Multiplexed Imaging, Pathology and Personalized Medicine: Keeping Sight of the Forest

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Tremendous efforts to appreciate the complexity of cancer and the specific threats and vulnerabilities of individual malignant tumors are underway, involving large-scale and high-resolution DNA, RNA and protein characterization. Results of these investigations may shape the kinds of tissue-based analytical tools that anatomic pathology will be called upon to develop and deploy. Increasingly, these often-multiplexed panels of molecular assays prove to be complex, expensive, proprietary, and hard to independently validate. But what if, in addition, these tests turn out to be focused on the trees and in fact are missing the forest? The interaction between cancers (highly mutable in their geno- and phenotypes) and their tissue environment (more stereotyped) has proven to be complex and informative. Recent data from multiple investigators suggest that host factors rather than specific tumor characteristics may determine clinical outcomes. How should these perspectives be reflected in research and clinical practice, particularly with respect to pathology-based tools? Perhaps a new focus on “stromics” is in order.
In addition, much of contemporary tumor profiling is undertaken in the context of current conventional or targeted therapies. What will happen if and when more general cancer treatments (equally applicable to tumors with differing tissue backgrounds and molecular phenotypes) become reality? Such developments may lead to simpler and lower-cost assays with improved impact on prognostic accuracy, treatment selection and outcome.

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  • CC3 was identified as a metastasis suppressor protein in vivo. This laboratory demonstrated that CC3 impairs apoptotic resistance of cells derived from aggressive tumors and inhibits production of angiogenic factors by these cells. CC3 expression was introduced into two breast carcinoma cell lines derived from metastatic tumors and expressing very low levels of this protein. Expression of exogenous CC3 lead to enhancement of their apoptotic responses to growth factors withdrawal and treatment with cytotoxic drugs. However, there was no effect of CC3 expression on angiogenic activity of breast cancer cells which was very low even prior to introduction of CC3. Because CC3 protein has no significant homologies to other known proteins, we have conducted analysis of cellular proteins that interact with CC3. Mass-spectrometric analysis of isolated protein revealed an amazing consistency among types of proteins that form complexes with CC3. Out of seven proteins identified, five belong to the family of importins of beta class and one is exportin, i.e. all these are proteins that serve as nuclear transport receptors. This amazing specificity in CC3 interactions pointed to a possibility that CC3 might be a factor involved in regulation of nuclear transport. Indeed, CC3 associates with nuclear envelope. Future work will focus on the mechanism through which CC3, via its possible role in nuclear transport, influences apoptosis.
  • Multiplexed Imaging, Pathology and Personalized Medicine: Keeping Sight of the Forest

