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New predictive biomarkers identified for checkpoint inhibitors: antigenicity and T-cell inflamed gene expression profile are complementary
The current development of immune checkpoint modulatory treatments has shown durable responses in the treatment of multiple cancer types.1 However, predictive biomarkers beyond PD-1 ligand 1 (PD-L1) expression and high microsatellite instability (MSI-H) to stratify patients and identify those who could benefit of these therapies are needed. In this sense, a recent study published in Science by Cristescu et al 2 describes the potential usefulness of combining the tumour mutational burden (TMB) and the T cell-inflamed gene expression profile (GEP) to jointly predict clinical response to pembrolizumab.
Both PD-L1 and the GEP represent a T cell-inflamed tumour microenvironment (TME), whereas TMB and MSI-H are indirect measures of tumour antigenicity generated by somatic tumour mutations. In the mentioned study, TMB was evaluated by whole-exome sequencing (WES) of germline and tumour DNA, and the T cell-inflamed GEP was analysed by targeted gene expression profiling of tumour RNA (with the NanoString platform) from formalin fixed, paraffin-embedded pretreatment samples. To assess the individual and joint clinical utility of TMB and GEP, patients were stratified in four biomarker-defined clinical response groups based on predefined cut-offs for TMB and GEP.
Objective response rates were strongest in patients with GEPhi TMBhi (37%–57%), moderate in those with GEPhi TMBlo (12% to 35%) and GEPlo TMBhi (11%–42%) and reduced or absent in those with GEPlo TMBlo (0%–9%). Additionally, longer PFS were seen in patients with higher levels of both TMB and GEP. Findings were comparable when TMB and PD-L1 expression were jointly assessed. Interestingly, more than 300 patient samples with advanced solid tumours and melanoma across 22 tumour types from four KEYNOTE clinical trials were included and analysed. Samples were assessed separately in four cohorts: one discovery, one pan-tumour validation and two single summary cohorts. Results were validated through transcriptome and exome TCGA database analyses including >6300 tumour samples. Within TCGA database, GEP and TMB again had a low correlation, demonstrating the potential to jointly stratify transcriptomic and genomic features across cancer types. Specific gene expression patterns reflective of TME biology showed significant associations with TMB, GEP or both. PD-L1 immunohistochemistry scores were also analysed showing that responses in patients who had both TMBhi and greater PD-L1 expression were greater than those in patients who had low levels of both TMB and PD-L1 expression.
In summary, this retrospective pan-tumour genomic study shows that TMB and inflammatory biomarkers (T cell-inflamed GEP and PD-L1 expression) can jointly stratify human cancers into responders and no responders to pembrolizumab analysing different features of the tumour immunobiology. From this rational basis, prospective studies are needed to validate their usefulness as biomarkers for precision cancer immunotherapy.
Actionable activating oncogenic ERRB2/HER2 transmembrane and juxtamembrane domain mutations
Amplification and overexpression of the HER2 gene is one of the main tumour drivers in breast and gastric cancer as well as in other solid tumours. Its established role as a potent oncogene has made HER2 a major target for therapy. While overexpression remains a major mechanism of HER2-driven tumorigenesis, recent large-scale sequencing efforts have identified oncogenic mutations in the extracellular domain and in the kinase domain. Mutations in the transmembrane domain and juxtamembrane domain have also been reported, although at a low frequency and their relevance in oncogenesis is not fully understood.
