Tissue slide-based microRNA characterization of tumors: how detailed could diagnosis become for cancer medicine?

Figures & data

Table 1. Variations and evolution of tissue slide-based assays for in situ detection of miRNAs^†.

miRNA probe	Tag	Label	Multiplex	Tissue	Procedure	Ref.
miR-9, miR-124, miR-125b	DIG	Chromo^NBT/BCIP	N/D	Brain	Manual	[51]
miR-21	FITC	Chromo^DAB	N/D	Colon	Manual	[71]
miR-150	DIG	Chromo^NBT/BCIP	N/D	Colon, Lymphoid	Manual	[106,118]
miR-21, miR-155	DIG	Chromo^NBT/BCIP	N/D	Pancreas	Manual	[72]
Let-7b, miR-205	DIG	Fluorescent		Breast^TMA	Manual	[103]
let-7a, let-7b, let-7c, let-7f, let-7g, miR-98	DIG	Chromo^DAB	N/D	Breast^TMA	Manual	[107]
let-7a, miR-21, miR-125b, miR-145, miR-205, miR-451	FITC	Fluorescent	N/D	Breast^TMA, Lung^TMA	Manual	[69,142,143]
miR-21, miR-126, miR-155, miR-182, miR-210	DIG2X	Chromo^NBT/BCIP	N/D	Lung^TMA	Manual	[82–86]
miR-146, miR-375	DIG2X	Fluorescent or Chromo^NBT/BCIP	N/D	Lung, Esophagus	Manual; 2-fluoro RNA probes	[87,88]
miR-200a	DIG2X	Chromo^NBT/BCIP	N/D	Breast^TMA	Manual	[89]
miR-21	DIG2X	Chromo^NBT/BCIP	N/D	Pancreas^TMA	Manual	[90]
Let-7a, miR-1, miR-21, miR-103, miR-124, miR-126, miR-143, miR-145, miR-205, miR-223	DIG2X	Chromo^NBT/BCIP	N/D	Brain, Breast, Colon	Manual	[77,78,91]
miR-205	DIG2X	Fluorescent	ISH/IFplex: 1 RNA + 1 protein	Prostate	Manual	[92]
miR-124	FITC	Fluorescent	ISH/IFplex: 1 RNA + 1 protein	Brain	Manual; HIER instead of PK	[99]
let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7glet-7i, miR-1, miR-7a, miR-9, miR-9*, miR-15a, miR-16, miR-21, miR-22, miR-26a, miR-26b, miR-27a, miR-28, miR-29a, miR-30a, miR-30d, miR-99a, miR-99b, miR-101a, miR-122, miR-124, miR-126*, miR-128a, miR-128b, miR-129, miR-130a, miR-133a, miR-132, miR-134, miR-135b, miR-140, miR-143, miR-153, miR-191, miR-370, miR-410, miR-434, miR-5p, miR-467a, miR-497, miR-744, miR-872	DIG	Fluorescent	IHC/ISHplex: 1 protein + 1 RNA	Mouse tissues	Manual; Post-fixation with EDC	[79]
miR-205, miR-375	FITC6X, Bio6X	Fluorescent	ISHplex: 3 RNAs	Skin	Manual; Post-fixation with EDC	[81]
miR-21, miR-34a, miR-92a, miR-205, miR-221	DIG	Fluorescent	ISH/IHCplex: 1 RNA + 1 protein	Breast^TMA Skin^TMA	Manual	[101,102]
miR-142–5p	DIG	Fluorescent	ISH/IFplex: 1 RNA + 2 proteins	Brain	Manual; EDC fixation post-HIER	[100]
miR-1, miR-125b, miR-199a	DIG2X	Fluorescent	ISH/IFplex: 1 RNA + 2 proteins	Heart	Manual	[93]
miR-21, miR-205	DIG2X	Fluorescent	ISH/IHCplex: 1 RNA + 1 protein	Breast, Colon	Semi-automated	[94]
let-7a, miR-10b, miR-21, miR-24, mir-34a, miR-34c-5p, miR-125b, miR-126, miR-141, miR-143, miR-145, miR-155, miR-196, miR-200b, miR-205, miR-214, miR-221, miR-375, miR-451	FITC2X, Bio2X, DIG2X, BrdU2X	Fluorescent	ISH/IHCplex: 4 RNAs + 1 protein 2 RNAs + 3 proteins 1 RNA + 4 proteins	Breast, Pancreas^FNA, Colon, Lung, Prostate	Semi-automated	[75,95,96]
miR-21	DIG2X	Chromo^NBT/BCIP	N/D	Colon	Fully- automated	[97,98]
miR-21, miR-126, miR-145	DIG2X	Chromo^NBT/BCIP	ISH/IHC2plex: 1 RNA + 1 protein	Mouse and rat tissues	Fully- automated	[80]
let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210	FITC2X	Fluorescent	ISH/IHC4plex: 2 RNAs + 2 proteins 1 RNAs + 3 proteins	Breast	Fully- automated	[76]

