Recurrent and novel fusions detected by targeted RNA sequencing as part of the diagnostic workflow of soft tissue and bone tumours.

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA-based sequencing (targeted RNA-seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA-seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA-seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA-seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA-seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH-positive analyses showed a lower percentage of rearrangement-positive nuclei (range 15-41%) compared to the concordant FISH-positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA-seq findings. For the EWSR1 FISH probe, we observed a gene-dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA-seq (22.9%). This study demonstrates an added value of targeted RNA-seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA-seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

Metabolic adaptation towards glycolysis supports resistance to neoadjuvant chemotherapy in early triple negative breast cancers.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care for patients with early-stage triple negative breast cancers (TNBC). However, more than half of TNBC patients do not achieve a pathological complete response (pCR) after NAC, and residual cancer burden (RCB) is associated with dismal long-term prognosis. Understanding the mechanisms underlying differential treatment outcomes is therefore critical to limit RCB and improve NAC efficiency. METHODS: Human TNBC cell lines and patient-derived organoids were used in combination with real-time metabolic assays to evaluate the effect of NAC (paclitaxel and epirubicin) on tumor cell metabolism, in particular glycolysis. Diagnostic biopsies (pre-NAC) from patients with early TNBC were analyzed by bulk RNA-sequencing to evaluate the predictive value of a glycolysis-related gene signature. RESULTS: Paclitaxel induced a consistent metabolic switch to glycolysis, correlated with a reduced mitochondrial oxidative metabolism, in TNBC cells. In pre-NAC diagnostic biopsies from TNBC patients, glycolysis was found to be upregulated in non-responders. Furthermore, glycolysis inhibition greatly improved response to NAC in TNBC organoid models. CONCLUSIONS: Our study pinpoints a metabolic adaptation to glycolysis as a mechanism driving resistance to NAC in TNBC. Our data pave the way for the use of glycolysis-related genes as predictive biomarkers for NAC response, as well as the development of inhibitors to overcome this glycolysis-driven resistance to NAC in human TNBC patients.

Predictive markers for pathological complete response (pCR) after neo-adjuvant chemotherapy in HER2-positive breast carcinoma.

OBJECTIVES: Patients with HER2-positive invasive breast cancer that is node-positive and/or larger than 3 cm are generally treated with neoadjuvant chemotherapy (NAC). We aimed to identify predictive markers for pathological complete response (pCR) after NAC in HER2-positive breast carcinoma. METHODS: Hematoxylin/eosin-stained slides of 43 HER2-positive breast carcinoma biopsies were histopathologically reviewed. Immunohistochemistry (IHC) was performed on pre-NAC biopsies, comprising HER2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, epidermal growth factor receptor (EGFR), mucin-4 (MUC4), p53 and p63. Dual-probe HER2 in situ hybridization (ISH) was performed to study the mean HER2 and CEP17 copy numbers. ISH and IHC data were retrospectively collected for a validation cohort, comprising 33 patients. RESULTS: Younger age at diagnosis, 3+ HER2 IHC scores, high mean HER2 copy numbers and high mean HER2/CEP17 ratios were significantly associated with an increased chance of achieving a pCR, and the latter two associations were confirmed in the validation cohort. No other immunohistochemical or histopathological markers were associated with pCR. CONCLUSIONS: This retrospective study of two community-based NAC-treated HER2-positive breast cancer patient cohorts identified high mean HER2 copy numbers as a strong predictor for pCR. Further studies on larger cohorts are required to determine a precise cut-point for this predictive marker.

Tumour-agnostic plasma assay for circulating tumour DNA predicts outcome in recurrent and/or metastatic squamous cell carcinoma of the head and neck treated with a PD-1 inhibitor.

