BACKGROUND:Cancer of unknown primary (CUP) accounts for 1-3% of newly diagnosed cancers and remains a biologically heterogeneous and clinically challenging entity. For decades, management has relied on empirical platinum-based chemotherapy, which offers a poor prognosis with a median overall survival typically below 1 year. Recent advances in comprehensive genomic profiling, liquid biopsy, functional imaging, and tumour-agnostic drug development may transform diagnostic and therapeutic paradigms for the management of CUP. SUMMARY:This review synthesises current and upcoming data presented at ESMO 2025 and the first International Cancer of Unknown Primary Meeting (ICUPM), integrated with recent peer-reviewed literature. We highlight emerging diagnostic strategies, including whole-genome and transcriptome sequencing, ctDNA-based tissue-of-origin and cancer signal origin assays, and novel imaging modalities applied to CUP. We further discuss the clinical impact of multimodal molecular tumour boards, early detection approaches, and tumour-agnostic treatment strategies, including KRAS-directed and T-cell receptor-engineered cellular therapies. KEY MESSAGES:(i) Whole-genome and transcriptome sequencing, particularly when integrated within multidisciplinary tumour boards, may substantially improve diagnostic precision and tissue-of-origin prediction and may further be associated with improved survival in CUP patients. (ii) Liquid biopsy-based assays provide complementary, minimally invasive approaches for molecular stratification and tissue-of-origin prediction, with faster turnaround times than classical tissue sequencing. (iii) Fibroblast activation protein inhibitor PET-CT shows promise in improving primary tumour and metastasis detection beyond FDG-PET-CT. (iv) Tumour-agnostic treatment strategies, e.g., KRAS-selective inhibitors and T-cell receptor-engineered T-cell therapy, may advance the groundwork for molecularly driven treatment for CUP.

Abstract Most patients with cancer of unknown primary (CUP) still receive platinum-based chemotherapy and have a poor prognosis, with overall survival of less than one year. Recent studies suggest improved outcomes with molecularly guided or site-specific therapies informed by molecular tissue profiling. Here, we analyzed ctDNA from 190 CUP patients using an integrated genomic and epigenomic assay to identify actionable alterations and predict tissue-of-origin (ToO). Integration of actionable biomarkers, ToO prediction and clinical data yielded diagnostic, prognostic or therapeutic information in 90% of unfavorable CUP cases and 88% of patients analyzed at first diagnosis. High ctDNA tumor fraction was associated with poorer prognosis in both favorable and unfavorable CUP. These findings highlight the clinical utility of ctDNA analysis for therapeutic decision-making in CUP and support its incorporation into the diagnostic work-up, particularly when tissue samples are unavailable or insufficient for molecular testing.

Oncological treatment in a substantial portion of patients with cancer of unknown primary (CUP) remains challenging due to limitations of conventional imaging and positron emission tomography/computed tomography with 18Fluor-fluorodeoxyglucose (18F-FDG-PET/CT). In head and neck-like CUP (HNCUP), several studies found significantly higher tracer-uptake and detection rates of primary tumors in 68Gallium-labeled fibroblast activation protein inhibitor-PET/CT (68Ga-FAPI-PET/CT). Here, we address a gap in CUP literature by retrospectively evaluating the diagnostic accuracy of both tracer in a head-to-head comparison of patients with single-site and oligometastatic extra-cervical CUP. 13 patients with extra-cervical CUP underwent both 18F-FDG- and 68Ga-FAPI-PET/CT. Suspicious PET-positive lesions were delineated using the volume of interest-technique (50