^†This table summarizes main technical features and capabilities of tissue-slide based assays for in situ detection of miRNAs in formalin-fixed paraffin-embedded normal and tumor tissues from various organ sites. Unless otherwise indicated, in situ miRNA detection was performed on whole-tissue sections. From the top to the bottom of the list, the evolution of this assay can be appreciated by the implementation of innovative features and technological advances, from multi-tag probes to fully automated performance.

Bio: Biotin; BrdU: Bromodeoxyuridine; Chromo^NBT/BCIP and Chromo^DAB: A chromogenic stain with alkaline phosphatase-mediated development of 4-nitroblue tetrazolium and 5-bromo-4-chloro-30-indolyl phosphate substrate or horseradish peroxidase-mediated development 3,3’-diaminobenzidine substrate, respectively; DIG: Digoxigenin; FITC: Fluorescein; FNA: Fine-needle aspirate; IHC: Immunohistochemistry (enzyme conjugated to antibody develops signal); ISH: In situ hybridization; IF: Immunofluorescence (signal directly conjugated to antibody); TMA: Tissue microarray.

Table 2. Potential clinical applications of miRNA-based in situ hybridization assays^†.

Cancer	Patient cohort	miRNA	Expressing cells	Informative cells	Clinical application	Ref.
Bladder	229 urothelial carcinoma cases: stage 0–1 (79%) versus stage II–IV (21%)	miR-34a	Epithelial/tumor cells^TMA	Tumor cells	Positive indicator for recurrence-free survival in stage 0–I cases [HR: 0.57; p = 0.04]	[Andrew AS, Cowper R, Marsit CJ, et al. Expression of tumor suppressive miRNA-34a is associated with a reduced risk of bladder cancer recurrence, Submitted]
Brain	193 glioma cases: grade I–III (19%) versus grade IV (81%, glioblastomas)	miR-21	Tumor cells, tumor-associated vessels	Tumor cells	Negative prognostic indicator [HR: 1.545; p = 0.049]	[105]
Breast	2033 population-based breast carcinoma cases: ER+ (84%) versus ER– (16%)	let-7b	Epithelial/tumor cells^TMA	Tumor cells	Positive prognostic indicator in ER+ and/or PR+ cases [HR: 0.79; p = 0.02]	[103]
	80 breast carcinoma cases: ER+ (44%) versus ER– (56%); stage I/II (56%) versus stage III/IV (44%)	let-7g	Luminal epithelial/tumor cells^TMA	Tumor cells	Positive prognostic indicator [p = 0.021; univariate]	[107]
	901 non-metastatic breast carcinoma cases: ER+ (76%) versus ER–(24%); stage I(68%) versus stage II (32%)	miR-21	Tumor cells, tumor-associated fibroblasts^TMA	Tumor cells or tumor-associated fibroblasts depending on molecular subtype	Negative prognostic indicator [HR: 1.96; p < 0.001]	[Mackenzie TA, Schwartz G, Calderone H, et al. Stromal expression of miR-21 identifies high-risk group in triple negative breast cancer, Submitted]
	102 breast carcinoma cases: ER+ (49.6%) versus ER– (65.7%)	miR-27	Tumor cells^TMA	Tumor cells	Negative prognostic indicator [HR: 3.57; p = 0.025]	[108]
	1686 population-based breast carcinoma cases: ER+ (81%) versus ER– (19%)	miR-205	Myoepithelial/tumor cells^TMA	Tumor cells	Positive prognostic indicator in ductal histology cases [HR: 0.77; p = 0.02]	[103]
	20 ER–, PR–, HER2– breast carcinoma cases		Myoepithelial/tumor cells^TMA	Tumor cells	Positive prognostic indicator in ER–, PR–, HER2– cases [trend only]	[69]
	377 breast carcinoma cases	miR-221	Tumor cells	Tumor cells	Positive prognostic indicator [HR: 0.70; p = 0.032]	[101]
Colon	129 stage II colon cancer cases	miR-21	Tumor cells, tumor-associated fibroblasts	Tumor-associated fibroblasts	Negative prognostic indicator [HR: 1.28; p = 0.012]	[97]
