Third-line treatment patterns in HER2-positive metastatic breast cancer: a retrospective analysis of real-world data in Canada.

There is an increasing demand for real-world data pertaining to the usage of cancer treatments, especially in settings where no standard treatment is specifically recommended. This study presents the first real-world analysis of third-line treatment patterns in HER2-positive metastatic breast cancer (mBC) patients in Canada. The purpose was to assess evolution of clinical practice and identify unmet needs in post-second-line therapy. Retrospective data from medical records of 66 patients who received third-line treatment before 31st October 2018, and data from 56 patients who received third-line treatment after this date, extracted from the Personalize My Treatment (PMT) cancer patient registry, were analyzed. In the first cohort, the study revealed heterogeneity in the third-line setting, with trastuzumab, lapatinib, and T-DM1 being the main treatment options. Even though data were collected before the wide availability of tucatinib, neratinib and trastuzumab deruxtecan in Canada, the PMT cohort revealed the emergence of new therapeutic combinations and a shift from lapatinib usage to T-DM1 choice was observed. These findings underscore the evolving nature of third-line treatment strategies in Canada, a facet that is intrinsically tied to the availability of new drugs. The absence of a consensus on post-second-line treatment highlights the pressing need for more efficient therapeutic alternatives beyond the currently available options. This study not only offers valuable insights into the present landscape of third-line treatment in Canada but validates the significance and effectiveness of the PMT registry as a tool for generating pan-Canadian real-world evidence in oncology and its capacity to provide information on evolution of therapeutic practices.

A Phase II Exploratory Study to Identify Biomarkers Predictive of Clinical Response to Regorafenib in Patients with Metastatic Colorectal Cancer Who Have Failed First-Line Therapy.

Single-agent regorafenib is approved in Canada for metastatic colorectal cancer (mCRC) patients who have failed previous lines of therapy. Identifying prognostic biomarkers is key to optimizing therapeutic strategies for these patients. In this clinical study (NCT01949194), we evaluated the safety and efficacy of single-agent regorafenib as a second-line therapy for mCRC patients who received it after failing first-line therapy with an oxaliplatin or irinotecan regimen with or without bevacizumab. Using various omics approaches, we also investigated putative biomarkers of response and resistance to regorafenib in metastatic lesions and blood samples in the same cohort. Overall, the safety profile of regorafenib seemed similar to the CORRECT trial, where regorafenib was administered as ≥ 2 lines of therapy. While the mutational landscape showed typical mutation rates for the top five driver genes (APC, KRAS, BRAF, PIK3CA, and TP53), KRAS mutations were enriched in intrinsically resistant lesions. Additional exploration of genomic-phenotype associations revealed several biomarker candidates linked to unfavorable prognoses in patients with mCRC using various approaches, including pathway analysis, cfDNA profiling, and copy number analysis. However, further research endeavors are necessary to validate the potential utility of these promising genes in understanding patients' responses to regorafenib treatment.

Copy number and transcriptome alterations associated with metastatic lesion response to treatment in colorectal cancer.

BACKGROUND: Therapeutic resistance is the main cause of death in metastatic colorectal cancer. To investigate genomic plasticity, most specifically of metastatic lesions, associated with response to first-line systemic therapy, we collected longitudinal liver metastatic samples and characterized the copy number aberration (CNA) landscape and its effect on the transcriptome. METHODS: Liver metastatic biopsies were collected prior to treatment (pre, n = 97) and when clinical imaging demonstrated therapeutic resistance (post, n = 43). CNAs were inferred from whole exome sequencing and were correlated with both the status of the lesion and overall patient progression-free survival (PFS). We used RNA sequencing data from the same sample set to validate aberrations as well as independent datasets to prioritize candidate genes. RESULTS: We identified a significantly increased frequency gain of a unique CN, in liver metastatic lesions after first-line treatment, on chr18p11.32 harboring 10 genes, including TYMS, which has not been reported in primary tumors (GISTIC method and test of equal proportions, FDR-adjusted p = 0.0023). CNA lesion profiles exhibiting different treatment responses were compared and we detected focal genomic divergences in post-treatment resistant lesions but not in responder lesions (two-tailed Fisher's Exact test, unadjusted p ≤ 0.005). The importance of examining metastatic lesions is highlighted by the fact that 15 out of 18 independently validated CNA regions found to be associated with PFS in this study were only identified in the metastatic lesions and not in the primary tumors. CONCLUSION: This investigation of genomic-phenotype associations in a large colorectal cancer liver metastases cohort identified novel molecular features associated with treatment response, supporting the clinical importance of collecting metastatic samples in a defined clinical setting.

