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CONTRIBUTORS
N.Y.R.Agar
BrighamandWomen’sHospital;Dana-FarberCancerInstitute,HarvardMedicalSchool,
Boston,MA,UnitedStates
M.Aichler
ResearchUnitAnalyticalPathology,HelmholtzZentrumMu€nchen,Neuherberg,Germany
P.M.Angel
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
G.Arentz
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS),UniversityofAdelaide,Adelaide,SA,Australia
B.Balluff
MaastrichtUniversity,MaastrichtMultiModalMolecularImaginginstitute(M4I),
Maastricht,TheNetherlands
M.Briggs
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS);ARCCentreforNanoscaleBioPhotonics(CNBP),Universityof
Adelaide,Adelaide,SA,Australia
E.Bruner
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
A.Buck
ResearchUnitAnalyticalPathology,HelmholtzZentrumMu€nchen,Neuherberg,Germany
J.Bunch
NationalPhysicalLaboratory,Teddington;SchoolofPharmacy,UniversityofNottingham,
Nottingham,UnitedKingdom
D.Calligaris
BrighamandWomen’sHospital,HarvardMedicalSchool,Boston,MA,UnitedStates
R.Casadonte
ProteopathGmbH,Trier,Germany
P.Chaurand
Universit(cid:2)edeMontr(cid:2)eal,Montreal,QC,Canada
R.R.Drake
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
M.Dufresne
Universit(cid:2)edeMontr(cid:2)eal,Montreal,QC,Canada
I.F.Dunn
BrighamandWomen’sHospital,HarvardMedicalSchool,Boston,MA,UnitedStates
ix
x Contributors
R.J.A.Goodwin
MassSpectrometryImagingGroup,PathologySciences,DrugSafety&Metabolism,
InnovativeMedicinesandEarlyDevelopment,AstraZeneca,Cambridge,UnitedKingdom
M.Hanselmann
HeidelbergCollaboratoryforImageProcessing(HCI),InterdisciplinaryCenterforScientific
Computing(IWR),UniversityofHeidelberg,Heidelberg,Germany
R.M.A.Heeren
MaastrichtUniversity,MaastrichtMultiModalMolecularImaginginstitute(M4I),
Maastricht,TheNetherlands
Y.-Y.Ho
AdelaideProteomicsCentre,SchoolofBiologicalSciences,UniversityofAdelaide,
Adelaide,SA,Australia
M.K.Hoffmann
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS),UniversityofAdelaide,Adelaide,SA,Australia
P.Hoffmann
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS),UniversityofAdelaide,Adelaide,SA,Australia
K.T.Huang
BrighamandWomen’sHospital,HarvardMedicalSchool,Boston,MA,UnitedStates
K.Huber
ResearchUnitAnalyticalPathology,HelmholtzZentrumMu€nchen,Neuherberg,Germany
E.E.Jones
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
J.Kriegsmann
ProteopathGmbH;InstituteofMolecularPathology;CenterforHistology,Cytologyand
MolecularDiagnostics,Trier,Germany
M.Kriegsmann
InstituteofPathology,UniversityofHeidelberg,Heidelberg,Germany
N.Lauzon
Universit(cid:2)edeMontr(cid:2)eal,Montreal,QC,Canada
E.Laws
BrighamandWomen’sHospital,HarvardMedicalSchool,Boston,MA,UnitedStates
R.Longuesp(cid:2)ee
ProteopathGmbH,Trier,Germany
S.Lou
LeidenUniversityMedicalCenter,Leiden,TheNetherlands
S.Ludy
BrighamandWomen’sHospital,HarvardMedicalSchool,Boston,MA,UnitedStates
Contributors xi
L.A.McDonnell
FondazionePisanaperlaScienzaONLUS,Pisa,Italy;LeidenUniversityMedicalCenter,
Leiden,TheNetherlands
D.F.McGinnity
DMPK,OncologyIMED,InnovativeMedicinesandEarlyDevelopment,AstraZeneca,
Cambridge,UnitedKingdom
J.S.McKenzie
ImperialCollegeLondon,London,UnitedKingdom
A.S.Mehta
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
P.Mittal
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS),UniversityofAdelaide,Adelaide,SA,Australia
N.H.Patterson
MassSpectrometryResearchCenter,VanderbiltUniversitySchoolofMedicine,Nashville,
TN,UnitedStates
T.W.Powers
MedicalUniversityofSouthCarolina,Charleston,SC,UnitedStates
S.Santagata
BrighamandWomen’sHospital;Dana-FarberCancerInstitute,HarvardMedicalSchool,
Boston,MA,UnitedStates
K.Schwamborn
InstituteofPathology,TechnischeUniversit€atMu€nchen(TUM),Munich,Germany
N.Strittmatter
DrugSafetyandMetabolism,AstraZeneca,Cambridge,UnitedKingdom
Z.Takats