BACKGROUND: Only 15-20% of recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) patients derive long-term benefit from nivolumab or pembrolizumab. We developed a circulating tumour DNA (ctDNA) tumour-agnostic assay aimed at the early prediction of single agent programmed cell death 1 (PD1) inhibitor efficacy in R/M SCCHN. PATIENTS AND METHODS: Our tumour-agnostic assay included 37 genes frequently mutated in R/M SCCHN and two HPV16 genes. Primary endpoint was the concordance between ctDNA kinetics (ΔctDNA) and the best overall response according to Response Evaluation Criteria in Solid Tumors version 1.1. ΔctDNA was defined as the difference in mean variant allele frequency (VAF) between the on-treatment sample harvested 6-10 weeks (FU1) after PD1 inhibitor initiation and the pre-treatment plasma sample (ΔctDNA = mean FU1 VAF - mean pre-treatment VAF). RESULTS: ctDNA was detected in 35/44 (80%) of the pre-treatment plasma samples. The concordance between ΔctDNA and imaging response was observed in 74%. Median progression-free survival was 8.6 months in the favourable ΔctDNA group and 2.5 months in the unfavourable ΔctDNA group (p = 0.057). Median overall survival (OS) was 18.1 and 8.2 months in the favourable and unfavourable ΔctDNA groups, respectively (p = 0.13). In patients with PD-L1 expressing SCCHN (Combined Positive Score ≥1), OS was significantly better in patients with favourable ΔctDNA compared with patients with unfavourable ΔctDNA: median OS was 41.5 and 8.4 months (p = 0.033), respectively. CONCLUSIONS: Tumour-agnostic ctDNA analysis for human papillomavirus (HPV)-negative and HPV-positive R/M SCCHN is feasible. ctDNA kinetics show promising results in predicting the efficacy of PD1 inhibitors in R/M SCCHN.

Tucatinib and Trastuzumab for Previously Treated Human Epidermal Growth Factor Receptor 2-Positive Metastatic Biliary Tract Cancer (SGNTUC-019): A Phase II Basket Study.

PURPOSE: To evaluate the efficacy and safety of tucatinib and trastuzumab in patients with previously treated human epidermal growth factor receptor 2-positive (HER2+) metastatic biliary tract cancer (mBTC). METHODS: SGNTUC-019 (ClinicalTrials.gov identifier: NCT04579380) is an open-label phase II basket study evaluating the efficacy and safety of tucatinib and trastuzumab in patients with HER2-altered solid tumors. In the biliary tract cancer cohort, patients had previously treated HER2 overexpressing or amplified (HER2+) tumors (identified with local testing) with no prior HER2-directed therapy. The primary end point was confirmed objective response rate (cORR) per investigator assessment. Patients were treated on a 21-day cycle with tucatinib (300 mg orally twice daily) and trastuzumab (8 mg/kg intravenously followed by 6 mg/kg every 3 weeks). RESULTS: Thirty patients were enrolled. As of data cutoff (January 30, 2023), the median duration of follow-up was 10.8 months. The cORR was 46.7% (90% CI, 30.8 to 63.0), with a disease control rate of 76.7% (90% CI, 60.6 to 88.5). The median duration of response and progression-free survival were 6.0 months (90% CI, 5.5 to 6.9) and 5.5 months (90% CI, 3.9 to 8.1), respectively. At data cutoff, 15 patients (50.0%) had died, and the estimated 12-month overall survival rate was 53.6% (90% CI, 36.8 to 67.8). The two most common treatment-emergent adverse events (TEAEs) were pyrexia (43.3%) and diarrhea (40.0%). Grade ≥3 TEAEs were reported in 18 patients (60.0%), with the most common being cholangitis, decreased appetite, and nausea (all 10.0%), which were generally not treatment related. TEAEs led to treatment regimen discontinuation in one patient, and there were no deaths due to TEAEs. CONCLUSION: Tucatinib combined with trastuzumab had clinically significant antitumor activity and was well tolerated in patients with previously treated HER2+ mBTC.

Long-lasting benefit on multimodal treatment combining osimertinib and stereotaxic radiotherapy for metastatic non-small cell lung cancer with the EGFR exon 20 insertion 773-774 HVdelinsLM: a case report.