Discovery of a putative blood-based protein signature associated with response to ALK tyrosine kinase inhibition.

BACKGROUND: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. METHODS: Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. RESULTS: Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 < PFS < 24 months (n = 10)] and poor responders [PFS ≤ 3 months (n = 5)]. Several proteins were identified as differentially expressed between long-term responders and poor responders, including DPP4, KIT and LUM. Next, using machine learning algorithms, we evaluated the classification potential of 40 proteins. Finally, by integrating the different analytic methods, we selected 22 proteins as potential candidates for a blood-based prognostic signature of response to crizotinib in NSCLC patients harboring ALK fusion. CONCLUSION: In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy.Trial registration ClinicalTrials.gov, NCT02041468. Registered 22 January 2014, https://clinicaltrials.gov/ct2/show/NCT02041468?term=NCT02041468&rank=1.

Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data.

: Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRASwildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRASwildtype and KRASG12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRASG12V. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In KRASG12V-mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRASG12V but 90% KRASwildtype. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.

Multiplexed real-time polymerase chain reaction cell-free DNA assay as a potential method to monitor stage IV colorectal cancer.

BACKGROUND: Liquid biopsy is a new area in cancer diagnostics that measures cell-free DNA in plasma from tumor that may serve as a monitoring tool in colorectal cancer patients. METHODS: Multiplexed real-time polymerase chain reaction based on multicopy retro-transposable elements (targeting 80 base pair and 265 base pair sequences and an internal-positive-control) was used to evaluate the ability of cell-free DNA concentration and DNA Integrity Index to discriminate cancer from healthy patients. A cohort of 40 healthy controls and 39 stage IV colorectal patient's plasma were interrogated. The potency of each biomarker was measured by using receiver operating characteristic curves and derived area under the curve measures. RESULTS: Significant differences in cell-free DNA concentration and DNA integrity index were observed between controls and stage IV patients with a limit of detection <0.1 pg/µL. Investigation of the ability of both biomarker candidates to differentiate cancer from healthy patients showed an area under the curve of 0.9891 and 0.9859 for 80 base pair and 265 amplicons respectively and 0.8603 for DNA integrity index-265/80. CONCLUSIONS: After establishing differences in cell-free DNA levels between healthy and treated and untreated stage IV patients, the multiplexed real-time polymerase chain reaction measurements of retro-transposable elements in cancer patient plasma potentially possess the ability to monitor therapy responsiveness in near real time.

Analysis of the Genomic Landscape in ALK+ NSCLC Patients Identifies Novel Aberrations Associated with Clinical Outcomes.

Rearrangements in the anaplastic lymphoma kinase (ALK) gene are found in approximately 5% of non-small cell lung carcinoma (NSCLC). Here, we present a comprehensive genomic landscape of 11 patients with ALK+ NSCLC and investigate its relationship with response to crizotinib. Using whole-exome sequencing and RNAseq data, we identified four rare ALK fusion partners (HIP1, GCC2, ERC1, and SLC16A7) and one novel partner (CEP55). At the mutation level, TP53 was the most frequently mutated gene and was only observed in patients with the shortest progression-free survival (PFS). Of note, only 4% of the genes carrying mutations are present in more than 1 patient. Analysis of somatic copy number aberrations (SCNA) demonstrated that a gain in EML4 was associated with longer PFS, and a loss of ALK or gain in EGFR was associated with shorter PFS. This study is the first to report a comprehensive view of the ALK+ NSCLC copy number landscape and to identify SCNA regions associated with clinical outcome. Our data show the presence of TP53 mutation as a strong prognostic indication of poor clinical response in ALK+ NSCLC. Furthermore, new and rare ALK fusion partners were observed in this cohort, expanding our knowledge in ALK+ NSCLC.

Low levels of IGFBP7 expression in high-grade serous ovarian carcinoma is associated with patient outcome.

