New techniques to identify the tissue of origin for cancer of unknown primary in the era of precision medicine: progress and challenges.

Despite a standardized diagnostic examination, cancer of unknown primary (CUP) is a rare metastatic malignancy with an unidentified tissue of origin (TOO). Patients diagnosed with CUP are typically treated with empiric chemotherapy, although their prognosis is worse than those with metastatic cancer of a known origin. TOO identification of CUP has been employed in precision medicine, and subsequent site-specific therapy is clinically helpful. For example, molecular profiling, including genomic profiling, gene expression profiling, epigenetics and proteins, has facilitated TOO identification. Moreover, machine learning has improved identification accuracy, and non-invasive methods, such as liquid biopsy and image omics, are gaining momentum. However, the heterogeneity in prediction accuracy, sample requirements and technical fundamentals among the various techniques is noteworthy. Accordingly, we systematically reviewed the development and limitations of novel TOO identification methods, compared their pros and cons and assessed their potential clinical usefulness. Our study may help patients shift from empirical to customized care and improve their prognoses.

Molecularly guided therapy versus chemotherapy after disease control in unfavourable cancer of unknown primary (CUPISCO): an open-label, randomised, phase 2 study.

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.

Site-specific therapy guided by a 90-gene expression assay versus empirical chemotherapy in patients with cancer of unknown primary (Fudan CUP-001): a randomised controlled trial.

BACKGROUND: Empirical chemotherapy remains the standard of care in patients with unfavourable cancer of unknown primary (CUP). Gene-expression profiling assays have been developed to identify the tissue of origin in patients with CUP; however, their clinical benefit has not yet been demonstrated. We aimed to evaluate the efficacy and safety of site-specific therapy directed by a 90-gene expression assay compared with empirical chemotherapy in patients with CUP. METHODS: This randomised controlled trial was conducted at Fudan University Shanghai Cancer Center (Shanghai, China). We enrolled patients aged 18-75 years, with previously untreated CUP (histologically confirmed metastatic adenocarcinoma, squamous cell carcinoma, poorly differentiated carcinoma, or poorly differentiated neoplasms) and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, who were not amenable to local radical treatment. Patients were randomly assigned (1:1) by the Pocock and Simon minimisation method to receive either site-specific therapy or empirical chemotherapy (taxane [175 mg/m2 by intravenous infusion on day 1] plus platinum [cisplatin 75 mg/m2 or carboplatin area under the curve 5 by intravenous infusion on day 1], or gemcitabine [1000 mg/m2 by intravenous infusion on days 1 and 8] plus platinum [same as above]). The minimisation factors were ECOG performance status and the extent of the disease. Clinicians and patients were not masked to interventions. The tumour origin in the site-specific therapy group was predicted by the 90-gene expression assay and treatments were administered accordingly. The primary endpoint was progression-free survival in the intention-to-treat population. The trial has been completed and the analysis is final. This study is registered with ClinicalTrials.gov (NCT03278600). FINDINGS: Between Sept 18, 2017, and March 18, 2021, 182 patients (105 [58%] male, 77 [42%] female) were randomly assigned to receive site-specific therapy (n=91) or empirical chemotherapy (n=91). The five most commonly predicted tissues of origin in the site-specific therapy group were gastro-oesophagus (14 [15%]), lung (12 [13%]), ovary (11 [12%]), cervix (11 [12%]), and breast (nine [10%]). At the data cutoff date (April 30, 2023), median follow-up was 33·3 months (IQR 30·4-51·0) for the site-specific therapy group and 30·9 months (27·6-35·5) for the empirical chemotherapy group. Median progression-free survival was significantly longer with site-specific therapy than with empirical chemotherapy (9·6 months [95% CI 8·4-11·9] vs 6·6 months [5·5-7·9]; unadjusted hazard ratio 0·68 [95% CI 0·49-0·93]; p=0·017). Among the 167 patients who started planned treatment, 46 (56%) of 82 patients in the site-specific therapy group and 52 (61%) of 85 patients in the empirical chemotherapy group had grade 3 or worse treatment-related adverse events; the most frequent of these in the site-specific therapy and empirical chemotherapy groups were decreased neutrophil count (36 [44%] vs 42 [49%]), decreased white blood cell count (17 [21%] vs 26 [31%]), and anaemia (ten [12%] vs nine [11%]). Treatment-related serious adverse events were reported in five (6%) patients in the site-specific therapy group and two (2%) in the empirical chemotherapy group. No treatment-related deaths were observed. INTERPRETATION: This single-centre randomised trial showed that site-specific therapy guided by the 90-gene expression assay could improve progression-free survival compared with empirical chemotherapy among patients with previously untreated CUP. Site-specific prediction by the 90-gene expression assay might provide more disease information and expand the therapeutic armamentarium in these patients. FUNDING: Clinical Research Plan of Shanghai Hospital Development Center, Program for Shanghai Outstanding Academic Leader, and Shanghai Anticancer Association SOAR PROJECT. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling.