Measurable residual disease (MRD) can predict relapse in patients with advanced myelodysplastic neoplasms (MDS) or acute myeloid leukemia (AML). We report the long-term efficacy and safety of MRD-guided preemptive azacitidine treatment to prevent relapse in the phase 2 RELAZA2 trial. Patients with MDS or AML after either intensive chemotherapy only or consecutive allogeneic stem cell transplantation were prospectively screened for imminent relapse by molecular MRD assessment. Patients who became MRD positive (MRDpos) during screening received azacitidine for up to 2 years to prevent relapse. The primary endpoint was the proportion of patients alive and relapse-free six months after azacitidine start. Of 357 patients screened, 119 (33.3%) became MRDpos, of whom 95 (79.8%) were eligible for azacitidine treatment. The primary endpoint was met; 60 (63%) patients were relapse-free (95% confidence interval 54-71%, P<0.0001) six months after azacitidine initiation with no new safety signals. Of 60 patients achieving MRD response during the first six cycles of azacitidine, 31 (52%) maintained response without hematological relapse for ≥2 years following azacitidine initiation. The median treatment-free duration following azacitidine discontinuation was 20.8 months; the longest ongoing response was 104 months. After a median follow-up of 6.6 years, 15 initial responders (25%) remained alive and in remission. Among screened patients who remained continuously MRDneg, 60-month overall survival and relapse-free survival were 88% and 79%, respectively. Continuously MRDneg patients display a very favorable prognosis. A majority of MRDpos patients can be effectively treated with azacitidine with potential long-term remission even after termination of azacitidine. Clinicaltrials.gov: NCT01462578.

CUPISCO (ClinicalTrials.gov identifier: NCT03498521) demonstrated longer progression-free survival (PFS) with comprehensive genomic profiling (CGP) and subsequent molecularly guided therapies (MGTs), versus standard platinum-based chemotherapy, in patients with previously untreated, unfavorable cancer of unknown primary (CUP) who reached disease control after induction chemotherapy (three cycles). We report efficacy and safety after >1 year of additional follow-up. Eligible patients were randomly assigned (3:1) to MGT (investigator-chosen after discussion in a molecular tumor board) or three further cycles of chemotherapy. The primary end point was PFS. Secondary end points included overall survival (OS) and safety. At data cutoff (December 6, 2024), 436 patients were randomly assigned (326 to MGT; 110 to chemotherapy). Median follow-up was 37.0 months (range, 0.0-67.8). Updated median PFS was 6.1 months (95% CI, 4.7 to 6.5) with MGT and 4.4 months (95% CI, 4.2 to 6.4) with chemotherapy (hazard ratio [HR], 0.75 [95% CI, 0.59 to 0.95]; P = .017); median OS was 15.2 months (95% CI, 13.9 to 18.4) and 12.8 months (95% CI, 9.8 to 15.4), respectively (HR, 0.79 [95% CI, 0.61 to 1.02]; P = .0689). No new safety signals were identified. These updated results aligned with the primary analysis, demonstrating the benefit of CGP with subsequent MGT and highlighting the importance of incorporating CGP at initial diagnosis to guide treatment decisions for patients with unfavorable CUP.

Cancer of unknown primary (CUP) is a metastatic malignancy for which a primary site of origin cannot be identified despite a thorough and standardized diagnostic work-up, and accounts for 1–3

