Integrative whole-genome and transcriptome analysis of HER2-amplified metastatic breast cancer.

BACKGROUND: In breast cancer, the advent of anti-HER2 therapies has made HER2+ tumors a highly relevant subgroup. However, the exact characteristics which prohibit clinical response to anti-HER2 therapies and drive disease progression are not yet fully known. Integrative whole-genome and transcriptomic sequencing data from both primary and metastatic HER2-positive breast cancer will enhance our understanding of underlying biological processes. METHODS: Here, we used WGS and RNA sequencing data of 700 metastatic breast tumors, of which 68 being HER2+, to search for specific genomic features of HER2+ disease and therapy resistance. Furthermore, we integrated results with transcriptomic data to associate tumors exhibiting a HER2+-specific gene expression profile with ERBB2 mutation status, prior therapy and relevant gene expression signatures. RESULTS: Overall genomic profiles of primary and metastatic HER2+ breast cancers were similar, and no specific acquired genomics traits connected to prior anti-HER2 treatment were observed. However, specific genomic features were predictive of progression-free survival on post-biopsy anti-HER2 treatment. Furthermore, a HER2-driven expression profile grouped HER2-amplified tumors with ERBB2-mutated cases and cases without HER2 alterations. The latter were reported as ER positive in primary disease, but the metastatic biopsy showed low ESR1 expression and upregulation of the MAPK pathway, suggesting transformation to ER independence. CONCLUSIONS: In summary, although the quantity of variants increased throughout HER2-positive breast cancer progression, the genomic composition remained largely consistent, thus yielding no new major processes beside those already operational in primary disease. Our results suggest that integrated genomic and transcriptomic analyses may be key in establishing therapeutic options.

Genomic Alterations Associated with Estrogen Receptor Pathway Activity in Metastatic Breast Cancer Have a Differential Impact on Downstream ER Signaling.

Mutations in the estrogen receptor gene (ESR1), its transcriptional regulators, and the mitogen-activated protein kinase (MAPK) pathway are enriched in patients with endocrine-resistant metastatic breast cancer (MBC). Here, we integrated whole genome sequencing with RNA sequencing data from the same samples of 101 ER-positive/HER2-negative MBC patients who underwent a tumor biopsy prior to the start of a new line of treatment for MBC (CPCT-02 study, NCT01855477) to analyze the downstream effects of DNA alterations previously linked to endocrine resistance, thereby gaining a better understanding of the associated mechanisms. Hierarchical clustering was performed using expression of ESR1 target genes. Genomic alterations at the DNA level, gene expression levels, and last administered therapy were compared between the identified clusters. Hierarchical clustering revealed two distinct clusters, one of which was characterized by increased expression of ESR1 and its target genes. Samples in this cluster were significantly enriched for mutations in ESR1 and amplifications in FGFR1 and TSPYL. Patients in the other cluster showed relatively lower expression levels of ESR1 and its target genes, comparable to ER-negative samples, and more often received endocrine therapy as their last treatment before biopsy. Genes in the MAPK-pathway, including NF1, and ESR1 transcriptional regulators were evenly distributed. In conclusion, RNA sequencing identified a subgroup of patients with clear expression of ESR1 and its downstream targets, probably still benefiting from ER-targeting agents. The lower ER expression in the other subgroup might be partially explained by ER activity still being blocked by recently administered endocrine treatment, indicating that biopsy timing relative to endocrine treatment needs to be considered when interpreting transcriptomic data.

Tumor-agnostic ctDNA levels by mFAST-SeqS in first-line HR-positive, HER2 negative metastatic breast cancer patients as a biomarker for survival.

This prospective cohort study reports aneuploidy score by mFast-SeqS as a strong prognostic marker in MBC patients. mFAST-SeqS is an affordable and easily implementable method for the assessment of total ctDNA levels and, as such, provides an alternative prognostic tool. One mixed cohort (cohort A, n = 45) starting any type of treatment in any line of therapy and one larger cohort (cohort B, n = 129) consisting of patients starting aromatase inhibitors (AI) as first-line therapy were used. mFAST-SeqS was performed using plasma of blood in which CTCs (CellSearch) were enumerated. The resulting aneuploidy score was correlated with categorized CTC count and associated with outcome. The aneuploidy score was significantly correlated with CTC count, but discordance was observed in 31.6% when applying cut-offs of 5. In both cohorts, aneuploidy score was a significant prognostic marker for both PFS and OS. In the Cox regression models, the HR for aneuploidy score for PFS was 2.52 (95% CI: 1.56-4.07), and the HR for OS was 2.37 (95% CI: 1.36-4.14). Results presented here warrant further investigations into the clinical utility of this marker in MBC patients.

