data were analyzed using bioinformatic tools to characterize mutation profiles, treatment recommendations, and their real-world implementation. Most patients (88.3%) had stage IV melanoma at the time of presentation and had received a median of three prior systemic treatment lines. Actionable treatment recommendations were formulated in 77.9% of eligible cases, yet only 33.7% of recommendations were implemented. Non-implementation was most commonly attributable to early patient death or regulatory barriers. Importantly, when recommended therapies were applied, patients experienced significantly improved progression-free survival (7.85 vs. 4.34 months; PFS ratio 1.8) and overall survival (10.64 vs. 5.06 months) compared with patients in whom recommended treatments were not implemented. Among patients with implemented MTB recommendations (n = 26), the median intra-patient PFS ratio was 1.68, and 14 of 26 patients (53.8%) achieved a PFS ratio ≥ 1.3. These findings indicate that MTBs frequently generate clinically actionable recommendations for metastatic melanoma, but late-stage referral substantially limits their real-world implementation. When applied, molecularly guided treatment strategies may confer meaningful clinical benefit, underscoring the importance of earlier integration of MTBs into melanoma care pathways.

Abstract Background This study aimed to assess the detection rate and spectrum of pathogenic variants (PVs) and candidate variants (variants of uncertain significance, VUS) in AVM patients. Methods In this retrospective multicenter cohort study, tissue and blood samples were collected from 114 patients with extracranial AVMs during treatment or when clinically indicated, for dedicated molecular genetic analyses. PVs (solved) and VUS were detected by targeted sequencing on DNA using gene panels analyzing genes suspected to be associated with AVMs. Unsolved cases were further categorized into unrestricted and restricted, with the latter reflecting methodological limitations. Subgroup analyses were carried out based on affected genes to explore associated genotype–phenotype correlations. Results PVs were identified in 80.7% (92/114) and VUS in 5.3% (6/114), resulting in a total detection rate of 86.0% (98/114). Unsolved cases accounted for 11.4% (13/114) including 6/13 (46.2%) with methodological restrictions. Somatic PVs were most frequent in KRAS (21.1%, 24/114), MAP2K1 (17.5%, 20/114), HRAS (8.8%, 10/114), and BRAF (7.9%, 9/114). Germline variants were found in RASA1 (7.0%, 8/114), PTEN (5.3%, 6/114), and EPHB4 (3.5%, 4/114). In a few cases, somatic variants in RASA1 (1.8%, 2/114) and PTEN (2.6%, 3/114) were identified. Additional PVs occurred in PIK3CA (3.5%, 4/114), SOS1 (2.6%, 3/114), GNAQ (1.8%, 2/114), and RIT1, RAF1, and GNA14 (each 0.9%, 1/114). Within the RAS/MAPK pathway, RAS variants (KRAS, HRAS) were linked to more severe clinical stages (65.6% vs. 40.0% MAP2K1 and 37.5% BRAF, p = 0.027) and higher relapse rates (55.6% vs. 43.8% MAP2K1 and 0% BRAF, p = 0.049). Germline variants showed a distinct distribution pattern with more syndromic presentations (61.1% vs. 15.6%, p < 0.001) compared to mosaic variants. Conclusions Broad and sensitive testing enables a high detection rate of causative variants in AVMs. PVs and VUS detected reveal a broader genetic spectrum than previously recognized. Somatic RAS PVs were associated with more advanced disease stages and higher relapse rates than MAP2K1 and BRAF variants, while germline variants were more frequently linked to syndromic patterns.

While pancreatic ductal adenocarcinoma (PDAC) carries a poor prognosis, a small fraction of patients show high microsatellite instability (MSI-H) and may respond to immune checkpoint inhibition. An MSI-H genotype is usually associated with deficiencies in the DNA mismatch-repair mechanism (MMRd). However, discordances between the mismatch-repair status by immunohistochemistry and the microsatellite status by molecular analyses have been noted. To date it is not clear whether PDAC patients with mutations in mismatch repair genes, which result in loss of protein expression (MMRd), who nonetheless retain microsatellite stability (MSS), can profit from checkpoint inhibitor therapy. Here, we present the case of a PDAC patient, diagnosed as MMRd/MSS, who responded to checkpoint inhibitor therapy after failing two lines of chemotherapy. Our data suggest that both MMR and microsatellite status should be determined in PDAC patients and that MMRd status alone, even in an MSS phenotype, can constitute an indication for checkpoint inhibitor therapy.