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique "fingerprint" that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low "cfDNA - high-molecular weight DNA ratio" has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in <1 week, and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist's toolbox in the diagnosis of CUPs.

Cancers of Unknown Primary Origin: Real-World Clinical Outcomes and Genomic Analysis at the European Institute of Oncology.

BACKGROUND: Cancer of unknown primary origin (CUP) poses a significant challenge due to poor clinical outcomes and limited treatment options. As such, further definition of clinicopathological factors and genomic profile to better adapt treatment strategies is required. METHODS: Medical records were interrogated to retrospectively include CUP with available clinical and genomics data at the European Institute of Oncology. Next-generation sequencing (NGS) included targeted panels. Statistical analyses were conducted with R Software 4.2.2. RESULTS: A total of 44 patients were included. With a median follow-up of 39.46 months (interquartile range [IQR] 35.98-47.41 months), median PFS (mPFS) to first-line regimen was 3.98 months (95% CI 3.22-5.98), with a clinical benefit rate of 26% (95% CI 14%-49%), and disease control rate (DCR) limited to 48.28%. Most patients (26 of 31, 83.87%) received platinum-doublet chemotherapy, with no statistically significant difference between first-line treatment regimens. Median OS (mOS) was 18.8 months (95% CI 12.3-39.9), with a 12-month OS rate of 66% (95% CI 50%-85%). All patients received comprehensive genomic profiling (CGP). For 11 patients, NGS was unsuccessful due to low sample quantity and/or quality. For the remaining, TP53 (n = 16, 48%) and KRAS (n = 10, 30%) represented the most altered (alt) genes. No microsatellite instability was observed (0 of 28), while 6 of 28 (21.43%) tumors carried high TMB (≥10 mutation per megabase). Eight of 33 tumors (24.2%) displayed at least one actionable alteration with potential clinical benefit according to ESCAT. Only 2 of them received targeted therapy matched to genomic alterations, with a combined mPFS of 2.63 months (95% CI 1.84-not evaluable) as third-line regimens. Six patients received anti-PD1/PD-L1 therapy, showing a meaningful mPFS of 13 months (95% CI 2.04-not evaluable). CONCLUSION: CUP exhibits poor prognosis with limited benefits from standard treatment regimens. A significant proportion of CUPs carry actionable alterations, underscoring the importance of genomic profiling to gather additional treatment opportunities. In addition, immunotherapy might represent a valuable treatment option for a subset of CUP. Finally, accurate definition of sequencing methods and platforms is crucial to overcome NGS failures.

The evolution of molecular management of carcinoma of unknown primary.

PURPOSE OF REVIEW: There is significant need to improve diagnostic and therapeutic options for patients with cancer of unknown primary (CUP). In this review, we discuss the evolving landscape of molecular profiling in CUP. RECENT FINDINGS: Molecular profiling is becoming accepted into the diagnostic work-up of CUP patients with tumour mutation profiling now described in international CUP guidelines. Although tissue-of-origin (ToO) molecular tests utilising gene-expression and DNA methylation have existed some time, their clinical benefit remains unclear. Novel technologies utilising whole genome sequencing and machine learning algorithms are showing promise in determining ToO, however further research is required prior to clinical application. A recent international clinical trial found patients treated with molecularly-guided therapy based on comprehensive-panel DNA sequencing had improved progression-free survival compared to chemotherapy alone, confirming utility of performing genomic profiling early in the patient journey. Small phase 2 trials have demonstrated that some CUP patients are responsive to immunotherapy, but the best way to select patients for treatment is not clear. SUMMARY: Management of CUP requires a multifaceted approach incorporating clinical, histopathological, radiological and molecular sequencing results to assist with identifying the likely ToO and clinically actionable genomic alternations. Rapidly identifying a subset of CUP patients who are likely to benefit from site specific therapy, targeted therapy and/or immunotherapy will improve patient outcomes.

