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Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

ABSTRACT: There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow-up of personalized cancer therapy, including immunotherapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n = 25) and benign lesions (n = 32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g., CA9, GZMB, IL-6, VEGFA, CXCL11, PDL1, and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g., CCL4, CCL8, CCL20, CXCL8, CXCL9, and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status, and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (P = 0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune-related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

SUBMITTER: Franzen B

PROVIDER: S-EPMC6360506 | biostudies-other | 2019 Feb

REPOSITORIES: biostudies-other

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Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

Franzén Bo B Alexeyenko Andrey A Kamali-Moghaddam Masood M Hatschek Thomas T Kanter Lena L Ramqvist Torbjörn T Kierkegaard Jonas J Masucci Giuseppe G Auer Gert G Landegren Ulf U Lewensohn Rolf R

Molecular oncology 20190107 2

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow-up of personalized cancer therapy, including immunotherapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FN ...[more]

PMID: 30451357

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Project description:INTRODUCTION. Gene expression profiling has emerged as a potentially useful tool for personalized management of breast cancer. Adequate RNA can be obtained for this scope from dedicated fine-needle aspiration cytology (FNAC), with the limitation that separate sampling prohibits direct comparison with the material used for cytology. The aim of this study was to investigate whether syringe leftovers of cytological smears obtained in diagnostic FNACs can be successfully used to extract RNA for reliable PCR- and microarray-based gene expression analysis. METHODS. Twenty-eight FNACs were performed under ultrasound guide. After preparing two smears for cytological diagnosis, the syringes were immediately rinsed with Trizol® to extract RNA from residual material. The abundance and type of cells obtained from the smears was recorded and compared to RNA amounts and quality as determined by spectrophotometry and RT-PCR. FNAC leftovers of eleven breast cancers of different size, histological type, grade and immunoprofile were then selected for gene expression profiling using oligonucleotide microarrays. RESULTS. FNAC leftovers yielded an average of over 10 micrograms of RNA, more proportional to the amount of cells on the smear than to the lesion size, and adequate for expression analysis in 26/28 cases. The samples chosen for microarray analysis were clustered according to four criteria: (i) gene expression profiles of all detected genes; (ii) gene expression profiles of a previously defined stromal gene signature; (iii) “biological scores”, related to diagnostic cytological variables; (iv) “technical scores”, related to RNA/cRNA/microarray quality controls. Clusters generated by both expression profiles partitioned the samples in well-distinguished subgroups that were highly overlapping with clusters obtained using biological scores. Conversely, poor overlap was observed with clusters based on technical scores, demonstrating that sample divergences in RNA quality and amounts due to acquisition, handling and processing did not severely influence gene expression profiling. CONCLUSIONS. The syringe leftover after the smearing procedures of FNAC samples is suitable for RNA banking even when taken from small, non-palpable breast lesions, which guarantees congruity between morphological and molecular analysis. RNA yield mostly correlates with cellularity of the smears, and gene expression profiles are consistent with the cytologic features, and therefore with tumour biology.

Project description:BackgroundHeterogeneity of immune gene expression patterns of luminal breast cancer (BC), which is clinically heterogeneous and overall considered as low immunogenic, has not been well studied especially in non-European populations. Here, we aimed at characterizing the immune gene expression profile of luminal BC in an Asian population and associating it with patient characteristics and tumor genomic features.MethodsWe performed immune gene expression profiling of tumor and adjacent normal tissue in 92 luminal BC patients from Hong Kong using RNA-sequencing data and used unsupervised consensus clustering to stratify tumors. We then used luminal patients from The Cancer Genome Atlas (TCGA, N = 564) and a Korean breast cancer study (KBC, N = 112) as replication datasets.ResultsBased on the expression of 130 immune-related genes, luminal tumors were stratified into three distinct immune subtypes. Tumors in one subtype showed higher level of tumor-infiltrating lymphocytes (TILs), characterized by T cell gene activation, higher expression of immune checkpoint genes, higher nonsynonymous mutation burden, and higher APOBEC-signature mutations, compared with other luminal tumors. The high-TIL subtype was also associated with lower ESR1/ESR2 expression ratio and increasing body mass index. The comparison of the immune profile in tumor and matched normal tissue suggested a tumor-derived activation of specific immune responses, which was only seen in high-TIL patients. Tumors in a second subtype were characterized by increased expression of interferon-stimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in TCGA and KBC, although the pattern was more similar in Asian populations. The germline APOBEC3B deletion polymorphism, which is prevalent in East Asian populations and was previously linked to immune activation, was not associated with immune subtypes in our study. This result does not support the hypothesis that the germline APOBEC3B deletion polymorphism is the driving force for immune activation in breast tumors in Asian populations.ConclusionOur findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification and a subset of luminal BC patients may benefit from checkpoint immunotherapy, at least in Asian populations.

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Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

Publications

Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

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