ObjectivesMolecular tumor boards (MTBs) have become an integral component of precision oncology, yet data on their real-world impact in urologic cancers are limited. This study aimed to characterize the molecular landscape of urologic malignancies presented to the MTB at the University Medical Center Freiburg and to evaluate the frequency and clinical relevance of genomic alterations across tumor entities.MethodsWe retrospectively analyzed 118 patients with histologically confirmed urologic tumors presented at the Freiburg MTB between Januar 2019 and December 2024. Comprehensive molecular profiling was performed using next-generation sequencing (TruSight Oncology 500 or whole exome sequencing). Data were analyzed for mutation frequency, tumor mutational burden (TMB), and co-occurrence patterns, and integrated with clinical data to guide therapy recommendations.ResultsSomatic mutations were identified in 90.6% of cases. Frequent alterations included TP53, BRCA2, KMT2D, and ATM, with DNA damage response and chromatin remodeling pathways commonly affected. Prostate cancers showed high rates of BRCA2 and APC co-mutations, indicating potential benefit from combined PARP and Wnt-targeted therapies. In bladder and upper tract urothelial carcinomas (UTUC), KMT2C co-occurred with genes such as SPTA1 and LRP1B, suggesting a hypermutated, immunoresponsive phenotype. Renal tumors frequently harbored alterations in VHL, PBRM1, and SETD2. Rare entities such as penile and testicular tumors displayed distinct mutation patterns, including BRCA1/2 and MMR gene alterations.ConclusionsComprehensive molecular profiling in a MTB setting reveals distinct and therapeutically relevant mutational patterns across urologic cancers. These data support the integration of MTBs into clinical workflows and highlight the potential of co-mutational signatures to guide personalized treatment strategies.

e17655 Background: Molecular tumor boards (MTBs) play a crucial role in personalized medicine in Germany, guiding the treatment of patients who have exhausted standard options. To generate evidence that can inform future therapeutic recommendations, data on molecular characteristics and treatment outcomes are needed. Methods: This study retrospectively analyzes the real-world data including the molecular landscape, MTB recommendations and their corresponding biomarkers as well as outcomes of patients with gynecologic malignancies (breast-, cervical-, endometrial-, ovarian-, vulvar cancer and uterine sarcoma who were discussed at the Molecular Tumor Board Freiburg (MTB-FR). Results: From 2019 to 2022, 262 cases of gynecologic malignancies (corresponding to 249 individual patients) were referred to the MTB-FR. N=229 cases (87.4%) received a recommendation for personalized diagnostics at the protein, DNA and/or transcriptomic level and were included in the analysis. This cohort was characterized as relatively young yet highly pretreated, with a significant unmet clinical need, as reflected by a mean age of 53.3 years and a mean of 3.1 prior lines of systemic treatment. Personalized diagnostics resulted in at least one treatment recommendation in n=170/229 (74.2%) cases, primarily off-label therapies. In 50.6% of cases, at least one potentially matching clinical trial could be identified. Molecularly informed therapy was implemented in n=63/170 cases (37.1%), while n=81/170 cases (47.6%) received none of the recommended treatments, predominantly due to deterioration in health status. In 15.3% of cases, the implementation of the recommended treatment was unclear due to loss to follow-up. The most prevalent implemented therapeutic agents were antibody-drug conjugates (25.7%) and immune checkpoint inhibitors (17.1%). 75.4% of implemented off-label treatments were supported by evidence from the same tumor entity (NCT level m1A-C). Clinical trials accounted for 12.9% of implemented treatments. In our retrospective analysis, patients who received an MTB-recommended therapy (n=61) exhibited a significantly longer median overall survival (OS) of 19 months compared to 6 months for those who did not receive an MTB-recommended therapy (n=59, log-rank p=0.0002). Conclusions: These results underscore the benefit of MTB referral in patients with gynecologic malignancies and highlight the importance of early intervention in this setting. Additional analysis and progression-free survival data are expected.