Patient with mediastinal carcinoma of unknown primary with RET fusion achieves durable response with RET inhibition.

Selective RET inhibitors have shown promise in thyroid cancer (TC) and nonsmall cell lung cancer (NSCLC) harboring RET fusions on next-generation sequencing (NGS), although rarity of the rearrangement has led to limited data for certain tumor types, such as carcinoma of unknown primary. We present a 65-year-old female with no history of malignancy, smoking or radiation exposure, who was found to have an anterior mediastinum malignancy of unknown primary, with metastases to supraclavicular lymph nodes. Core biopsy of the mediastinum revealed poorly differentiated carcinoma, while a biopsy of the thyroid revealed atypia of indeterminate significance (Bethesda III). PD-L1 immunohistochemistry was positive (90%), and liquid NGS revealed mutations in TP53 and the TERT promoter (c.-124C>T), as well as a CCDC6-RET fusion. This genetic profile resembled an anaplastic TC vs. NSCLC primary, although thymic primary and poorly differentiated TC remained on the differential. The patient was initiated on selpercatinib, which was held after 3 weeks due to thrombocytopenia and hypertension. At a reduced dosage, patient developed transaminitis, and selpercatinib was switched to pralsetinib. Brain MRI showed a nonenhancing temporal lobe signal abnormality, which on biopsy proved to be glioblastoma (GBM) with TERT promoter c.-124C>T mutation and FGFR3-TACC3 fusion by NGS. Pralsetinib was held during adjuvant chemoradiation for the GBM, and again for 4 weeks due to pneumonitis that resolved with steroids, and pralsetinib was restarted at a reduced dose. The patient has since demonstrated a stable reduction of the mediastinal mass for >15 months with RET inhibition therapy, despite several treatment interruptions.

Head and neck squamous cell carcinomas of unknown primary: Can ancillary studies help identify more primary tumor sites?

A subset of head and neck squamous cell carcinomas present solely as metastatic disease in the neck and are of unknown primary origin (SCCUP). Most primary tumors will ultimately be identified, usually in the oropharynx. In a minority of cases, the primary site remains elusive. Here, we examine the role of ancillary testing, including mutational signature analysis (MSA), to help identify likely primary sites in such cases. Twenty-two cases of SCCUP in the neck, collected over a 10-year period, were classified by morphology and viral status; including human papillomavirus (HPV) testing by p16 immunohistochemistry (IHC) and RT-qPCR, as well as Epstein-Barr virus (EBV) testing by EBER-ISH. CD5 and c-KIT (CD117) IHC was done to evaluate for possible thymic origin in all virus-negative cases. Whole exome sequencing, followed by MSA, was used to identify UV signature mutations indicative of cutaneous origin. HPV was identified in 12 of 22 tumors (54.5%), favoring an oropharyngeal origin, and closely associated with nonkeratinizing tumor morphology (Fisher's exact test; p = 0.0002). One tumor with indeterminant morphology had discordant HPV and p16 status (p16+/HPV-). All tumors were EBV-negative. Diffuse expression of CD5 and c-KIT was identified in 1 of 10 virus-negative SCCUPs (10%), suggesting a possible ectopic thymic origin rather than a metastasis. A UV mutational signature, indicating cutaneous origin, was identified in 1 of 10 (10%) virus-negative SCCUPs. A cutaneous auricular primary emerged 3 months after treatment in this patient. Primary tumors became clinically apparent in 2 others (1 hypopharynx, 1 hypopharynx/larynx). Thus, after follow-up, 6 tumors remained unclassifiable as to the possible site of origin (27%). Most SCCUPs of the neck in our series were HPV-associated and thus likely of oropharyngeal origin. UV signature mutation analysis and additional IHC for CD5 and c-KIT for possible thymic origin may aid in further classifying virus-negative unknown primaries. Close clinical inspection of hypopharyngeal mucosa may also be helpful, as a subset of primary tumors later emerged at this site.