In an important article recently published by Pahuja et al in Cancer Cell,3 exomes deriving from 111.176 patients, representing 54 disease groups and about 400 cancer types were analysed. HER2 mutations were detected in 3851 tumours (3.5%). The role of different transmembrane domain and juxtamembrane domain mutations was investigated. These included G660D, V659E, R678Q, Q709L, V659 and G660 as well as part of the N-terminal S656-xxx-G660 motif in the HER2 transmembrane domain. Those mutations are considered important for receptor dimerisation, kinase activation and signalling. A striking relationship between the chemical nature of the transmembrane domain mutations and the potency with which they activate HER2 was observed. In vitro, trastuzumab, was found to be effective in blocking cell proliferation and survival of HER2 V659E, G660D, G660R, L663P, R678Q and Q709L while pertuzumab was not. Despite the J juxtamembrane domain plays a critical role in EGFR activation and is highly conserved in HER2,4 5 no benefit was obtained when cetuximab or erlotinib were used. Among the small-molecule kinase inhibitors tested, neratinib, afatinib and lapatinib were found to be active in blocking cell viability and survival. To evaluate the potential role also of germline mutations, WES using DNA obtained from peripheral blood samples from the three patients with NSCLC belonging to the same family was performed. In one patient, a G660D mutation was confirmed in paraffin embedded tissue. After erlotinib failure, this patient was treated as fourth line with afatinib. A 21% reduction in the tumour volume was observed after afatinib treatment. This control effect was lasted for over 15 months. These results suggest that the HER2 G660D germline mutation was the main driver in this specific tumour, indicating that patients with this molecular alteration can benefit from HER2-targeted therapy. Although further research is needed, this investigation opens the possibility for new treatments for patients diagnosed with solid tumours harbouring HER2 mutations, contributing to precision medicine in oncology.
Short-term patient-derived ovarian cancer organoids are useful to assess sensitivity to PARP inhibitors and other targetable DNA repair alterations
Precision Oncology goes beyond complex molecular or genomic studies to detect potential actionable targets. It also includes the use of patient-derived xenografts in mice and organoids to facilitate functional studies, which may complement or even improve on molecular tests for determining a predictive value on the response to certain anticancer agents. Organoids are three-dimensional cell cultures directly obtained from tumour tissues, which are essentially easier, faster and cheaper to develop than patient-derived organoids. Other potential advantages of organoids include the representation of clonal heterogeneity as well as the microenvironment containing immune cells.
In an elegant study, performed by a group of investigators belonging to different centres from the University of Harvard in Boston and the University of Washington at Seattle, short-term organoid cultures were generated from 22 patients with high-grade serous ovarian cancer.6 The authors hypothesised that patient-derived organoids could be useful to assess DNA repair activity and to determine which repair defects could confer sensitivity to some DNA repair drugs, as single agents or in combinations. In this series, organoids were generated from solid tumours extracted from primary, metastatic or recurrent sites. All parent tumours and derived organoids presented common features of high-grade serous ovarian cancers, such as nuclear pleomorphism, dense chromatin and prominent nucleoli. After WES, a median of 98.2% of mutations identified in the tumours were also found in their derived organoids. The high level of multidimensional concordance between tumours and high-grade serous ovarian cancer derived makes them a good model for evaluating DNA damage repair defects.
The organoid cultures were exposed to the PARP inhibitor olaparib due to the fact that olaparib sensitivity relates to homologous recombination defects (HRD), but also to carboplatin, a well-known replication fork stalling agent, and other CHK1 inhibitors or ATR inhibitors. Only 6% of the organoids tested were olaparib sensitive, indicating a low rate of HRD, despite the initial molecular findings. However, sensitivity to carboplatin, CHK1 or ATM inhibitors or even gemcitabine was much higher and was seen in 41%, 47%, 44% and 82% of them, respectively. This finding also indicates that HRD are not required to observe a response to the other agents. The functional results derived from organoid experiments indicate that a BRCA1/2 mutation is neither needed nor sufficient for conferring PARP sensitivity. The authors also underline how functional assays may predict responses to treatment. Moreover, organoids do exhibit clonal heterogeneity and functional assays may be useful to address the effects of heterogeneity in tumour response, even helping to select rational drug combinations, by defining potential synergies on antagonism of different anticancer agents of clinical interest.
These organoids match genetically and functionally those tumours from which they are derived. Although further prospective trials are needed, the current study strongly suggests that organoids derived from high-grade serous ovarian tumours, which do properly grow in only 7–10 days, could be a very useful tool to predict clinical response of individual patients, tailoring more efficiently the treatment of individual patients. These organoids match genetically and functionally those tumours from which they are derived.
Contributors All authors contributed equally.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; internally peer reviewed.
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