	764 stage II colon cases		Tumor cells, tumor-associated fibroblasts	Tumor-associated fibroblasts	Negative prognostic indicator [HR: 1.92; p = 0.008]	[98]
	89 metastatic colorectal cancer cases treated with first-line capecitabine and oxaliplatin (XELOX)	miR-126	Endothelial cells in primary tumor tissues	Endothelial cells	Positive predictive indicator of response to XELOX treatment [p < 0.0001]	[104]
					Positive prognostic indicator	[104]
	185 colorectal cases	miR-150	Epithelial/Tumor cells	Tumor cells	Positive prognostic indicator [HR: 0.18; p < 0.01]	[106]
					Positive predictive indicator of response to adjuvant chemotherapy	[106]
Esophagus	249 esophageal squamous cell carcinoma cases with paired N/T	miR-375	Esophageal cells/Tumor cells^TMA	Tumor cells	Positive prognostic indicator [p = 0.04, univariate]	[88]
Lung	335 NSCLC cases: stage I (47%), stage II (40%), stage III (13%); node negative (69%) versus node positive (31%)	miR-21	Tumor cells, tumor-associated fibroblasts^TMA	Tumor cells	Positive prognostic indicator in node positive cases [HR: 0.49; p = 0.027]	[85]
			Tumor cells	Tumor-associated fibroblasts	Negative prognostic indicator [p = 0.002, univariate]	[85]
	312 NSCLC cases: stage I (47%), stage II (40%), stage III (13%)	miR-126	Tumor cells and others	Tumor cells	Negative prognostic indicator [HR: 1.78; p = 0.01]	[83]
	43 NSCLC cases	miR-146	Tumor cells	Tumor cells	Negative prognostic indicator [HR: 10.56; p = 0.003]	[87]
	320 NSCLC cases: adenocarcinoma (34%, AC) versus SCC (66%) carcinomas; node negative (69%) versus node positive (21%)	miR-155	Tumor cells^TMA	Tumor cells	Negative prognostic indicator in AC cases [HR: 1.87; p = 0.047]	[82]
					Positive prognostic indicator in node positive SCC cases [HR: 0.45; p = 0.039]	[82]
	305 NSCLC cases: stage I (47%), stage II (42%), stage III (9%); adeno- (37%), squamous cell (62.6%, SCC), large cell (0.4%) carcinomas	miR-182	Tumor cells and stromal cells^TMA	Tumor cells	Positive prognostic indicator in stage II cases [HR: 0.5; p = 0.02]	[84]
					Positive prognostic indicator in SCC cases [HR: 0.57; p = 0.048]	[84]
	259 NSCLC cases: stage I (47%), stage II (40%), stage III (13%)	miR-210	Tumor cells and stromal cells^TMA	Stromal cells	Positive prognostic indicator [HR: 1.9; p = 0.013]	[86]
Lymphoid	36 lymphoid cases: 28 BL versus 8 reactive lymphoid hyperplasia	miR-150	Lymphoid/Tumor cells	Tumor cells	Differential diagnostic marker for BL [p = 0.001]	[118]
Pancreas	106 cases: benign (10%) versus PDAC (90%)	miR-10b	Tumor cells, others cell types^FNA	Tumor cells	Differential diagnostic marker for PDAC [p = 0.001]	[95]
	95 PDAC cases: stage I/II (52%) versus stage III/IV (48%)		Tumor cells, others cell types^FNA	Tumor cells	Negative prognostic indicator [HR: 3.1; p = 0.032]	[95]
	145 PDAC cases: stage I (37%) versus stage II (62%)	miR-21	Tumor cells, tumor-associated fibroblasts^TMA	Tumor-associated fibroblasts	Negative prognostic indicator [HR: 1.4; p = 0.004]	[90]
	80 PDAC cases		Tumor cells^TMA	Tumor cells	Negative prognostic indicator in node negative cases [p = 0.037, univariate]	[70]
Prostate	169 prostatic cancer cases	miR-21	Tumor cells	Tumors	Negative indicator for biochemical recurrence [p < 0.001, univariate]	[144]
Skin	297 melanoma cases: 105 training set + 192 validation set	miR-205	Tumor cells^TMA	Tumor cells	Positive prognostic indicator [HR: 0.511; p = 0.048 – validation set]	[102]
	16 cases: four BCC versus 12 MCC	miR-205	Tumor cells	Tumor cells	Differential diagnostic marker for BCC	[81]
		miR-375	Tumor cells	Tumor cells	Differential diagnostic marker for MCC	[81]