Utility of next-generation sequencing in the diagnosis of metastatic melanoma: A case report.

During routine dermatologic examination, a 77-year-old male was noted to have a firm blue subcutaneous nodule on his right lateral upper back. His past medical history included metastatic melanoma of unknown primary involving right and left axillary lymph nodes, treated with ipilimumab/nivolumab with complete response, and subsequent primary uveal melanoma. The subcutaneous nodule was located near his previous right axillary scar for metastatic melanoma. Excision of the nodule showed a plexiform neoplasm involving mid and deep dermis composed of spindle and epithelioid atypical cells admixed with numerous melanophages. Central necrosis was present. Immunohistochemical studies revealed the tumor cells to be diffusely positive for HMB45, with retained expression of BAP1 and p16. The tumor cells were negative for PRAME, nuclear expression of ß-catenin, LEF1, and BRAF V600E. Molecular studies demonstrated BAP1 and GNA11 somatic mutations, a profile different from that exhibited by his prior melanoma. Collectively, these data were interpreted as a metastasis from uveal melanoma and not a recurrence of his metastatic likely cutaneous melanoma after complete response to immunotherapy. This case emphasizes the importance of molecular studies for definitive diagnosis in challenging clinical situations, especially when there is discordance among histopathological, immunohistochemical, and molecular studies. Integration of clinical, histopathological, and molecular features is warranted.

PD-L1 expression and microsatellite instability (MSI) in cancer of unknown primary site.

BACKGROUND: Cancer of unknown primary site (CUP) is a heterogeneous group of tumors for which the origin remains unknown. Clinical outcomes might be influenced by regulatory processes in its microenvironment. Microsatellite instability (MSI) is a predictive biomarker for cancer immunotherapy and its status, as well as co-occurrence with PD-L1 expression, is poorly evaluated. We aim to evaluate the expression of PD-L1 and the status of MSI in CUP and their possible associations with clinical-pathological features. METHODS: The combined positive score (CPS) PD-L1 expression was evaluated by immunohistochemistry. MSI status was assessed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. RESULTS: Among the 166 cases, MSI analysis was conclusive in 120, with two cases being MSI positive (1.6%). PD-L1 expression was positive in 18.3% of 109 feasible cases. PD-L1 expression was significantly associated with non-visceral metastasis and a dominance of nodal metastasis. The median overall survival (mOS) was 3.7 (95% CI 1.6-5.8) months and patients who expressed PD-L1 achieved a better mOS compared to those who did not express PD-L1 (18.7 versus 3.0 months, p-value: < .001). ECOG-PS equal to or more than two and PD-L1 expression were independent prognostic factors in multivariate analysis (2.37 and 0.42, respectively). CONCLUSION: PD-L1 is expressed in a subset (1/5) of patients with CUP and associated with improved overall survival, while MSI is a rare event. There is a need to explore better the tumor microenvironment as well as the role of immunotherapy to change such a bad clinical outcome.

Impact of the Identification of Nonhuman Genetic Signatures in the Diagnosis and Management of Carcinoma of Unknown Primary.

This report presents the case of a 62-year-old woman who was diagnosed in 1999 with stage I cervical carcinoma treated by surgical resection. In 2021, she presented to the emergency department with a complaint of predominantly right-sided lower back pain. A CT scan of the lumbosacral region revealed a bone lesion in the L5 vertebra and retroperitoneal lymphadenopathies suggestive of malignancy. Histology of the L5 vertebra biopsy showed a poorly differentiated carcinoma with an inconclusive immunophenotypic profile. Treatment for carcinoma of unknown primary was started with a combination of carboplatin and paclitaxel every 21 days. A genomic study of the biopsy specimen performed on the FoundationOne CDx platform identified a nonhuman genetic signature compatible with HPV. The presence of HPV 18 DNA in the specimen was confirmed by PCR-reverse dot blot, and the immunophenotypic profile was expanded, revealing strong and diffuse p16 expression, thus corroborating the molecular findings. In view of these findings, the case was reclassified as a recurrence of the cervical adenocarcinoma that had been diagnosed and treated 23 years earlier. Based on the new results, and according to first-line cervical carcinoma protocols, bevacizumab at 15 mg/kg every 21 days was added to her chemotherapy regimen. The identification of HPV DNA sequences by next-generation sequencing facilitated the correct diagnosis and led to a modification of the first-line therapeutic approach.