Breast cancer genomes from CHEK2 c.1100delC mutation carriers lack somatic TP53 mutations and display a unique structural variant size distribution profile.

BACKGROUND: CHEK2 c.1100delC was the first moderate-risk breast cancer (BC) susceptibility allele discovered. Despite several genomic, transcriptomic and functional studies, however, it is still unclear how exactly CHEK2 c.1100delC promotes tumorigenesis. Since the mutational landscape of a tumor reflects the processes that have operated on its development, the aim of this study was to uncover the somatic genomic landscape of CHEK2-associated BC. METHODS: We sequenced primary BC (pBC) and normal genomes of 20 CHEK2 c.1100delC mutation carriers as well as their pBC transcriptomes. Including pre-existing cohorts, we exhaustively compared CHEK2 pBC genomes to those from BRCA1/2 mutation carriers, those that displayed homologous recombination deficiency (HRD) and ER- and ER+ pBCs, totaling to 574 pBC genomes. Findings were validated in 517 metastatic BC genomes subdivided into the same subgroups. Transcriptome data from 168 ER+ pBCs were used to derive a TP53-mutant gene expression signature and perform cluster analysis with CHEK2 BC transcriptomes. Finally, clinical outcome of CHEK2 c.1100delC carriers was compared with BC patients displaying somatic TP53 mutations in two well-described retrospective cohorts totaling to 942 independent pBC cases. RESULTS: BC genomes from CHEK2 mutation carriers were most similar to ER+ BC genomes and least similar to those of BRCA1/2 mutation carriers in terms of tumor mutational burden as well as mutational signatures. Moreover, CHEK2 BC genomes did not show any evidence of HRD. Somatic TP53 mutation frequency and the size distribution of structural variants (SVs), however, were different compared to ER+ BC. Interestingly, BC genomes with bi-allelic CHEK2 inactivation lacked somatic TP53 mutations and transcriptomic analysis indicated a shared biology with TP53 mutant BC. Moreover, CHEK2 BC genomes had an increased frequency of > 1 Mb deletions, inversions and tandem duplications with peaks at specific sizes. The high chromothripsis frequency among CHEK2 BC genomes appeared, however, not associated with this unique SV size distribution profile. CONCLUSIONS: CHEK2 BC genomes are most similar to ER+ BC genomes, but display unique features that may further unravel CHEK2-driven tumorigenesis. Increased insight into this mechanism could explain the shorter survival of CHEK2 mutation carriers that is likely driven by intrinsic tumor aggressiveness rather than endocrine resistance.

GRHL2-controlled gene expression networks in luminal breast cancer.

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract.

HER2-low breast cancer shows a lower immune response compared to HER2-negative cases.

Currently, the human epidermal growth factor receptor 2 (HER2) status of breast cancer is classified dichotomously as negative or positive to select patients for HER2-targeted therapy. However, with the introduction of novel treatment options, it is important to get more insight in the biology of cancers with low HER2 expression. Therefore, we studied several clinicopathologic characteristics in relation to the level of HER2 expression (HER2- versus HER2low). We used a well-documented cohort of breast cancer patients (n = 529), with available tissue microarrays and Affymetrix mRNA expression data. HER2 status was scored as negative (immunohistochemistry 0) or low (immunohistochemistry 1 + or 2 + without amplification). We associated HER2 status with several clinicopathologic characteristics, gene-expression data and survival, stratified for estrogen receptor (ER) status. Overall, breast cancers were scored as HER2- (n = 429) or HER2low (n = 100). Within the ER+ cohort (n = 305), no significant associations were found between the HER2 groups and clinicopathologic features. However, HER2low tumors showed several differentially expressed genes compared to HER2- cases, including genes that are associated with worse outcome and depletion of immunity. In ER- cases (n = 224), HER2low status was significantly associated with increased regional nodal positivity, lower density of tumor infiltrating lymphocyte and a lower protein expression of Ki-67 and EGFR compared to HER2- cases. After multivariate analysis, only density of tumor infiltrating lymphocytes remained significantly associated with HER2low status (P = 0.035). No difference in survival was observed between HER2low and HER2- patients, neither in the ER+ nor ER- cohort. In conclusion, our data suggests that HER2low breast cancer is associated with a lower immune response compared to HER2- breast cancer.

Lost by Transcription: Fork Failures, Elevated Expression, and Clinical Consequences Related to Deletions in Metastatic Colorectal Cancer.