^†This table summarizes clinical characteristics of patient cohorts in which altered miRNA expression was determined by tissue slide-based assays. Statistically significant associations between contextual information of the tumor compartment and the levels of miRNA expression and clinical parameters are reported. Positive prognostic indicators denote that high levels of the miRNAs are positively correlated with good outcome (HR < 1) and, conversely, negative prognostic indicators indicate that high levels of the miRNAs are negatively correlated with good outcome (HR > 1). Unless otherwise indicated, HR for outcome refers to cancer-specific or overall survival in all comers as determined by multivariate COX regression analyses. Unless otherwise indicated, in situ miRNA detection was performed on whole-tissue sections.

BCC: Basal cell carcinoma; BL: Burkitt lymphoma; HR: Hazard ratio; FNA: Fine-needle aspirate; MCC: Merkel cell carcinoma; NSCLC: Non-small-cell lung cancer; PDAC: Pancreatic ductal adenocarcinoma; SCC: Squamous cell carcinoma; TMA: Tissue microarray.

Figure 1. Automated platform for high-content marker analysis of tumor tissues. (A) Key components of in situ miRNA detection assay. (B) Purposes of key components and the steps they take part in, from tissue digestion to covalently linked deposition of fluorescent labels. (C) Virtual staining rendition of a 9plex assay that co-detects three RNA markers (two miRNAs + control rRNA) and six protein markers (three cell type-specific + three functional/prognostic markers); black dots indicate nuclei revealed by counterstaining with DAPI. Top left, representative miR-21 stain (green) on an ER⁻PR⁻HER2⁻ breast cancer case in the first round of HRP-mediated deposition of reactive substrate conjugated with a fluorochrome. Sequential rounds of HRP-mediated deposition of substrates conjugated with spectrally unique fluorochromes are carried out to detect probes against other RNA markers with appropriate anti-hapten primary and secondary antibody combinations or protein markers with appropriate primary and secondary antibody combinations. Each new staining round requires a peroxide block step to inactive HRP from the preceding round.

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