Tissue of origin prediction for cancer of unknown primary using a targeted methylation sequencing panel.

RATIONALE: Cancer of unknown primary (CUP) is a group of rare malignancies with poor prognosis and unidentifiable tissue-of-origin. Distinct DNA methylation patterns in different tissues and cancer types enable the identification of the tissue of origin in CUP patients, which could help risk assessment and guide site-directed therapy. METHODS: Using genome-wide DNA methylation profile datasets from The Cancer Genome Atlas (TCGA) and machine learning methods, we developed a 200-CpG methylation feature classifier for CUP tissue of origin prediction (MFCUP). MFCUP was further validated with public-available methylation array data of 2977 specimens and targeted methylation sequencing of 78 Formalin-fixed paraffin-embedded (FFPE) samples from a single center. RESULTS: MFCUP achieved an accuracy of 97.2% in a validation cohort (n = 5923) representing 25 cancer types. When applied to an Infinium 450 K array dataset (n = 1052) and an Infinium EPIC (850 K) array dataset (n = 1925), MFCUP achieved an overall accuracy of 93.4% and 84.8%, respectively. Based on MFCUP, we established a targeted bisulfite sequencing panel and validated it with FFPE sections from 78 patients of 20 cancer types. This methylation sequencing panel correctly identified tissue of origin in 88.5% (69/78) of samples. We also found that the methylation levels of specific CpGs can distinguish one cancer type from others, indicating their potential as biomarkers for cancer diagnosis and screening. CONCLUSION: Our methylation-based cancer classifier and targeted methylation sequencing panel can predict tissue of origin in diverse cancer types with high accuracy.

A cfDNA methylation-based tissue-of-origin classifier for cancers of unknown primary.

Cancers of Unknown Primary (CUP) remains a diagnostic and therapeutic challenge due to biological heterogeneity and poor responses to standard chemotherapy. Predicting tissue-of-origin (TOO) molecularly could help refine this diagnosis, with tissue acquisition barriers mitigated via liquid biopsies. However, TOO liquid biopsies are unexplored in CUP cohorts. Here we describe CUPiD, a machine learning classifier for accurate TOO predictions across 29 tumour classes using circulating cell-free DNA (cfDNA) methylation patterns. We tested CUPiD on 143 cfDNA samples from patients with 13 cancer types alongside 27 non-cancer controls, with overall sensitivity of 84.6% and TOO accuracy of 96.8%. In an additional cohort of 41 patients with CUP CUPiD predictions were made in 32/41 (78.0%) cases, with 88.5% of the predictions clinically consistent with a subsequent or suspected primary tumour diagnosis, when available (23/26 patients). Combining CUPiD with cfDNA mutation data demonstrated potential diagnosis re-classification and/or treatment change in this hard-to-treat cancer group.

Image-based deep learning model using DNA methylation data predicts the origin of cancer of unknown primary.

Cancer of unknown primary (CUP) is a rare type of metastatic cancer in which the origin of the tumor is unknown. Since the treatment strategy for patients with metastatic tumors depends on knowing the primary site, accurate identification of the origin site is important. Here, we developed an image-based deep-learning model that utilizes a vision transformer algorithm for predicting the origin of CUP. Using DNA methylation dataset of 8,233 primary tumors from The Cancer Genome Atlas (TCGA), we categorized 29 cancer types into 18 organ classes and extracted 2,312 differentially methylated CpG sites (DMCs) from non-squamous cancer group and 420 DMCs from squamous cell cancer group. Using these DMCs, we created organ-specific DNA methylation images and used them for model training and testing. Model performance was evaluated using 394 metastatic cancer samples from TCGA (TCGA-meta) and 995 samples (693 primary and 302 metastatic cancers) obtained from 20 independent external studies. We identified that the DNA methylation image reveals a distinct pattern based on the origin of cancer. Our model achieved an overall accuracy of 96.95 % in the TCGA-meta dataset. In the external validation datasets, our classifier achieved overall accuracies of 96.39 % and 94.37 % in primary and metastatic tumors, respectively. Especially, the overall accuracies for both primary and metastatic samples of non-squamous cell cancer were exceptionally high, with 96.79 % and 96.85 %, respectively.

