Showing papers by "Oscar M. Rueda published in 2021"

Landscapes of cellular phenotypic diversity in breast cancer xenografts and their impact on drug response

Abstract: The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.

Abstract: DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors. Understanding the molecular mechanisms underlying DNA methylation in cancer and its clinical relevance remains crucial. Here, the authors study RRBS-based profiles of 1538 breast tumours and 244 normal breast tissues from the METABRIC cohort and report epigenomic instability and cis-regulatory effects.

Deciphering the signaling network of breast cancer improves drug sensitivity prediction.

Abstract: One goal of precision medicine is to tailor effective treatments to patients' specific molecular markers of disease. Here, we used mass cytometry to characterize the single-cell signaling landscapes of 62 breast cancer cell lines and five lines from healthy tissue. We quantified 34 markers in each cell line upon stimulation by the growth factor EGF in the presence or absence of five kinase inhibitors. These data-on more than 80 million single cells from 4,000 conditions-were used to fit mechanistic signaling network models that provide insight into how cancer cells process information. Our dynamic single-cell-based models accurately predicted drug sensitivity and identified genomic features associated with drug sensitivity, including a missense mutation in DDIT3 predictive of PI3K-inhibition sensitivity. We observed similar trends in genotype-drug sensitivity associations in patient-derived xenograft mouse models. This work provides proof of principle that patient-specific single-cell measurements and modeling could inform effective precision medicine strategies.

NRG1 fusions in breast cancer

Abstract: NRG1 gene fusions may be clinically actionable, since cancers carrying the fusion transcripts can be sensitive to tyrosine kinase inhibitors. The NRG1 gene encodes ligands for the HER2(ERBB2)-ERBB3 heterodimeric receptor tyrosine kinase, and the gene fusions are thought to lead to autocrine stimulation of the receptor. The NRG1 fusion expressed in the breast cancer cell line MDA-MB-175 serves as a model example of such fusions, showing the proposed autocrine loop and exceptional drug sensitivity. However, its structure has not been properly characterised, its oncogenic activity has not been fully explained, and there is limited data on such fusions in breast cancer. We analysed genomic rearrangements and transcripts of NRG1 in MDA-MB-175 and a panel of 571 breast cancers. We found that the MDA-MB-175 fusion—originally reported as a DOC4(TENM4)-NRG1 fusion, lacking the cytoplasmic tail of NRG1—is in reality a double fusion, PPP6R3-TENM4-NRG1, producing multiple transcripts, some of which include the cytoplasmic tail. We hypothesise that many NRG1 fusions may be oncogenic not for lacking the cytoplasmic domain but because they do not encode NRG1’s nuclear-localised form. The fusion in MDA-MB-175 is the result of a very complex genomic rearrangement, which we partially characterised, that creates additional expressed gene fusions, RSF1-TENM4, TPCN2-RSF1, and MRPL48-GAB2. We searched for NRG1 rearrangements in 571 breast cancers subjected to genome sequencing and transcriptome sequencing and found four cases (0.7%) with fusions, WRN-NRG1, FAM91A1-NRG1, ARHGEF39-NRG1, and ZNF704-NRG1, all splicing into NRG1 at the same exon as in MDA-MB-175. However, the WRN-NRG1 and ARHGEF39-NRG1 fusions were out of frame. We identified rearrangements of NRG1 in many more (8% of) cases that seemed more likely to inactivate than to create activating fusions, or whose outcome could not be predicted because they were complex, or both. This is not surprising because NRG1 can be pro-apoptotic and is inactivated in some breast cancers. Our results highlight the complexity of rearrangements of NRG1 in breast cancers and confirm that some do not activate but inactivate. Careful interpretation of NRG1 rearrangements will therefore be necessary for appropriate patient management.

Positive correlation between transcriptomic stemness and PI3K/AKT/mTOR signaling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype

Abstract: A PI3Kα-selective inhibitor has recently been approved for use in breast tumors harboring mutations in PIK3CA, the gene encoding p110α. Preclinical studies have suggested that the PI3K/AKT/mTOR signaling pathway influences stemness, a dedifferentiation-related cellular phenotype associated with aggressive cancer. However, to date, no direct evidence for such a correlation has been demonstrated in human tumors. In two independent human breast cancer cohorts, encompassing nearly 3,000 tumor samples, transcriptional footprint-based analysis uncovered a positive linear association between transcriptionally-inferred PI3K/AKT/mTOR signaling scores and stemness scores. Unexpectedly, stratification of tumors according to PIK3CA genotype revealed a "biphasic" relationship of mutant PIK3CA allele dosage with these scores. Relative to tumor samples without PIK3CA mutations, the presence of a single copy of a hotspot PIK3CA variant was associated with lower PI3K/AKT/mTOR signaling and stemness scores, whereas the presence of multiple copies of PIK3CA hotspot mutations correlated with higher PI3K/AKT/mTOR signaling and stemness scores. This observation was recapitulated in a human cell model of heterozygous and homozygous PIK3CAH1047R expression. Collectively, our analysis (1) provides evidence for a signaling strength-dependent PI3K-stemness relationship in human breast cancer; (2) supports evaluation of the potential benefit of patient stratification based on a combination of conventional PI3K pathway genetic information with transcriptomic indices of PI3K signaling activation.

FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1

Abstract: FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44–4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models. FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Germline APOBEC3B deletion increases somatic hypermutation in Asian breast cancer that is associated with Her2 subtype, PIK3CA mutations, and immune activation

Abstract: A 30-kb deletion that eliminates the coding region of APOBEC3B (A3B) is >5 times more common in women of Asian compared to European descent. This polymorphism creates a chimera with the APOBEC3A (A3A) coding region and A3B 3'UTR, and is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between the A3B deletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germline A3B deletion polymorphism leads to expression of the A3A-B hybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless of A3B germline status, were associated with the Her2 molecular subtype and PIK3CA mutations. Compared to non-hypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germline A3B deletion polymorphism, via the A3A-B hybrid isoform, contributes to APOBEC-mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless of A3B background, may be an important clinical biomarker for Asian breast cancers. This article is protected by copyright. All rights reserved.

Predicting COVID-19 progression from diagnosis to recovery or death linking primary care and hospital records in Castilla y León (Spain).

Abstract: This paper analyses COVID-19 patients' dynamics during the first wave in the region of Castilla y Leon (Spain) with around 2.4 million inhabitants using multi-state competing risk survival models. From the date registered as the start of the clinical process, it is assumed that a patient can progress through three intermediate states until reaching an absorbing state of recovery or death. Demographic characteristics, epidemiological factors such as the time of infection and previous vaccinations, clinical history, complications during the course of the disease and drug therapy for hospitalised patients are considered as candidate predictors. Regarding risk factors associated with mortality and severity, consistent results with many other studies have been found, such as older age, being male, and chronic diseases. Specifically, the hospitalisation (death) rate for those over 69 is 27.2% (19.8%) versus 5.3% (0.7%) for those under 70, and for males is 14.5%(7%) versus 8.3%(4.6%)for females. Among patients with chronic diseases the highest rates of hospitalisation are 26.1% for diabetes and 26.3% for kidney disease, while the highest death rate is 21.9% for cerebrovascular disease. Moreover, specific predictors for different transitions are given, and estimates of the probability of recovery and death for each patient are provided by the model. Some interesting results obtained are that for patients infected at the end of the period the hazard of transition from hospitalisation to ICU is significatively lower (p < 0.001) and the hazard of transition from hospitalisation to recovery is higher (p < 0.001). For patients previously vaccinated against pneumococcus the hazard of transition to recovery is higher (p < 0.001). Finally, internal validation and calibration of the model are also performed.

TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis

Abstract: Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Transcriptomically-inferred PI3K activity and stemness show a counterintuitive correlation with PIK3CA genotype in breast cancer

Abstract: A PI3Kα-selective inhibitor has recently been approved for use in breast tumours harbouring mutations in PIK3CA, the gene encoding PI3Kα. Preclinical studies have suggested that the PI3K/AKT/mTORC1 signalling pathway influences stemness, a dedifferentiation-related cellular phenotype associated with aggressive cancer. No direct evidence for such a correlation has been demonstrated to date in human tumours. In two independent human breast cancer cohorts, encompassing nearly 3,000 tumour samples, transcriptional footprint-based analysis uncovered a positive linear association between transcriptionally-inferred PI3K signalling scores and stemness scores. Unexpectedly, stratification of tumours according to PIK3CA genotype revealed a “biphasic” relationship of mutant PIK3CA allele dosage with these scores. Relative to tumour samples without PIK3CA mutations, the presence of a single copy of a hotspot PIK3CA variant was associated with lower PI3K signalling and stemness scores, whereas tumours with multiple copies of PIK3CA hotspot mutations showed higher PI3K signalling and stemness scores. This observation was recapitulated in a human cell model of heterozygous and homozygous PIK3CAH1047R expression. Collectively, our analysis provides evidence for a signalling strength-dependent PI3K-stemness relationship in human breast cancer, which may aid future patient stratification for PI3K-targeted therapies.