This report describes the case of a 25-year-old female patient with multicentric infantile myofibromatosis since early infancy, superficial capillary malformations and congenital hypoplasia of the third and fourth finger of her right hand. All known lesions were located in the upper extremities, the chest and the upper back. A pathogenic, gain-of-function platelet-derived growth factor receptor-beta (PDGFRB) variant (p.N666K, c.1998 C > A) was detected in two myofibromas and in a capillary malformation on the upper back, but not in DNA obtained from blood mononuclear cells. Thus, PDGFRB mosaicism appears to account for the patient’s myofibromas and capillary malformations, supporting a broad spectrum of PDGFRB-driven anomalies ranging from myofibromas to vascular malformations.

Immune-related adverse events (irAEs) in cancer patients receiving immune checkpoint inhibitors (ICIs) cause morbidity and necessitate cessation of treatment. Comparing irAE treatments, we find that anti-tumor immunity is preserved in mice after extracorporeal photopheresis (ECP) but reduced with glucocorticosteroids, TNFα blockade, and α4β7-integrin inhibition. Local adiponectin production elicits a tissue-specific effect by reducing pro-inflammatory T cell frequencies in the colon while sparing tumor-specific T cell development. A prospective phase-1b/2 trial (EudraCT-No.2021-002073-26) with 14 patients reveals low ECP-related toxicity. Overall response rate for all irAEs is 92% (95% confidence interval [CI]: 63.97%-99.81%); colitis-specific complete remission rate is 100% (95% CI: 63.06%-100%). Glucocorticosteroid dosages could be reduced for all patients after ECP therapy. The ECP-adiponectin axis reduces intestinal tissue-resident memory T cell activation and CD4+IFN-γ+ T cells in patients with ICI-induced colitis without evidence of loss of anti-tumor immunity. In conclusion, we identify adiponectin as an immunomodulatory molecule that controls ICI-induced irAEs without blocking anti-tumor immunity.

BACKGROUND:A modified grass allergen subcutaneous immunotherapy (SCIT) product with MicroCrystalline Tyrosine and monophosphoryl lipid-A as an adjuvant system (Grass MATA MPL [PQ Grass]) is being developed as short-course treatment of grass-pollen allergic rhinitis (SAR) and/or rhinoconjunctivitis. We sought to evaluate the combined symptom and medication score (CSMS) of the optimized cumulative dose of 27,600 standardized units (SU) PQ Grass in a field setting prior to embarking on a pivotal Phase III trial. METHODS:In this exploratory, randomized, double-blind, placebo-controlled trial subjects were enrolled across 14 sites (Germany and the United States of America). Six pre-seasonal subcutaneous injections of PQ Grass (using conventional or extended regimens) or placebo were administered to 119 subjects (aged 18-65 years) with moderate-to-severe SAR with or without asthma that was well-controlled. The primary efficacy endpoint was CSMS during peak grass pollen season (GPS). Secondary endpoints included Rhinoconjunctivitis Quality of Life Questionnaire standardized (RQLQ-S) and allergen-specific IgG4 response. RESULTS:The mean CSMS compared to placebo was 33.1% (p = .0325) and 39.5% (p = .0112) for the conventional and extended regimens, respectively. An increase in IgG4 was shown for both regimens (p < .01) as well as an improvement in total RQLQ-S for the extended regimen (mean change -0.72, p = .02). Both regimens were well-tolerated. CONCLUSIONS:This trial demonstrated a clinically relevant and statistically significant efficacy response to PQ Grass. Unprecedented effect sizes were reached for grass allergy of up to ≈40% compared to placebo for CSMS after only six PQ Grass injections. Both PQ Grass regimens were considered equally safe and well-tolerated. Based on enhanced efficacy profile extended regime will be progressed to the pivotal Phase III trial.