BACKGROUND: Insulin-like growth factor binding protein 7 (IGFBP7) has been suggested to act as a tumour suppressor gene in various human cancers, yet its role in epithelial ovarian cancer (EOC) has not yet been investigated. We previously observed that IGFBP7 was one of several genes found significantly upregulated in an EOC cell line model rendered non-tumourigenic as consequence of genetic manipulation. The aim of the present study was to investigate the role of IGFBP7 in high-grade serous ovarian carcinomas (HGSC), the most common type of EOC. METHODS: We analysed IGFBP7 gene expression in 11 normal ovarian surface epithelial cells (NOSE), 79 high-grade serous ovarian carcinomas (HGSC), and seven EOC cell lines using a custom gene expression array platform. IGFBP7 mRNA expression profiles were also extracted from publicly available databases. Protein expression was assessed by immunohistochemistry of 175 HGSC and 10 normal fallopian tube samples using tissue microarray and related to disease outcome. We used EOC cells to investigate possible mechanisms of gene inactivation and describe various in vitro growth effects of exposing EOC cell lines to human recombinant IGFBP7 protein and conditioned media. RESULTS: All HGSCs exhibited IGFBP7 expression levels that were significantly (p = 0.001) lower than the mean of the expression value of NOSE samples and that of a whole ovary sample. IGFBP7 gene and protein expression were lower in tumourigenic EOC cell lines relative to a non-tumourigenic EOC cell line. None of the EOC cell lines harboured a somatic mutation in IGFBP7, although loss of heterozygosity (LOH) of the IGFBP7 locus and epigenetic methylation silencing of the IGFBP7 promoter was observed in two of the cell lines exhibiting loss of gene/protein expression. In vitro functional assays revealed an alteration of the EOC cell migration capacity. Protein expression analysis of HGSC samples revealed that the large majority of tumour cores (72.6%) showed low or absence of IGFBP7 staining and revealed a significant correlation between IGFBP7 protein expression and a prolonged overall survival (p = 0.044). CONCLUSION: The low levels of IGFPB7 in HGSC relative to normal tissues, and association with survival are consistent with a purported role in tumour suppressor pathways.

Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics.

BACKGROUND: The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer. METHOD: The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested. RESULTS: BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing. Spectral karyotyping revealed a largely normal diploid karyotype (in greater than 95% of cells) with a visibly shorter chromosome 20 contig. High density SNP array analysis also revealed few genomic anomalies in BIN-67 cells, which included loss of heterozygosity of an estimated 16.7 Mb interval on chromosome 20. SNP array analyses of four SCCOHT samples also indicated a low frequency of genomic anomalies in the majority of cases. Although resistant to platinum chemotherapeutic drugs, BIN-67 cell viability in vitro was reduced by > 75% after infection with oncolytic viruses. CONCLUSIONS: These results show that SCCOHT differs from high-grade serous carcinomas by exhibiting few chromosomal anomalies and lacking TP53 mutations. Although BIN-67 cells are resistant to standard chemotherapeutic agents, their sensitivity to oncolytic viruses suggests that their therapeutic use in SCCOHT should be considered.

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer.

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.

The genomic landscape of TP53 and p53 annotated high grade ovarian serous carcinomas from a defined founder population associated with patient outcome.

High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.

DeltaNp63 is essential for epidermal commitment of embryonic stem cells.

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.

Embryonic stem cells as a cellular model for neuroectodermal commitment and skin formation.

Embryonic stem (ES) cells can be differentiated into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells were able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1(+) neural precursors to undergo specific apoptosis while inducing epidermal differentiation through DeltaNp63 gene activation. We further demonstrated that DeltaNp63 enhances ES-derived ectodermal cell proliferation and is necessary for epidermal commitment. This unique cellular model further provides a powerful tool for identifying the molecular mechanisms controlling normal skin development and for investigating p63-ectodermal dysplasia human congenital pathologies.

Key role of p63 in BMP-4-induced epidermal commitment of embryonic stem cells.

In vivo studies, transgenic and knock-out mice have demonstrated that p63 isoforms play pivotal roles in ectodermal and epidermal development but their respective function remains highly controversial. Since embryonic stem (ES) cells can be differentiated into many cell types, they represent an effective tool to recapitulate in vitro the main steps of embryonic development. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells and clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1(+) neural precursors to undergo specific apoptosis while inducing epidermal differentiation through DeltaNp63 gene activation. DeltaNp63 is not required for ectodermal fate but enhances ES-derived ectodermal cell proliferation and epidermal commitment. This unique cellular model should further provide a powerful tool for identifying the molecular mechanisms controlling normal skin development and in p63-ectodermal dysplasia human congenital pathologies.

[Embryonic stem cells and epidermal commitment]. / Cellules souches embryonnaires et lignage épidermique.

Embryonic stem (ES) cells can be cultured indefinitely, differentiated into many cell types in vitro, thus providing a potentially unlimited supply of cells for cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells are able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. This model demons- trates that ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation, clarifies the role of the morphogen BMP-4 in the binary neuroectodermal choice and provides a powerful tool for the study of molecular mechanisms controlling skin development and multipotent epidermal stem cell properties. Its potential for cutaneous cell therapy and dermatocosmetological applications is discussed.