3081 Background: MTAP , a key enzyme in the polyamine pathway breaks down 5’Deoxy-5-Methylthioadenosine ( MTA ) into methionine and adenine. MTAP loss reduces adenine and accumulates MTA, which inhibits protein arginine methyltransferase 5 ( PRMT5 ). This suggests MTAP loss cancers may respond to PRMT5 inhibition. Methionine adenosyl transferase 2α ( MAT-2A ) is a primary producer of donor S-adenosylmethionine ( SAM ) and the depletion of MAT-2A has antiproliferative effect in cancers with MTAP loss. Based on the synthetic lethality concept, MTAP loss is being used as a biomarker for accrual in multiple trials with PRMT5 and MAT-2A inhibitors. We queried the genomic landscape of MTAP loss in patients with nsCUP. Methods: DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue of 7,440 nsCUP cases from 2020 to 2024 underwent hybrid capture-based comprehensive genomic profiling (CGP) to assess all classes of genomic alterations (GA). All cases underwent central pathology review to confirm that at the time of sequencing, a primary site for the cases was not established. Microsatellite instability (MSI) status and tumor mutational burden (TMB) were derived from the CGP data. Programmed death-ligand 1 (PD-L1) was determined by immunohistochemistry (IHC) using the DAKO 22C3 system. Results: 853 (11.5%) of nsCUP cases had either complete or partial MTAP loss with 0.7% 1 exon, 1.2% 2 exons, 2.9% 3 exons, 5.1% 4 exons, 0.5% 5 exons, 2.5% 6 exons, 32.8% 7 exons and 54.3% 8 exons lost. The median age of the MTAP loss patients was higher (68 vs 65; p<.0001) and the gender distributions were similar (52% to 54% female; not significant (NS)). Cyclin-dependent kinase inhibitor 2A (CDKN2A) loss co-occurred in 99.8% in patients with MTAP loss. MSI-high status was uncommon in both MTAP loss vs MTAP wildtype (0.4% vs 0.7%; NS). The MTAP wildtype group had higher tumor mutational burden (TMB) > 10 mutations/mb (15.7% vs 11.1%; p=.0004) and TMB >20 mutations/Mb (5.4% vs 3.5%; p=.017) rates. MTAP loss nsCUPs had higher frequencies of KRAS GA and KRAS G12C, whereas MTAP wildtype cases had greater frequencies of ERBB2 , PTEN , MET and EGFR GA (Table). GA in BRCA1 / 2 and FGFR2 were similar in both groups. GA in ALK , RET , ROS1 , RET and TRK were extremely uncommon in both groups (all less than 1%). Conclusions: At 11.5%, nsCUP features a relatively high frequency of MTAP loss, with the vast majority involving either all (8 of 8) or nearly all (7 of 8) exons. MTAP loss patients are slightly older and have reduced TMB levels which may impact their responsiveness to immunotherapy-based combination regimens with PRMT5/MAT-2A inhibitors. Clinical trials for the development of targeted therapies to use PRMT5 inhibition and MAT-2A in nsCUP are warranted. nsCUP MTAP Loss (N=853) nsCUP MTAP wildtype ( N=6,587) P value KRAS all/G12C 45.9%/7.6% 31.2%/4.2% <.0001/<.0001 ERBB2 all/amp only 6.7%/4.3% 10.9%/8.1% <.0001/<.0001 PIK3CA 6.2% 7.1% NS BRAF 6.0% 5.0% NS FGFR2 4.3% 4.0% NS PTEN 4.1% 6.1% .02 BRCA1 / 2 1.8%/2.3% 2.1%/2.4% NS/NS MET 2.1% 4.6% .0004 EGFR 2.6% 4.1% .031

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation.

Chromosomal instability is a major driver of intratumoral heterogeneity (ITH), promoting tumor progression. In the present study, we combined structural variant discovery and nucleosome occupancy profiling with transcriptomic and immunophenotypic changes in single cells to study ITH in complex karyotype acute myeloid leukemia (CK-AML). We observed complex structural variant landscapes within individual cells of patients with CK-AML characterized by linear and circular breakage-fusion-bridge cycles and chromothripsis. We identified three clonal evolution patterns in diagnosis or salvage CK-AML (monoclonal, linear and branched polyclonal), with 75% harboring multiple subclones that frequently displayed ongoing karyotype remodeling. Using patient-derived xenografts, we demonstrated varied clonal evolution of leukemic stem cells (LSCs) and further dissected subclone-specific drug-response profiles to identify LSC-targeting therapies, including BCL-xL inhibition. In paired longitudinal patient samples, we further revealed genetic evolution and cell-type plasticity as mechanisms of disease progression. By dissecting dynamic genomic, phenotypic and functional complexity of CK-AML, our findings offer clinically relevant avenues for characterizing and targeting disease-driving LSCs.