Insertion mutations in exon 20 of the epidermal growth factor receptor gene (EGFR exon20ins) are rare, heterogeneous alterations observed in non-small cell lung cancer (NSCLC). With a few exceptions, they are associated with primary resistance to established EGFR tyrosine kinase inhibitors (TKIs). As patients carrying EGFR exon20ins may be eligible for treatment with novel therapeutics—the bispecific antibody amivantamab, the TKI mobocertinib, or potential future innovations—they need to be identified reliably in clinical practice for which quality-based routine genetic testing is crucial. Spearheaded by the German Quality Assurance Initiative Pathology two international proficiency tests were run, assessing the performance of 104 participating institutes detecting EGFR exon20ins in tissue and/or plasma samples. EGFR exon20ins were most reliably identified using next-generation sequencing (NGS). Interestingly, success rates of institutes using commercially available mutation-/allele-specific quantitative (q)PCR were below 30% for tissue samples and 0% for plasma samples. Most of these mutation-/allele-specific (q)PCR assays are not designed to detect the whole spectrum of EGFR exon20ins mutations leading to false negative results. These data suggest that NGS is a suitable method to detect EGFR exon20ins in various types of patient samples and is superior to the detection spectrum of commercially available assays.

Despite major advances in molecular profiling and classification of primary brain tumors, personalized treatment remains limited for most patients. Here, we explored the feasibility of individual molecular profiling and the efficacy of biomarker-guided therapy for adult patients with primary brain cancers in the real-world setting within the molecular tumor board Freiburg, Germany. We analyzed genetic profiles, personalized treatment recommendations, and clinical outcomes of 102 patients with 21 brain tumor types. Alterations in the cell cycle, BRAF, and mTOR pathways most frequently led to personalized treatment recommendations. Molecularly informed therapies were recommended in 71% and implemented in 32% of patients with completed molecular diagnostics. The disease control rate following targeted treatment was 50% and the overall response rate was 30%, with a progression-free survival 2/1 ratio of at least 1.3 in 31% of patients. This study highlights the efficacy of molecularly guided treatment and the need for biomarker-stratified trials in brain cancers.

INTRODUCTION:Personalized medicine poses great opportunities and challenges. While the therapeutic landscape markedly expands, descriptions about status, clinical implementation and real-world benefits of precision oncology and molecular tumor boards (MTB) remain sparse, particularly in the field of genitourinary (GU) cancer. Hence, this study characterized urological MTB cases to better understand the potential role of MTB in uro-oncology. METHODS:We analyzed patients with complete data sets being reviewed at an MTB from January 2019 to October 2022, focusing on results of molecular analysis and treatment recommendations. RESULTS:We evaluated 102 patients with GU cancer with a mean patient age of 61.7 years. Prostate cancer (PCa) was the most frequent entity with 52.9% (54/102), followed by bladder cancer (18.6%, 19/102) and renal cell carcinoma (14.7%, 15/102). On average, case presentation at MTB took place 54.9 months after initial diagnosis and after 2.7 previous lines of therapy. During the study period, 49.0% (50/102) of patients deceased. Additional MTB-based treatment recommendations were achieved in a majority of 68.6% (70/102) of patients, with a recommendation for targeted therapy in 64.3% (45/70) of these patients. Only 6.7% (3/45) of patients - due to different reasons - received the recommended MTB-based therapy though, with 33% (1/3) of patients reaching disease control. Throughout the MTB study period, GU cancer case presentations and treatment recommendations increased, while the time interval between initial presentation and final therapy recommendation were decreasing over time. CONCLUSION:Presentation of uro-oncological patients at the MTB is a highly valuable measure for clinical decision-making. Prospectively, earlier presentation of patients at the MTB and changing legislative issues regarding comprehensive molecular testing and targeted treatment approval might further improve patients' benefits from comprehensive molecular diagnostics.

INTRODUCTION:Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis. METHODS:To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability. RESULTS:The mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences. CONCLUSION:Continuous optimization of bioinformatic workflows and participating in round robin tests are recommended.

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.