Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B ...evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably.
Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3
, CD4
, CD8
, CD20
, CD68
, FOXP3
) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures.
The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82).
There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.
Abstract
We retrospectively assessed association of stromal tumor-infiltrating lymphocytes (sTILs) with clinical outcomes and molecular variables reportedly predictive of trastuzumab-benefit in ...National Surgical Adjuvant Breast and Bowel Project B-31 (N = 2130). sTILs were assessed in 1581 eligible B-31 cases utilizing all available hematoxylin and eosin slides. Mean concordance between main reviewer and six other pathologists was 90.8% in 100 cases. Cox regressions were used to calculate hazard ratios (HRs). In chemotherapy and trastuzumab added to chemotherapy arms, increases in sTILs, as a semicontinuous variable (combined arms HR = 0.42, 95% confidence interval = 0.27 to 0.64, two-sided P < .001) or as lymphocyte-predominant breast cancer with more than 50% sTILs (combined arms HR = 0.65, 95% confidence interval = 0.49 to 0.86, two-sided P = .003) were statistically significantly associated with improved disease-free survival. There was no association of sTILs with trastuzumab benefit. However, higher sTILs were statistically significantly associated with higher trastuzumab benefit groups by 8-gene prediction model (two-sided P < .001). Neither PIK3CA mutations nor Fc-gamma-receptor polymorphisms were associated with sTILs. sTILs may have utility as a prognostic biomarker identifying HER2-positive early breast cancer at low recurrence risk.
A recent comprehensive whole genome analysis of a large breast cancer cohort was used to link known and novel drivers and substitution signatures to the transcriptome of 266 cases. Here, we validate ...that subtype-specific aberrations show concordant expression changes for, for example, TP53, PIK3CA, PTEN, CCND1 and CDH1. We find that CCND3 expression levels do not correlate with amplification, while increased GATA3 expression in mutant GATA3 cancers suggests GATA3 is an oncogene. In luminal cases the total number of substitutions, irrespective of type, associates with cell cycle gene expression and adverse outcome, whereas the number of mutations of signatures 3 and 13 associates with immune-response specific gene expression, increased numbers of tumour-infiltrating lymphocytes and better outcome. Thus, while earlier reports imply that the sheer number of somatic aberrations could trigger an immune-response, our data suggests that substitutions of a particular type are more effective in doing so than others.
Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a ...method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (
< 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of
,
, and
in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of
was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear,
levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that
is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.
To infer the prognostic value of simultaneous androgen receptor (
) and
profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of
...signaling inhibitors (ARSi).
Between March 2014 and April 2017, we recruited patients with mCRPC (
= 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight
splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of
and
was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models.
Overall, no single
perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS.
inactivation harbored independent prognostic value HR 1.88; 95% confidence interval (CI), 1.18-3.00;
= 0.008, and outperformed ARV expression and detection of genomic
alterations. Using Cox coefficient analysis of clinical parameters and
status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months;
< 0.0001), which was validated in an independent mCRPC cohort (
= 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months;
< 0.0001).
In an all-comer cohort, tumor burden estimates and
outperform any
perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.
Abstract Background Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). ...However, a large fraction of nonresponding patients are AR-V7–negative. Objective To investigate if a comprehensive liquid biopsy–based AR profile may improve patient stratification in the context of second-line endocrine therapy. Design, setting, and participants Peripheral blood was collected from patients with CRPC ( n = 30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Outcome measurements and statistical analysis Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Results and limitations Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424–14.41; p = 0.0105). Six out of 17 poor responders were AR-V7–negative, but four carried other AR perturbations. Conclusions Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Patient summary Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has ...the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited.
A combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients.
ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays.
ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Propofol is a potent and widely used central-acting sedative drug. It has been implicated in the development of a usually fatal syndrome characterized by metabolic acidosis, rhabdomyolysis, ...ventricular arrhythmia, and Brugada-like cardiac rhythm disturbances, all leading to cardiac and renal failure. The investigators describe a 12-year-old male patient with a fatal case of this so-called propofol infusion syndrome. Postmortem investigation showed not only the well-known myocytolysis in skeletal and cardiac muscle but also not previously demonstrated widespread fat accumulation in the myocardium. In conclusion, this cardiac fat accumulation illustrates the proposed underlying pathophysiology of impaired (muscular) free fatty acid utilization.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer with high metastatic potential. Most patients have lymph node involvement at the time of diagnosis and 1/3 of ...the patients have distant metastases. In a previous study, we demonstrated that IBC is a distinct form of breast cancer in comparison with non-IBC. The aim of this study was to investigate the presence of the different molecular subtypes in our data set of 16 IBC and 18 non-IBC specimen. Therefore, we selected an 'intrinsic gene set' of 144 genes, present on our cDNA chips and common to the 'intrinsic gene set' described by Sorlie et al. PNAS, 2003. This set of genes was tested for performance in the Norway/Stanford data set by unsupervised hierarchical clustering. Expression centroids were then calculated for the core members of each of the five subclasses in the Norway/Stanford data set and used to classify our own specimens by calculating Spearman correlations between each sample and each centroid. We identified the same cell-of-origin subtypes in IBC as those already described in non-IBC. The classification was in good agreement with immunohistochemical data for estrogen receptor protein expression and cytokeratin 5/6 protein expression. Confirmation was done by an alternative unsupervised hierarchical clustering method. The robustness of this classification was assessed by an unsupervised hierarchical clustering with an alternative gene set of 141 genes related to the cell-of-origin subtypes, selected using a discriminating score and iterative random permutation testing. The contribution of the different cell-of-origin subtypes to the IBC phenotype was investigated by principal component analysis. Generally, the combined ErbB2-overexpressing and basal-like cluster was more expressed in IBC compared to non-IBC, whereas the combined luminal A, luminal B and normal-like cluster was more pronounced in non-IBC compared to IBC. The presence of the same molecular cell-of-origin subtypes in IBC as in non-IBC does not exclude the specific molecular nature of IBC, since gene lists that characterize IBC and non-IBC are entirely different from gene lists that define the different cell-of-origin subtypes, as evidenced by principal component analysis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We ...found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
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