Use of molecular pathology in the evaluation of precision animal modelsKeith MansfieldDirectorDiscovery and Investigative PathologyNovartis Institute for Biomedical Research

Discovery and Investigative Pathology

Molecular pathologyKey element in precision medicine

• Molecular pathology is focused on the study and diagnosis of disease through the examination of molecules (generally DNA, RNA and protein) within organs, tissues or bodily fluids.

• It attempts to understand the molecular basis of morphological changes and requires a firm foundation in anatomic pathology.

• It is multi-disciplinary in nature and integrates genomics, genetics, proteomics, and physiology with morphology and utilizes a number of tools including immunohistochemistry and in situ hybridization.

• Molecular pathology tools are frequently used in the clinical diagnosis and management of cancer patients and the discipline is one of the foundations of precision medicine.

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Molecular localization Immunohistochemistry automated work flow

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• Molecular localization is the ability to define the spatial distribution of molecular entities in tissue.

• Immunohistochemistry has been automated on several platforms increasing efficiency and improving reproducibility.

• Commercial antibody quality and specificity vary widely and appropriate controls are required to qualify reagents.

• Assay validation may include WB, flow cytometry, staining of cell lines and tissues with known levels of expression, peptide blocking experiments, correlation with mRNA data and reproducibility assays.

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Molecular localization In situ hybridization• In situ hybridization techniques using traditional riboprobes, DNA probes

or LNA probes or next generation probe technology (Affymetrix/Panomics(ViewRNA) and Advanced Cell Diagnostics (RNAscope)) may be adapted to autostaining platforms.

• Next generation technologies can detect splice variants and SNPs.

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Variant 1 Variant 2

RNAscope/ViewRNA• Detects single mRNA molecules and provide

quantitative data at single cell resolution.• Provides quantitative measure of RNA per cell.• Easily multiplexed

Splice variant analysis

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Highly multiplexed IHC assaysNext-gen IHC

• Imaging mass cytometry/Laser Ablation Inductively Coupled Plasma MS Imaging

• Highly multiplexed (20-30 markers) performed on a single slide.

• Utilizes rare metal labels of antibodies and maintains their 2D spatial orientation allowing virtual reconstruction.

• Requires significant resources to validate protocols.

• Bioinformatics are key to extracting data to the fullest extent.

V. Dubost, NIBR

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BiobankingComparative molecular pathogenesis

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• Biobanks containing well curated collections of normal and diseased human biological samples as well as samples from preclinical species and precision models can be used for target and model validation.

• Comparison of diseased human samples to samples from precision models can determine similarities and differences in morphologic and molecular alterations.

• This comparison may inform the selection of models that most closely parallel aspects of the human disease to be investigated.

• Comparisons assists in experimental design of future studies and may reduce the total number of animals needed to complete studies.

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Molecular localizationComparative molecular pathogenesis

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Upregulation of p22phox and Nox2 mRNA in CCl4 liver fibrosis model

Alterations in p22phox and Nox2 mRNA not detected in septal fibroblasts in human cirrhosis

Control mouse liver

Mouse model

Normal human liver

Cirrhotic human liver

Mouse Human

NADPH oxidases contribute to TGF-β1-induced α-SMA production and mediate TGF-β1-induced fibroblast differentiation into myofibroblasts.

• Side by side evaluation of normal and diseased human and animal tissues can inform the comparative relevance of animal models and their potential limitations.

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Molecular localizationComparative molecular pathology of pancreatic adenocarcinoma

Stromal component• Neoplastic EMT• Cancer associated fibroblasts• Vascular endothelium• Lymphatic endothelium• Pericytes• Adipocytes

Immune component• T and B lymphocytes• Tregs, NK and NKT cells• Tumor associated macrophages• Myeloid suppressor cells• Tumor-associated neutrophils• Myeloid dendritic cells

Epithelial component• Neoplastic cells• Residual acinar and ductular

pancreatic cells

How do mouse models reproduce elements of this microenvironment?