Tracing unknown tumor origins with a biological-pathway-based transformer model.

Cancer of unknown primary (CUP) represents metastatic cancer where the primary site remains unidentified despite standard diagnostic procedures. To determine the tumor origin in such cases, we developed BPformer, a deep learning method integrating the transformer model with prior knowledge of biological pathways. Trained on transcriptomes from 10,410 primary tumors across 32 cancer types, BPformer achieved remarkable accuracy rates of 94%, 92%, and 89% in primary tumors and primary and metastatic sites of metastatic tumors, respectively, surpassing existing methods. Additionally, BPformer was validated in a retrospective study, demonstrating consistency with tumor sites diagnosed through immunohistochemistry and histopathology. Furthermore, BPformer was able to rank pathways based on their contribution to tumor origin identification, which helped to classify oncogenic signaling pathways into those that are highly conservative among different cancers versus those that are highly variable depending on their origins.

Comprehensive biomarker diagnostics of unfavorable cancer of unknown primary to identify patients eligible for precision medical therapies.

PURPOSE: Current guidelines recommend combination chemotherapy for treatment of patients with unfavorable cancer of unknown primary (CUP). Biomarker-guided targeted therapies may offer additional benefit. Data on the feasibility and effectiveness of comprehensive genomic biomarker profiling of CUP in a standard clinical practice setting are limited. METHODS: This analysis included 156 patients with confirmed unfavorable CUP diagnosis according to ESMO guidelines, who were treated at the West German Cancer Center, Essen, Germany, from 2015 to 2021. Clinical parameters and outcome data were retrieved from the electronic hospital information system. Genomic biomarker analyses were performed in formalin-fixed paraffin-embedded tumor tissue whenever possible using the QIAseq Multimodal-Pancancer-Panel. RESULTS: Non-squamous histologies, high tumor burden, and age above 60 years associated with poor survival outcome. Tissue availability restricted comprehensive biomarker analyses to 50 patients (32%), reflecting a major limitation in the real-world setting. In those patients a total of 24 potentially actionable alterations were identified in 17 patients (34% of profiled patients, 11% of total population). The most prevalent biomarkers were high tumor mutational burden and BRCA-mutations. CONCLUSION: In a real-world setting precision medicine for patients with CUP is severely restricted by tissue availability, and a limited spectrum of actionable alterations. Progress for patients may require emphasizing the need for sufficient biopsies, and prospective exploration of blood-based biomarker profiling.

Head and neck cancer of unknown primary: unveiling primary tumor sites through machine learning on DNA methylation profiles.

BACKGROUND: The unknown tissue of origin in head and neck cancer of unknown primary (hnCUP) leads to invasive diagnostic procedures and unspecific and potentially inefficient treatment options for patients. The most common histologic subtype, squamous cell carcinoma, can stem from various tumor primary sites, including the oral cavity, oropharynx, larynx, head and neck skin, lungs, and esophagus. DNA methylation profiles are highly tissue-specific and have been successfully used to classify tissue origin. We therefore developed a support vector machine (SVM) classifier trained with publicly available DNA methylation profiles of commonly cervically metastasizing squamous cell carcinomas (n = 1103) in order to identify the primary tissue of origin of our own cohort of squamous cell hnCUP patient's samples (n = 28). Methylation analysis was performed with Infinium MethylationEPIC v1.0 BeadChip by Illumina. RESULTS: The SVM algorithm achieved the highest overall accuracy of tested classifiers, with 87%. Squamous cell hnCUP samples on DNA methylation level resembled squamous cell carcinomas commonly metastasizing into cervical lymph nodes. The most frequently predicted cancer localization was the oral cavity in 11 cases (39%), followed by the oropharynx and larynx (both 7, 25%), skin (2, 7%), and esophagus (1, 4%). These frequencies concord with the expected distribution of lymph node metastases in epidemiological studies. CONCLUSIONS: On DNA methylation level, hnCUP is comparable to primary tumor tissue cancer types that commonly metastasize to cervical lymph nodes. Our SVM-based classifier can accurately predict these cancers' tissues of origin and could significantly reduce the invasiveness of hnCUP diagnostics and enable a more precise therapy after clinical validation.