Among the structural variants observed in metastatic colorectal cancer (mCRC), deletions (DELs) show a size preference of ~10 kb-1 Mb and are often found in common fragile sites (CFSs). To gain more insight into the biology behind the occurrence of these specific DELs in mCRC, and their possible association with outcome, we here studied them in detail in metastatic lesions of 429 CRC patients using available whole-genome sequencing and corresponding RNA-seq data. Breakpoints of DELs within CFSs are significantly more often located between two consecutive replication origins compared to DELs outside CFSs. DELs are more frequently located at the midpoint of genes inside CFSs with duplications (DUPs) at the flanks of the genes. The median expression of genes inside CFSs was significantly higher than those of similarly-sized genes outside CFSs. Patients with high numbers of these specific DELs showed a shorter progression-free survival time on platinum-containing therapy. Taken together, we propose that the observed DEL/DUP patterns in expressed genes located in CFSs are consistent with a model of transcription-dependent double-fork failure, and, importantly, that the ability to overcome the resulting stalled replication forks decreases sensitivity to platinum-containing treatment, known to induce stalled replication forks as well. Therefore, we propose that our DEL score can be used as predictive biomarker for decreased sensitivity to platinum-containing treatment, which, upon validation, may augment future therapeutic choices.

Identifying Transcripts with Tandem Duplications from RNA-Sequencing Data to Predict BRCA1-Type Primary Breast Cancer.

Patients with cancers that are deficient for homologous recombination repair (HRD) may benefit from PARP inhibitor treatment. Therefore, methods that identify such cancers are crucial. Using whole genome sequencing data, specific genomic scars derived from somatic mutations and genomic rearrangements can identify HRD tumors, with only BRCA1-like HRD cancers profoundly displaying small (<10 kb) tandem duplications (TDs). In this manuscript we describe a method of detecting BRCA1-type HRD in breast cancer (BC) solely from RNA sequencing data by identifying TDs surfacing in transcribed genes. We find that the number of identified TDs (TD-score) is significantly higher in BRCA1-type vs. BRCA2-type BCs, or vs. HR-proficient BCs (p = 2.4 × 10-6 and p = 2.7 × 10-12, respectively). A TD-score ≥2 shows an 88.2% sensitivity (30 out of 34) to detect a BRCA1-type BC, with a specificity of 64.7% (143 out of 221). Pathway enrichment analyses showed genes implicated in cancer to be affected by TDs of which PTEN was found significantly more frequently affected by a TD in BRCA1-type BC. In conclusion, we here describe a novel method to identify TDs in transcripts and classify BRCA1-type BCs with high sensitivity.

Apolipoprotein B mRNA-Editing Catalytic Polypeptide-Like-Induced Protein Changes in Estrogen Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Throughout Disease Progression.

PURPOSE: Apolipoprotein B mRNA-Editing Catalytic Polypeptide-like (APOBEC) enzymes are mutagenic factors contributing to tumor progression and therapy resistance. However, the effects of APOBEC-induced protein changes have not been systematically assessed. Here, we describe the effects of APOBEC on the coding sequence in primary and metastatic estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer (BC). METHODS: We determined the enrichment of amino acid (AA) changes resulting from APOBEC mutagenesis in 323 primary BC tumors and 424 metastatic breast cancer (mBC) lesions via comparison with a simulated mutational genomic landscape not under selection pressure. We subsequently explored genes with recurrent APOBEC-associated AA changes and investigated the clonality of individual APOBEC-associated mutations. Using public sequencing data from an independent primary BC and mBC cohort, we further confirm our findings by reporting genes having these enriched AA changes in an APOBEC context. RESULTS: Our analysis demonstrated that several APOBEC-derived AA changes are significantly enriched compared with a simulated AA change distribution drawn at random. Among the enriched AA changes, Glutamate(E)>Lysine(K) and Glutamate(E)>Glutamine(Q) were mostly found at hotspots in oncogenes, whereas termination codons (Glutamine[Q]>STOP[X] and Serine[S]>STOP[X]) occurred in tumor suppressor genes and, mostly, not at hotspot locations. These mutations are found in genes contributing to BC initiation, eg, introduction of termination codons in TP53, MAP3K1, and CDH1 (in lobular BC) and two oncogenic hotspots in PIK3CA (p.E542K and p.E545K). In endocrine-resistant BC, we observed APOBEC-induced termination codons at ER-modulating genes, KMT2C, ARID1A, NF1, and ZFHX3. Finally, in mBC, compared with single PIK3CA mutations, dual PIK3CA mutations occurred more frequently in an APOBEC context (Fisher's exact P < .001). CONCLUSION: Our results show that APOBEC mutagenesis recurrently targets various known drivers of BC initiation, progression, and endocrine resistance.

Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer.

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh-frozen tumor tissue of 239 patients with stage I-III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group-regularized logistic ridge regression with post hoc group-weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.

Spatial immunophenotypes predict response to anti-PD1 treatment and capture distinct paths of T cell evasion in triple negative breast cancer.

Only a subgroup of triple-negative breast cancer (TNBC) responds to immune checkpoint inhibitors (ICI). To better understand lack of response to ICI, we analyze 681 TNBCs for spatial immune cell contextures in relation to clinical outcomes and pathways of T cell evasion. Excluded, ignored and inflamed phenotypes can be captured by a gene classifier that predicts prognosis of various cancers as well as anti-PD1 response of metastatic TNBC patients in a phase II trial. The excluded phenotype, which is associated with resistance to anti-PD1, demonstrates deposits of collagen-10, enhanced glycolysis, and activation of TGFß/VEGF pathways; the ignored phenotype, also associated with resistance to anti-PD1, shows either high density of CD163+ myeloid cells or activation of WNT/PPARγ pathways; whereas the inflamed phenotype, which is associated with response to anti-PD1, revealed necrosis, high density of CLEC9A+ dendritic cells, high TCR clonality independent of neo-antigens, and enhanced expression of T cell co-inhibitory receptors.

Transcriptomic Properties of HER2+ Ductal Carcinoma In Situ of the Breast Associate with Absence of Immune Cells.

The identification of transcriptomic alterations of HER2+ ductal carcinoma in situ (DCIS) that are associated with the density of tumor-infiltrating lymphocytes (TILs) could contribute to optimizing choices regarding the potential benefit of immune therapy. We compared the gene expression profile of TIL-poor HER2+ DCIS to that of TIL-rich HER2+ DCIS. Tumor cells from 11 TIL-rich and 12 TIL-poor DCIS cases were micro-dissected for RNA isolation. The Ion AmpliSeq Transcriptome Human Gene Expression Kit was used for RNA sequencing. After normalization, a Mann-Whitney rank sum test was used to analyze differentially expressed genes between TIL-poor and TIL-rich HER2+ DCIS. Whole tissue sections were immunostained for validation of protein expression. We identified a 29-gene expression profile that differentiated TIL-rich from TIL-poor HER2+ DCIS. These genes included CCND3, DUSP10 and RAP1GAP, which were previously described in breast cancer and cancer immunity and were more highly expressed in TIL-rich DCIS. Using immunohistochemistry, we found lower protein expression in TIL-rich DCIS. This suggests regulation of protein expression at the posttranslational level. We identified a gene expression profile of HER2+ DCIS cells that was associated with the density of TILs. This classifier may guide towards more rationalized choices regarding immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy.

Circular RNA in Chemonaive Lymph Node Negative Colon Cancer Patients.

Circular RNAs (circRNAs) appear important in tumor progression of colon cancer (CC). We identified an extensive catalog of circRNAs in 181 chemonaive stage I/II colon tumors, who underwent curative surgery between 2007 and 2014. We identified circRNAs from RNAseq data, investigated common biology related to circRNA expression, and studied the association between circRNAs and relapse status, tumor stage, consensus molecular subtypes (CMS), tumor localization and microsatellite instability (MSI). We identified 2606 unique circRNAs. 277 circRNAs (derived from 260 genes) were repeatedly occurring in at least 20 patients of which 153 showed a poor or even negative (R < 0.3) correlation with the expression level of their linear gene. The circular junctions for circSATB2, circFGD6, circKMT2C and circPLEKHM3 were validated by Sanger sequencing. Multiple correspondence analysis showed that circRNAs were often co-expressed and that high diversity in circRNAs was associated with favorable disease-free survival (DFS), which was confirmed by Cox regression analysis (Hazard Ratio (HR) 0.60, 95% CI 0.38-0.97, p = 0.036). Considering individual circRNAs, absence of circMGA was significantly associated with relapse, whereas circSATB2, circNAB1, and circCEP192 were associated with both MSI and CMS. This study represents a showcase of the potential clinical utility of circRNAs for prognostic stratification in patients with stage I-II colon cancer and demonstrated that high diversity in circRNAs is associated with favorable DFS.

Differential reprogramming of breast cancer subtypes in 3D cultures and implications for sensitivity to targeted therapy.

Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.

Whole genome sequencing of metastatic colorectal cancer reveals prior treatment effects and specific metastasis features.

In contrast to primary colorectal cancer (CRC) little is known about the genomic landscape of metastasized CRC. Here we present whole genome sequencing data of metastases of 429 CRC patients participating in the pan-cancer CPCT-02 study (NCT01855477). Unsupervised clustering using mutational signature patterns highlights three major patient groups characterized by signatures known from primary CRC, signatures associated with received prior treatments, and metastasis-specific signatures. Compared to primary CRC, we identify additional putative (non-coding) driver genes and increased frequencies in driver gene mutations. In addition, we identify specific genes preferentially affected by microsatellite instability. CRC-specific 1kb-10Mb deletions, enriched for common fragile sites, and LINC00672 mutations are associated with response to treatment in general, whereas FBXW7 mutations predict poor response specifically to EGFR-targeted treatment. In conclusion, the genomic landscape of mCRC shows defined changes compared to primary CRC, is affected by prior treatments and contains features with potential clinical relevance.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Metabotropic glutamate receptor 1 is associated with unfavorable prognosis in ER-negative and triple-negative breast cancer.

New therapies are an urgent medical need in all breast cancer subgroups. Metabotropic glutamate receptor 1 (mGluR1) is suggested as a potential new molecular target. We examined the prevalence mGluR1 expression in different clinically relevant breast cancer subgroups and determined its association with prognosis. In this retrospective cohort, 394 consecutive primary breast cancer tissues were incorporated into a tissue microarray and immunohistochemically stained for mGluR1. The prevalence of mGluR1 protein expression in different breast cancer subgroups was evaluated and correlated with metastasis-free survival (MFS) and overall survival (OS). In total, 56% (n = 219) breast cancer tissues had mGluR1 expression. In estrogen receptor (ER)-negative tumors, 31% (n = 18/58) had mGluR1 expression that was significantly associated with MFS (HR 5.00, 95% CI 1.03-24.35, p = 0.046) in multivariate analysis, independently from other prognostic factors. Of the 44 triple-negative breast cancer (TNBC), 25% (n = 11) expressed mGluR1. mGluR1 expression in TNBC was significantly associated with shorter MFS (HR 8.60, 95% CI 1.06-20.39, p = 0.044) and with poor OS (HR 16.07, 95% CI 1.16-223.10, p = 0.039). In conclusion, mGluR1 is frequently expressed in breast cancer. In ER-negative breast cancer and in TNBC mGluR1 protein expression is an unfavorable prognostic marker. This study provides rationale to explore mGluR1 as a novel target for breast cancer treatment, especially for the more aggressive TNBC.

Differential quantities of immune checkpoint-expressing CD8 T cells in soft tissue sarcoma subtypes.

INTRODUCTION: Local T-cell immunity is recognized for its contribution to the evolution and therapy response of various carcinomas. Here, we investigated characteristics of tumor-infiltrating lymphocytes (TILs), as well as T-cell evasive mechanisms in different soft tissue sarcoma (STS) subtypes. METHODS: Liposarcoma, gastrointestinal stromal tumor (GIST), leiomyosarcoma, myxofibrosarcoma and pleomorphic sarcomas were assessed for T-cell numbers and phenotypes using flow cytometry. Next-generation sequencing was used to analyze T-cell receptor repertoire, mutational load, immune cell frequencies, and expression of immune-related genes. RESULTS: GIST, myxofibrosarcoma and pleomorphic sarcoma showed high numbers of CD8+ TILs, with GIST having the lowest fraction of effector memory T cells. These TILs coexpress the immune checkpoints PD1, TIM3, and LAG3 in myxofibrosarcoma and pleomorphic sarcoma, yet TILs coexpressing these checkpoints were near negligible in GIST. Fractions of dominant T-cell clones among STS subtypes were lowest in GIST and liposarcoma, whereas mutational load was relatively low in all STS subtypes. Furthermore, myeloid-derived cells and expression of the costimulatory ligands CD86, ICOS-L and 41BB-L were lowest in GIST when compared with other STS subtypes. CONCLUSION: STS subtypes differ with respect to number and phenotypical signs of antitumor responsiveness of CD8+ TILs. Notably, GIST, myxofibrosarcoma and pleomorphic sarcoma harbor high numbers of CD8+ T cells, yet in the GIST microenvironment, these T cells are less differentiated and non-exhausted, which is accompanied with a relatively low expression of costimulatory ligands.