ImperialCollegeLondon,London,UnitedKingdom
A.Walch
ResearchUnitAnalyticalPathology,HelmholtzZentrumMu€nchen,Neuherberg,Germany
L.Winderbaum
AdelaideProteomicsCentre,SchoolofBiologicalSciences,UniversityofAdelaide,
Adelaide,SA,Australia
C.Zhang
AdelaideProteomicsCentre,SchoolofBiologicalSciences;InstituteforPhotonicsand
AdvancedSensing(IPAS),UniversityofAdelaide,Adelaide,SA,Australia
PREFACE
ItwasthepioneeringworkperformedinthelaboratoryofRichardCaprioli
thatledtothecurrentwidespreadapplicationofimagingmassspectrometry
tocancerresearch(Caprioli,Farmer,&Gile,1997).Usingspatiallyresolved
matrix-assistedlaserdesorption/ionization(MALDIimagingmassspectrom-
etry) they demonstrated how the distributions of hundreds of proteins in a
tissue section could be simultaneously recorded, without prior knowledge
and without labeling. Shortly after this first technical report it was demon-
stratedhowthetechniquecouldrevealdifferentialproteinexpressionprofiles
throughouttumorsamples,includingproteinslocalizedinthetumor’sinfil-
tratingzone(Stoeckli,Chaurand,Hallahan,&Caprioli,2001).
Imaging mass spectrometry has grown rapidly, in terms of application
andtechnology.Newtechnologiesnowenablehighspeed,highspatialres-
olution, and high mass resolution analysis; new tissue preparation methods
enable the label-free analysis of diverse molecular classes, including hor-
mones,peptides,proteins,proteinisoforms(Chapter2),lipids(Chapter3),
glycans(Chapter4),metabolites(Chapter5),anddrugs(aswellasthedrug’s
metabolites)(Chapter6);newdataregistrationmethodsenabletheseamless
integration of imaging mass spectrometry with histopathological analysis
andinvivoimaging,inbothtwoandthreedimensions;andfinallynewdata
analysisroutineshaveenabledallofthesetoolstobeusedtoinvestigatethe
molecular changes that accompany cancer development. While earlier
applications of imaging mass spectrometry focused on the identification of
diagnosticbiomarkers,thetechniquehasdevelopedrapidlyandisnowused
toidentifybiomarkersofsurvival,metastaticstatus,hormonereceptorstatus,
microvascularinvasion,andpatientresponsetotherapy;thetechniqueisalso
used to investigate intratumor heterogeneity, from tumor cell density,
tumorcelltype,andevensubpopulationphenotypeperspectives(Chapter8),
and intraoperatively to rapidly assess tumor margins during surgery
(Chapters 9 and 10).
In part the developments in imaging mass spectrometry reflect the
demandsoftheapplicationareas,aswellastheactiveinvolvementofscien-
tists from different disciplines. Pharmaceutical analysis (Chapter 6) is now
established as one of the principal application areas of imaging mass
spectrometry owing to its ability to help reduce the cost of developing
and testing new drugs and drug formulations.
xiii
xiv Preface
The active involvement of pathologists in the application of imaging
mass spectrometry is of paramount importance (Chapters 1 and 7); it is
thecombinationofhistologyandimagingmassspectrometry,enablingmass
spectralprofilesofspecifichistopathologicalfeaturestobeobtained,thatis,
theuniquestrengthofthetechniqueforcancerresearch.Inthisissueaseries
ofreviewscoverhowthetechniquecanbeappliedincancerresearch,both
fromadiagnosticandbiologicalperspective.Thewidearrayofbiomolecular
targets central to cancer development and progression that can be detected
in cancer tissues and emerging applications of imaging mass spectrometry
technology are also evaluated.
RICHARD R. DRAKE
LIAM A. MCDONNELL
REFERENCES
Caprioli,R.M.,Farmer,T.B.,&Gile,J.(1997).Molecularimagingofbiologicalsamples:
LocalizationofpeptidesandproteinsusingMALDI-TOFMS.AnalyticalChemistry,69,
4751–4760.
Stoeckli, M., Chaurand, P., Hallahan, D. E., & Caprioli, R. M. (2001). Imaging mass
spectrometry: A new technology for the analysis of protein expression in mammalian
tissues.NatureMedicine,7,493–496.