Utility of UV Signature Mutations in the Diagnostic Assessment of Metastatic Head and Neck Carcinomas of Unknown Primary.

BACKGROUND: Metastatic carcinoma of unknown primary origin to the head and neck lymph nodes (HNCUP) engenders unique diagnostic considerations. In many cases, the detection of a high-risk human papillomavirus (HR-HPV) unearths an occult oropharyngeal squamous cell carcinoma (SCC). In metastatic HR-HPV-independent carcinomas, other primary sites should be considered, including cutaneous malignancies that can mimic HR-HPV-associated SCC. In this context, ultraviolet (UV) signature mutations, defined as ≥ 60% C→T substitutions with ≥ 5% CC→TT substitutions at dipyrimidine sites, identified in tumors arising on sun exposed areas, are an attractive and underused tool in the setting of metastatic HNCUP. METHODS: A retrospective review of institutional records focused on cases of HR-HPV negative HNCUP was conducted. All cases were subjected to next generation sequencing analysis to assess UV signature mutations. RESULTS: We identified 14 HR-HPV negative metastatic HNCUP to either the cervical or parotid gland lymph nodes, of which, 11 (11/14, 79%) had UV signature mutations, including 4 (4/10, 40%) p16 positive cases. All UV signature mutation positive cases had at least one significant TP53 mutation and greater than 20 unique gene mutations. CONCLUSION: The management of metastatic cutaneous carcinomas significantly differs from other HNCUP especially metastatic HR-HPV-associated SCC; therefore, the observation of a high percentage of C→T with CC →TT substitutions should be routinely incorporated in next generation sequencing reports of HNCUP. UV mutational signatures testing is a robust diagnostic tool that can be utilized in daily clinical practice.

Clinicopathologic features and genomic profiling of female axillary lymph node metastases from adenocarcinoma or poorly differentiated carcinoma of unknown primary.

PURPOSE: Axillary lymph node metastases from adenocarcinoma or poorly differentiated carcinoma of unknown primary (CUPAx) is a rare disease in women. This retrospective study intended to examine the clinicopathological features of CUPAx and compared CUPAx genetically with axillary lymph node metastases from breast cancer (BCAx), investigating differences in their biological behavior. METHODS: We conducted the clinical and prognostic analysis of 58 CUPAx patients in West China Hospital spanning from 2009 to 2021. Gemonic profiling of 12 CUPAx patients and 16 BCAx patients was conducted by the FoundationOne CDx (F1CDx) platform. Moreover, we also compared the gene mutation spectrum and relevant pathways between the two groups and both TCGA and COSMIC databases. RESULTS: The majority of the 58 CUPAx patients were HR-/HER2- subtype. Most patients received mastectomy combined radiotherapy (50 Gy/25f). CUPAx patients who received mastectomy instead of breast-conserving surgery had a more favorable overall prognosis. Radiotherapy in chest wall/breast and supraclavicular/infraclavicular fossa was the independent prognostic factor (HR = 0.05, 95%CI = 0.00-0.93, P = 0.04). In 28 sequencing samples (CUPAx, n = 12, BCAx, n = 16) and 401 TCGA-BRCA patients, IRS2 only mutated in CUPAx (33.33%) but amplified in BCAx (11.11%) and TCGA-BRCA (1.5%). Pathway analysis revealed that BCAx had more NOTCH pathway mutations than CUPAx. Enrichment analysis showed that CUPAx enriched more in mammary development and PML bodies than BCAx, but less in the positive regulation of kinase activity. CONCLUSIONS: More active treatment methods, like chemotherapy, mastectomy and postoperative radiotherapy, could improve the prognosis of CUPAx. The differential mutation genes of CUPAx and BCAx might be associated with their respective biological behaviors like invasiveness and prognosis.