A non-small-cell-lung-cancer patient with cerebral metastasis presenting an atypical exon 20 mutation in the EGFR gene had a long-lasting tumor cotrol on mulimodal treatment with osimertinib and stereotaxic radiotherapy on oligoprogressing lesions. Most exon-20 mutations are resistant to first, second and third generation EGFR-directed TKI. This case was discussed on our molecular tumour board. As the more specific exon-20 targeted therapies were not yet available and as sporadic short responses on the third generation EGFR-directed TKI, osimertinib had been described, the patient started osimertinib. She had a prolonged tumoral response on Osimertinib. The patient is still asymptomatic up to 32 months after initiating the medication. This case confirms that not all exon20 EGFR mutations are equal to osimertinib and that the localization of the exon 20 insertion mutation is probably important to consider when treating EGFR mutated NSCLC. The long-term clinical benefit can be maintained through stereotactic radiotherapy on focal progressive lesions.

Predictive Biomarkers of Response to Neoadjuvant Chemotherapy in Breast Cancer: Current and Future Perspectives for Precision Medicine.

Pathological complete response (pCR) after neoadjuvant chemotherapy in patients with early breast cancer is correlated with better survival. Meanwhile, an expanding arsenal of post-neoadjuvant treatment strategies have proven beneficial in the absence of pCR, leading to an increased use of neoadjuvant systemic therapy in patients with early breast cancer and the search for predictive biomarkers of response. The better prediction of response to neoadjuvant chemotherapy could enable the escalation or de-escalation of neoadjuvant treatment strategies, with the ultimate goal of improving the clinical management of early breast cancer. Clinico-pathological prognostic factors are currently used to estimate the potential benefit of neoadjuvant systemic treatment but are not accurate enough to allow for personalized response prediction. Other factors have recently been proposed but are not yet implementable in daily clinical practice or remain of limited utility due to the intertumoral heterogeneity of breast cancer. In this review, we describe the current knowledge about predictive factors for response to neoadjuvant chemotherapy in breast cancer patients and highlight the future perspectives that could lead to the better prediction of response, focusing on the current biomarkers used for clinical decision making and the different gene signatures that have recently been proposed for patient stratification and the prediction of response to therapies. We also discuss the intratumoral phenotypic heterogeneity in breast cancers as well as the emerging techniques and relevant pre-clinical models that could integrate this biological factor currently limiting the reliable prediction of response to neoadjuvant systemic therapy.

Case Report Series: Aggressive HR Deficient Colorectal Cancers Related to BRCA1 Pathogenic Germline Variants.

Objective: The link between BRCA1 and homologous recombination deficiency (HRD) in cancer has gained importance with the emergence of new targeted cancer treatments, while the available data on the role of the gene in colorectal cancer (CRC) remain contradictory. The aim of this case series was to elucidate the role of known pathogenic BRCA1 variants in the development of early-onset CRC. Design: Patients were evaluated using targeted next generation sequencing, exome sequencing and chromosomal microarray analysis of the paired germline and tumor samples. These results were used to calculate the HRD score and the frequency of mutational signatures in the tumors. Results: Three patients with metastatic CRC were heterozygous for a previously known BRCA1 nonsense variant. All tumors showed remarkably high HRD scores, and the HRD-related signature 3 had the second highest contribution to the somatic pattern of variant accumulation in the samples (23% in 1 and 2, and 13% in sample 3). Conclusions: A BRCA1 germline pathogenic variant can be involved in CRC development through HRD. Thus, BRCA1 testing should be considered in young patients with a personal history of microsatellite stable CRC as this could further allow a personalized treatment approach.

Liquid Biopsy to Detect Minimal Residual Disease: Methodology and Impact.

One reason why some patients experience recurrent disease after a curative-intent treatment might be the persistence of residual tumor cells, called minimal residual disease (MRD). MRD cannot be identified by standard radiological exams or clinical evaluation. Tumor-specific alterations found in the blood indirectly diagnose the presence of MRD. Liquid biopsies thus have the potential to detect MRD, allowing, among other things, the detection of circulating tumor DNA (ctDNA), circulating tumor cells (CTC), or tumor-specific microRNA. Although liquid biopsy is increasingly studied, several technical issues still limit its clinical applicability: low sensitivity, poor standardization or reproducibility, and lack of randomized trials demonstrating its clinical benefit. Being able to detect MRD could give clinicians a more comprehensive view of the risk of relapse of their patients and could select patients requiring treatment escalation with the goal of improving cancer survival. In this review, we are discussing the different methodologies used and investigated to detect MRD in solid cancers, their respective potentials and issues, and the clinical impacts that MRD detection will have on the management of cancer patients.