Tumour mutational burden (TMB), defined as the total number of somatic non-synonymous mutations present within the cancer genome, varies across and within cancer types. A first wave of retrospective and prospective research identified TMB as a predictive biomarker of response to immune-checkpoint inhibitors and culminated in the disease-agnostic approval of pembrolizumab for patients with TMB-high tumours based on data from the Keynote-158 trial. Although the applicability of outcomes from this trial to all cancer types and the optimal thresholds for TMB are yet to be ascertained, research into TMB is advancing along three principal avenues: enhancement of TMB assessments through rigorous quality control measures within the laboratory process, including the mitigation of confounding factors such as limited panel scope and low tumour purity; refinement of the traditional TMB framework through the incorporation of innovative concepts such as clonal, persistent or HLA-corrected TMB, tumour neoantigen load and mutational signatures; and integration of TMB with established and emerging biomarkers such as PD-L1 expression, microsatellite instability, immune gene expression profiles and the tumour immune contexture. Given its pivotal functions in both the pathogenesis of cancer and the ability of the immune system to recognize tumours, a profound comprehension of the foundational principles and the continued evolution of TMB are of paramount relevance for the field of oncology.

BACKGROUND: Cancer of unknown primary (CUP) is defined as a primary metastatic malignancy, in which the primary tumor remains elusive in spite of a comprehensive diagnostic workup. The frequency and prognostic value of circulating tumor cells (CTCs), which are considered to be the source of metastasis, has not yet been systematically evaluated in CUP. METHODS: A total of 110 patients with a confirmed diagnosis of CUP according to the European Society for Medical Oncology (ESMO) guidelines, who presented to our clinic between July 2021 and May 2023, provided blood samples for CTC quantification using CellSearch methodology. CTC counts were correlated with demographic, clinical, and molecular data generated by comprehensive genomic profiling of tumor tissue. RESULTS: CTCs were detected in 26% of all patients at initial presentation to our department. The highest CTC frequency was observed among patients with unfavorable CUP (35.5%), while patients with single-site/oligometastatic CUP harbored the lowest CTC frequency (11.4%). No statistically significant association between CTC positivity and the number of affected organs (P = 0.478) or disease burden (P = 0.120) was found. High CTC levels (>= 5 CTCs/7.5 mL; 12/95 analyzed patients) predicted for adverse overall survival compared to negative or low CTC counts (6-months overall survival rate 90% vs 32%, log-rank P < 0.001; HR 5.43; 95% CI 2.23-13.2). CTC dynamics were also prognostic for overall survival by landmark analysis (log-rank P < 0.001, HR 10.2, 95% CI 1.95-52.9). CONCLUSIONS: CTC frequency is a strong, independent predictor of survival in patients with CUP. CTC quantification provides a useful prognostic tool in the management of these patients.

BACKGROUND:Patients with unfavourable subset cancer of unknown primary (CUP) have a poor prognosis when treated with standard platinum-based chemotherapy. Whether first-line treatment guided by comprehensive genomic profiling (CGP) can improve outcomes is unknown. The CUPISCO trial was designed to inform a molecularly guided treatment strategy to improve outcomes over standard platinum-based chemotherapy in patients with newly diagnosed, unfavourable, non-squamous CUP. The aim of the trial was to compare the efficacy and safety of molecularly guided therapy (MGT) versus standard platinum-based chemotherapy in these patients. This was to determine whether the inclusion of CGP in the initial diagnostic work-up leads to improved outcomes over the current standard of care. We herein report the primary analysis. METHODS:CUPISCO was a phase 2, prospective, randomised, open-label, active-controlled, multicentre trial done at 159 sites in 34 countries outside the USA. Patients with central eligibility review-confirmed disease (acceptable histologies included adenocarcinoma and poorly differentiated carcinoma) and an Eastern Cooperative Oncology Group performance status of 0 or 1, evaluated by CGP, who reached disease control after three cycles of standard first-line platinum-based chemotherapy were randomly assigned 3:1 via a block-stratified randomisation procedure to MGT versus chemotherapy continuation for at least three further cycles. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. The study is registered with ClinicalTrials.gov, NCT03498521, and follow-up is ongoing. FINDINGS:From July 10, 2018, to Dec 9, 2022, 636 (42%) of 1505 screened patients were enrolled. Median follow-up in the treatment period was 24·1 months (IQR 11·6-35·6). Of 438 patients who reached disease control after induction chemotherapy, 436 were randomly assigned: 326 (75%) to the MGT group and 110 (25%) to the chemotherapy group. Median progression-free survival in the intention-to-treat population was 6·1 months (95% CI 4·7-6·5) in the MGT group versus 4·4 months (4·1-5·6) in the chemotherapy group (hazard ratio 0·72 [95% CI 0·56-0·92]; p=0·0079). Related adverse event rates per 100-patient-years at risk were generally similar or lower with MGT versus chemotherapy. INTERPRETATION:In patients with previously untreated, unfavourable, non-squamous CUP who reached disease control after induction chemotherapy, CGP with subsequent MGTs resulted in longer progression-free survival than standard platinum-based chemotherapy. On the basis of these results, we recommend that CGP is performed at initial diagnosis in patients with unfavourable CUP. FUNDING:F Hoffmann-La Roche.