    1. 1. Multiplexed Imaging, Pathology andPersonalized Medicine: Keeping Sight ofthe ForestRichard Levenson, MDDept. of Pathology andLaboratory MedicineUC Davis Medical Center
    2. 2. Themes1.The stroma (not the epithelium) may be key(…“STROMICS”)2.“Systems biology” is most relevant at structural,intercellular and tissue levels3.Bonus: glimpse of novel structural (target: collagen)and multiplexed (target: heterogeneity) imagingtechnologies
    3. 3. Are we looking inthe right places?How might a focus onmicro-environmentand host factors affectdiagnosis andtreatment strategies?
    4. 4. Normal breast
    5. 5. Invasivesquamous cellcarcinoma
    6. 6. Gabriella dental x-ray and CT: loss of boneNov, 2011 Feb, 2012
    7. 7. 14 monthslater…
    8. 8. Implications:New therapies will need new companion diagnostics—or perhaps none…Molecular phenotypes in spatial context can beinformativeComing up:Multiplexed imagingSelection of targets
    9. 9. 12How well could we understand the battle of Waterloo…if we blenderized the battlefield?
    10. 10. Spectral imaging variantsPARISSNUANCE/VECTRALCTFHSi-440cAOTFRebellionIMS
    11. 11. Vectra: Evaluating EMT in FFPE-Tissue SectionsC. Hoyt, PerkinElmer, Inc.; C. Gagen & R. Wetzel, Cell Signaling TechnologyDAPI E-cadherin vimentin
    12. 12. Raw color imageImmunofluorescence with quantum dotsDAPIPR (585-nm QDot)ER (655-nm QDot)Convert to brightfieldDAPI and autofluorescence
    13. 13. 16From Mouse to Microscope: Breast cancer and Cy5-anti-Her2 antibodyCourtesy Roche, Penzberg, Germany
    14. 14. 17Four nuclear signals can be distinguished usingchromogens in brightfieldHematoxylinKi67—redcell cyclepHH3—browncell cycleCC3—grayantiangiogenesismet. suppressor
    15. 15. Great tools and reagents…So what compartments andcomponents should we belooking at?(hint: stroma)
    16. 16. Nature Medicine, 2008Top 200 gene expressionlevels most differentbetween microdissectednormal and tumor-associated stroma.Automatic clustering gavethese recurrence-freesurvival curves
    17. 17. 26-gene stromal signature is independent of all majorclinical and pathological variables:• ER• HER2• lymph node status• age• grade• tumor sizeNOT PREDICTIVE!
    18. 18. C-Path: ‘Omic’s approach to theanalysis of tumor morphologyAH Beck, …, D Koller. Systematicanalysis of breast cancer morphologyuncovers stromal features associated withsurvival. Science Translational Medicine2011. 3, 108ra113.
    19. 19. Seventy-gene prognosis signatureSizeInvasiveness gene signatureMastectomyWound response signatureERBB2 molecular subtypeGradeBasal molecular subtypeERLymph nodeGenomic grade indexLuminal A molecular subtypeLuminal B molecular subtypeChemotherapyNOTSIGNIFICANT:Classic tumor characteristics are not predictiveAndy Beck
    20. 20. • C-Path score• Hypoxia• AgeWhat is significant:Just 3 stromal features predictedoutcome better than 8 epithelial featuresMoreover:Andy Beck
    21. 21. Untreated Treated2009
    22. 22. Tumor heterogeneity: Stromal environmentaffects stemness and HER2-positivity(Stem cell marker)In this case,HER2-induction NOTdue to geneamplificationImplications forHER2 IHC andISH assays?Wicha 2013
    23. 23. What about structuralproperties of the stroma?Can we detect them intissue slices?
    24. 24. Boyd et al. Breast Cancer Research 2011 13:223OR 1 1.7 2.1 2.4 4.7Mammographic density affects the risk of breast cancer0 <10 <25<50 <75 >75
    25. 25. 28Stromal rearrangement around cancer can reflect biologicalpropertiesLinear collagen strandsoriented tangentially (0degrees)Linear collagen strandsoriented at 90 degreesNon-invasivetumorLocallyinvasivetumor(Second harmonic generation imaging)Provenzano et al., BMC 2008
    26. 26. TACS-2 TACS-3Locker and Segal commentary on Conklin et al, AJP, 2011Risk independent of:GradeSizeAgeER/PR/HER-2node statusConklin et al, AJP, 2011DISEASE-FREE SURVIVAL
    27. 27. SHG: complex, expensive
    28. 28. Inexpensive collagen detection
    29. 29. Tissue microarray sample: low magnification
    30. 30. Orientation of fibers viewed with color mapBile duct in cirrhotic liver
    31. 31. Sneak preview: Subcellular-resolution multiplexed MSConventional imaging mass spec: spatial resolution ~35 microns(Images by Richard Caprioli, keynotespeaker at a previous Tucson Symposium)
    32. 32. Sneak preview: Subcellular-resolution multiplexed MS
    33. 33. Hematoxylin and Ki-67 imaged via mass specSpatial resolution < 1 micron
    34. 34. Large field of view, high-resolution mass-spec image
    35. 35. So what’s coming?•Stromics•“Companion diagnostics” regime notgoing away …(so ER/PR/Her2 etc. will be around for a while)•… but disruptive therapies will alter the diagnosticlandscape•Spatially resolved, highly multiplexed subcellular-scale molecular and structural profiling is feasible•New tools--and new paradigms—should allow us tosee the leaves, the trees and the forest.
    36. 36. Prognosis: encouraging
    37. 37. The Venezuelan PoodleMoth is a possible newspecies of moth discoveredin 2009 by Dr. Arthur Ankerof Bishkek, Kyrgyzstan.
    38. 38. Snapshot collagen birefringenceimage of an H&E-stained breastcancer sample
    39. 39. Notes• Instead of using canonical cell surface markers to identify leukemiastem cells (LSCs), Lagadinou et al. apply a functional approach… ofROS measurements: …both novel and potentially of great utility.• Cell Stem Cell. 2013 Jan 15.• BCL-2 Inhibition Targets Oxidative Phosphorylation and SelectivelyEradicates Quiescent Human Leukemia Stem Cells.• The Bcl-2 gene has been implicated in a number of cancers, includingmelanoma, breast, prostate, chronic lymphocytic leukemia, and lungcarcinomas.• “We demonstrate here that it is feasible to eradicate resistant LSCpopulations by targeting their unique metabolic dependencies.”
    40. 40. Simple antigen-positivity measurements in bulk tumorsmay be simplisticYellow =HER2(+) stemcells (not bulkpopulation)Wicha, 2013
    41. 41. FT-SHG: tissue-scale featureschange optics, then:P-SHG: molecular organization(6 measurements required)N.B.: Half the malignant stroma is“normal,” and localized featuresimportant, so imaging is necessary
    42. 42. -1012345670 10 20 30 40 50 60 70 80 90 100Tumor Transplantation (days)TumorBurden(cm3)Left Inguinal (Control rat)Right Axillary (Control rat)Left Axillary (Control rat)Right Axillary (Cured rat)Left Axillary (Cured rat)DeathEffect ofimmunophotonictherapy onprimary andmetastatic tumorsin ratsPrimaryMetsCD8 cell attacking a tumor cell
    43. 43. Tumor Immunotherapy: Stage 4 breast cancer
    44. 44. Biology of tumor progression meets biology ofaging

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