Successful treatment with yttrium-90 microspheres in a metastatic breast cancer patient and sclerosing cholangitis.

Breast cancer is the most common malignancy occurring in women worldwide. More than 90% of patients present with localized disease are treated with curative intent; however, recurrence can occur with development of metastatic lesions. Frequently associated with extra-hepatic lesions, localized treatments (surgery or stereotaxic body radiotherapy) are rarely proposed in liver lesions. 90Y radioembolization has extensively been evaluated in colorectal cancer, but its role in breast cancer with isolated liver metastases remains largely unknown. Pre-existing liver diseases are known risk factors for 90Y induced liver toxicity. Not considered as an excluding factor for this treatment, data are limited regarding its safe use with cholangitis. We report a successful control of liver metastases by 90Y radioembolization in a breast cancer patient.

Tumor apelin and obesity are associated with reduced neoadjuvant chemotherapy response in a cohort of breast cancer patients.

Obesity is a known factor increasing the risk of developing breast cancer and reducing disease free survival. In addition to these well-documented effects, recent studies have shown that obesity is also affecting response to chemotherapy. Among the multiple dysregulations associated with obesity, increased level of the apelin adipokine has been recently shown to be directly involved in the association between obesity and increased breast cancer progression. In this study, we analyzed in a retrospective cohort of 62 breast cancer patients the impact of obesity and tumoral apelin expression on response to neoadjuvant chemotherapy. In the multivariate logistic regression, obesity and high tumoral apelin expression were associated with a reduced response to NAC in our cohort. However, obesity and high tumoral apelin expression were not correlated, suggesting that those two parameters could be independently associated with reduced NAC response. These findings should be confirmed in independent cohorts.

Preclinical Evaluation of the Association of the Cyclin-Dependent Kinase 4/6 Inhibitor, Ribociclib, and Cetuximab in Squamous Cell Carcinoma of the Head and Neck.

Epidermal growth factor receptor (EGFR) overexpression is observed in 90% of human papillomavirus (HPV)-negative squamous cell carcinomas of the head and neck (SCCHN). Cell cycle pathway impairments resulting in cyclin-dependent kinase (CDK) 4 and 6 activation, are frequently observed in SCCHN. We investigated the efficacy of ribociclib, a CDK4/6 inhibitor, in combination with cetuximab, a monoclonal antibody targeting the EGFR, in HPV-negative SCCHN patient-derived tumor xenograft (PDTX) models. The combination of cetuximab and ribociclib was not significantly more active than cetuximab monotherapy in all models investigated. In addition, the combination of cetuximab and ribociclib was less active than ribociclib monotherapy in the cetuximab-resistant PDTX models. In these models, a significant downregulation of the retinoblastoma (Rb) protein was observed in cetuximab-treated mice. We also observed Rb downregulation in the SCCHN cell lines chronically exposed and resistant to cetuximab. In addition, Rb downregulation induced interleukin 6 (Il-6) secretion and the Janus kinase family member/signal transducer and activator of transcription (JAK/STAT) pathway activation that might be implicated in the cetuximab resistance of these cell lines. To conclude, cetuximab is not an appropriate partner for ribociclib in cetuximab-resistant SCCHN models. Our work has significant clinical implications since the combination of anti-EGFR therapy with CDK4/6 inhibitors is currently being investigated in clinical trials.

Predictive markers for pathological complete response after neo-adjuvant chemotherapy in triple-negative breast cancer.