•Treating ALK+ NSCLC in pregnancy is delicate due to scarce data on tyrosine kinase inhibitors (TKI) fetotoxicity.•Postpartum period might render ALK+ NSCLC patients especially vulnerable to toxicity.•This is the first case of globus pallidus necrosis while on TKI therapy in ALK+ NSCLC.•Patients with previous pneumonitis from other TKI can tolerate exposure to brigatinib.

Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138pos plasma cells. The protocol comprises steps for electron microscopy sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining. For complete details on the use and execution of this protocol, please refer to Dittrich et al.1

<p>Extended experimental procedures Supplementary Fig. S1. Effect of CP-673451 and Crenolanib on centrosome amplification in U2OS cells. Supplementary Fig. S2. Crenolanib prolongs of mitotic duration. Supplementary Fig. S3. CP-673451 activates cofilin in a dose-dependent manner in a variety of cancer cell lines. Supplementary Fig. S4. Inhibition of cofilin phosphorylation by LIMKi3 and Damnacanthal. Supplementary Fig. S5. Immunoblot analysis showing transient overexpression of different cofilin constructs and representative images of transfection efficiencies in U2OS-EGFP-PLK4 cells. Supplementary Fig. S6. Validation of SSH knock-down by real-time PCR. Supplementary Fig. S7. Effect of CP-673451 on SSH1 phosphatase activity. Supplementary Fig. S8. Immunoblot analysis of different cell lines showing that CP-673451 increases Akt and MEK1/2 phosphorylation under normal cultivation conditions. Supplementary Table S2. Average percentages of multipolar telophases in various cell lines after 24 hours of exposure to CP-673451 or crenolanib.</p>

PDF file - 69K, GF-15 decreases tumor growth in a HT29 xenograft mouse model

Individuals with germ line variants associated with hereditary hematopoietic malignancies (HHMs) have a highly variable risk for leukemogenesis. Gaps in our understanding of premalignant states in HHMs have hampered efforts to design effective clinical surveillance programs, provide personalized preemptive treatments, and inform appropriate counseling for patients. We used the largest known comparative international cohort of germline RUNX1, GATA2, or DDX41 variant carriers without and with hematopoietic malignancies (HMs) to identify patterns of genetic drivers that are unique to each HHM syndrome before and after leukemogenesis. These patterns included striking heterogeneity in rates of early-onset clonal hematopoiesis (CH), with a high prevalence of CH in RUNX1 and GATA2 variant carriers who did not have malignancies (carriers-without HM). We observed a paucity of CH in DDX41 carriers-without HM. In RUNX1 carriers-without HM with CH, we detected variants in TET2, PHF6, and, most frequently, BCOR. These genes were recurrently mutated in RUNX1-driven malignancies, suggesting CH is a direct precursor to malignancy in RUNX1-driven HHMs. Leukemogenesis in RUNX1 and DDX41 carriers was often driven by second hits in RUNX1 and DDX41, respectively. This study may inform the development of HHM-specific clinical trials and gene-specific approaches to clinical monitoring. For example, trials investigating the potential benefits of monitoring DDX41 carriers-without HM for low-frequency second hits in DDX41 may now be beneficial. Similarly, trials monitoring carriers-without HM with RUNX1 germ line variants for the acquisition of somatic variants in BCOR, PHF6, and TET2 and second hits in RUNX1 are warranted.