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H&E Stroma T cells B cells ProliferationMacrophageEpithelium

Molecular localizationComparative molecular pathology of pancreatic adenocarcinoma

Vimentin CD3 CD19 Ki67CD68cytokeratin

Vimentin CD3 B220 Ki67F4/80cytokeratin

Vimentin CD3 B220 Ki67F4/80cytokeratin

CD3 B220 Ki67F4/80cytokeratin

CD3 B220 F4/80 Ki67

Vimentin

Vimentin

Patient sample: pancreatic

adenocarcinoma

Xenograft: PANC cell line in

NSG mouse

Syngeneic: engineered pancreatic

organoid mouse

Syngeneic: engineered pancreatic

organoid in NSG mouse

Xenograft: HPAFII cell line in NSG mouse

cytokeratin

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Digital pathologyWork flow

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ArchiveArchive

Traditional work flow

Rep

ort

• Digital archive with metadata • Available for virtual

microscopy, image analysis, data mining and other bioinformatic approaches

Rep

ort

Digital pathology work flow

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Image analysisWhole slide image analysis

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Perform IHC/ISH and slide scan

Image analysis soft ware trained to identify tissue

typesCell segmentation and

staining intensity scoredQuantitative data

exported and integrated with sample metadata

CD3 IHC

• Digital pathology facilitates whole slide image analysis.

• Quantitative measures of staining intensity, cell number, cell morphology and relative spatial distribution of specific cell types can be obtained.

Digitized slide Cell segmentation markupTissue segmentation markup

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Tissue microarraysMolecular profiling of tissue types

• Tissue microarrays (TMA) can be used for IHC and ISH on automated staining platforms to examine variation in target biology.

• 24-96 cores can be arrayed on an individual slide.

• Custom TMAs can be constructed to array tissues from a single species, disease process or organ from multiple species.

• Quality control of source material is paramount and may be lacking in some commercially available TMAs.

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Image analysisTissue microarrays

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age

Ana

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Perform IHC and scan Tissue microarray

Image analysis soft ware identifies and assigns cores

Cell segmentation and staining

intensity scored

Data heat map produced from

Image analysis H-score

Quantitative H-scores can be integrated with

sample metadata

• Quantitative image analysis techniques can be applied to tissue microarrays to facilitate high through put analysis of molecular localization studies and integration with sample metadata

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Image analysisSpatial resolution of target expression

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Scale (spot no.)

Control

Treatment

In situ hybridization with single mRNA molecule resolution

Individual dot density converted to intensity heatmap to assist in data visualization

• Image analysis techniques can assist in the spatial resolution of target mRNA expression.

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Genomic profilingIntegration with molecular localization

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• Genomic expression profiling can be coupled with morphologic interpretation of tissue to assist in elucidating the pathogenesis of disease.

• Paired samples are routinely taken for genomic profiling and processing for histological analysis.

• Analysis is complex as changes in overall gene expression may result from alterations in individual cells and from changes in the cellular composition of tissue.

• Molecular localization studies can be used to confirm expression changes and cellular source.

Target A

Target B

Target A Target B

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Genomic profilingCell-type related signatures can be extracted from a complex tissue

Kidney: an heterogenous tissue

Nephrin 1

Podocyte

Clcnkb Nphs1

Cldn5 Pdpn

Clic3 Podxl

Cst3 Sema3g

Ddn Sparc

Gja5 Tnnc1

Nes Wt1

Glomerularendothelial cellAdamts5Anxa1CpeEdnrbLyve1OgnPlatTjp1

MesangialcellActn1Anxa2Anxa5Col4a1Col4a2CygbFlnaTagln2Vim

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PodocyteClcnkbCldn5Clic3Cst3DdnGja5NesNphs1PdpnPodxlSema3gSparcTnnc1Wt1

Glomerularendothelial cell

Adamts5Anxa1CpeEdnrbLyve1OgnPlatTjp1

MesangialcellActn1Anxa2Anxa5Col4a1Col4a2CygbFlnaTagln2Vim

Genomic profilingCell-type related signatures are validated by Immuno-histochemistry and in situ hybridization

Ogn

Anxa5

Pdpn

PodxlSema3g

Tjp1

Cst3

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Summary• Molecular pathology advances our understanding of comparative

pathogenesis of human disease and relevant animal models.

• It is a rapidly advancing field that requires the integration of traditional anatomic pathology skills with molecular biology and bioinformaticapproaches.

• Interrogation of tissues from precision models must be made in conjunction with evaluation of human diseased tissues to better understand the model’s relevance and limitations.

• Future advances will build on of the use of bioinformatics, highly multiplexed localization assays and computational interrogation of digital slide databases.

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AcknowledgementsNovartis Institutes for Biomedical Research• C Buono

• K Wharton

• C Saravanan

• D Stiehl

• P Moulin

• V Dubost

• Z Chen

• J Judge

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