A combination of Sox10 and GATA3 was previously identified as a marker for metastatic triple-negative breast cancer (TNBC), but it is uncertain whether their expression is associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). This study investigates the predictive value of clinicopathological characteristics, as well as protein expression of Sox10, GATA3, p53 and p63, in a consecutive series of TNBC patients treated with NAC. Archived hematoxylin & eosin stained slides of core biopsies and resection specimens from 35 TNBC patients were reviewed. The following clinicopathological characteristics were determined at the biopsy level: age at diagnosis, cancer type, Nottingham grade, lympho-vascular invasion, syncytial growth, necrosis, clear cell differentiation, myxoid peritumor stroma, stromal tumor-infiltrating lymphocytes (sTILs) and presence of an in situ component. The MD Anderson residual cancer burden (RCB) score and corresponding RCB class were determined. Immunohistochemistry for Sox10, p53, GATA3 and p63 was performed at the biopsy level. sTILs, either as a continuous or as a dichotomous variable, were the only parameter that was significantly associated with pCR in univariable and multivariable analyses. Assessment of sTILs showed moderate to good interobserver agreement. High sTILs (≥40%) were significantly associated with increased pCR rates, and this association was observer-independent. This retrospective study of a consecutive community-based cohort of TNBC patients confirms that sTILs are a robust, observer-independent predictor for therapeutic response after NAC. The combination of Sox10, GATA3 and p53 immunoreactivity is unlikely to harbor any predictive value for pCR in TNBC.

Tumor sequencing is useful to refine the analysis of germline variants in unexplained high-risk breast cancer families.

BACKGROUND: Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. METHODS: Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. RESULTS: Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. CONCLUSION: Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.

Liquid biopsy for mutational profiling of locoregional recurrent and/or metastatic head and neck squamous cell carcinoma.

OBJECTIVES: The molecular landscape of head and neck squamous cell carcinoma (HNSCC) harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to (i) characterize the mutational landscape of recurrent/metastatic HNSCC using a comprehensive gene panel and (ii) estimate the concordance between DNA mutations identified from circulating tumor DNA (ctDNA) and matched tumor tissues. MATERIALS AND METHODS: Targeted next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) of 39 patients with locoregional recurrent (n = 19) and/or metastatic (n = 20) HNSCC. Tumor biopsy (n = 18) was sequenced using the same technique. RESULTS: ctDNA was detected in 51% of patients (20/39) with a higher probability of detection in metastatic than locoregional recurrent disease (70% versus 30%, p = 0.025). 81% and 58% of the tissue tumor variants were not detected in plasma when considering all patients and only metastatic patients with detectable ctDNA, respectively. In a multivariate analysis, the likelihood of detecting the tissue tumor variant in plasma was related to metastatic status (p = 0.012), tumor variant allele frequency (p < 0.001) and ctDNA quantity (p < 0.001). 26% of the variants were detected only in liquid and not in the solid biopsy. Three patients without an available tumor sample had plasma containing three different potentially actionable PIK3CA mutations. CONCLUSION: CtDNA detection and characterization using targeted NGS is feasible in metastatic HNSCC. Liquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants not found in matched tumor tissue.

Routine use of gene panel testing in hereditary breast cancer should be performed with caution.

Breast cancer is the most frequent cancer occurring in women. Ten percent of these cancers are considered hereditary. Among them, 30% are attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. Other genes of lower penetrance are also known, explaining together up to 40% of the hereditary risk of breast cancer. New techniques, such as next-generation sequencing, allow the simultaneous analysis of multiple genes in a cost-effective way. As a logical consequence, gene panel testing is entering clinical practice with the promise of personalized care. We however advocate that gene panel testing is not ready for non-specialist clinical use, as it generates many variants of unknown significance and includes more genes than are presently considered clinically useful. We hereby review the data for each gene that can change the risk management of patients carrying a pathogenic variant.

Rapid and fatal acute heart failure induced by pazopanib.

Tyrosine kinase inhibitors, represented by sunitinib, sorafenib, axitinib and pazopanib, are emerging molecules harbouring antitumoural efficacy in multiple neoplasia. We report the case of a 51-year-old woman with right thoracic sarcoma who developed fatal heart failure on pazopanib. The patient had no cardiovascular risk factor, except previous exposure to anthracycline, and her cardiac function was normally controlled before initiating the pazopanib. Despite a rapid tumour response, fatigue rapidly appeared, requiring treatment interruption 2 weeks after pazopanib introduction. After clinical improvement, the pazopanib was reintroduced at reduced dose; however, a few days later, our patient was admitted for worsening dyspnoea and fatigue. Pulmonary embolism was excluded as was pleuropericardial effusion. Brain natriuretic peptide was the only laboratory abnormality, and echocardiography revealed acute and severe heart failure. The patient died despite pazopanib arrest and inotropic support.