CHAPTER ONE
The Importance of Histology and
Pathology in Mass Spectrometry
Imaging
K. Schwamborn1
InstituteofPathology,TechnischeUniversit€atMu€nchen(TUM),Munich,Germany
1Correspondingauthor:e-mailaddress:[email protected]
Contents
1. ImportanceofPathology 2
2. PossibleErrorsCausedbyTissueInherentFactors—WhyHistologyIsImportant
forSupervisedAnalysis 4
3. PossibleErrorsCausedbySampleInherentFactors—SmallPretherapeutic
Biopsies 9
4. PossibleErrorsCausedbySamplePreparations—ArtifactsAreNotYourFriend 12
5. PossibleErrorsCausedbyIll-DefinedSampleGroupsintheTrainingSet—KeepIt
BlackandWhite/ShadesofGrayAreNotWelcome 16
6. Conclusion 19
References 20
Abstract
Massspectrometryimaging(MSI)hasbecomeavaluabletoolincancerresearch.Even
more,duetoitscapabilitytodirectlylinkmolecularchangeswithhistology,itholdsthe
prospecttorevolutionizetissue-baseddiagnostics.Inordertolearntowalkbeforerun-
ning, however, information obtained through classical histology should not be
neglectedbutratherusedtoitsfullcapacityandintegratedwithmassspectrometry
data to lead to a superior molecular histology synthesis. In order to achieve this,
pathomorphological analyseshavetobeintegrated intoMSIanalysesrightfromthe
beginningtoavoiderrorsandpitfallsofMSIapplicationpossiblyleadingtoincorrect
orimprecisestudyoutcomes.Sucherrorscanbecausedbydifferentsampleortissue
inherentfactorsorthroughfactorsinsamplepreparation.Futurestudiesshould,there-
fore,aimforacomprehensiveincorporationofhistologyandpathologycharacteristics
toensurethegenerationofhigh-qualitydatainMSItoexploititsfullcapacityintissue-
basedbasicandtranslationalresearch.
AdvancesinCancerResearch,Volume134 #2017ElsevierInc. 1
ISSN0065-230X Allrightsreserved.
http://dx.doi.org/10.1016/bs.acr.2016.11.001
2 K.Schwamborn
1. IMPORTANCE OF PATHOLOGY
The old saying that “a picture is worth a thousand words” is doubt-
lesslyalsotrueinscience.Interpretationofhistomorphologicalchangeshas
substantially contributed to the understanding of physiological and patho-
logicalbiologicalprocessesindifferentorgans.Recently,however,thefocus
hasshiftedtowardaddingadditionalmolecularcomponentstosuchconven-
tionalimagingtechniques.Thisso-calledmolecularhistologyissupposedto
reveal the underlying biochemistry of tissues and organs, while simulta-
neously providing additional information on how therapeutics or toxins
influencethefunctionormisfunctionofanorgan(Vachet,2015).However,
correct interpretation of these molecular “pictures” can only be done in
conjunction with simultaneous interpretation of the underlying conven-
tional histomorphological “picture” by trained pathologists/morphologist.
Having a profound knowledge and understanding of the biological basis
of disease is the key for correct interpretation of tissue phenotypes. While,
forexample,biologistandotherscientistscanfulfilsomeofthoseneeds,only
a trained pathologist/morphologist is able to integrate the morphological,
clinical,andmoleculardimensionsofastudieddisease.Abiologistcancer-
tainlylearnspecificmorphologyinterpretationinselected(patho)physiolog-
icalsettings;however,forafullscalecomprehensiveinterpretationusuallya
trainedanatomistorpathologistisneeded,whohastrainedontheinterpre-
tationofthewholespectrumofpossiblehistomorphologicaltissuechanges
overseveralyears/decades.Nevertheless,sinceonlyafewtrainedmorphol-
ogistsarededicatedtoresearchandevenlessareworkinginthefieldofmass
spectrometryimaging(MSI),theinterfacebetweenbasicresearchanddiag-
nosticmorphologyisprobablytheweakestlinkinthewholeinterpretation
chaininmolecularimaging.Ithasbecomeevident,however,thatinorder
toadvancethetranslationofbiomarkerdiscoveryintodiagnosticandther-
apeuticapplication,morphologistsandespeciallypathologistshavetoplaya
major role in this endeavor (Salto-Tellez, James, & Hamilton, 2014).
Histology, the study of the microscopic structure of tissues, is an
integral part of daily pathology routine. According to the Oxford English
Dictionary, pathology is the science of the causes and effects of diseases
(Stevenson,2010);inparticular,however,theterm“pathology”alsostands
forasubspecialtyofmedicinethatdealswiththelaboratoryexaminationof
samplesofbodytissuesfordiagnosticpurposes(Stevenson,2010).Pathology
bridges science and medicine and underpins every aspect of patient care,
ApplicationsofMassSpectrometryImagingtoCancer 3
from diagnostic testing, quality control, and treatment advice to the use of
cutting-edgegenetictechnologies andthepreventionofdisease.Theanal-
ysisofcytology,biopsy,orresectionspecimenoftenrepresentsthefinalstep
indiagnosticprocessesandhasremainedthelinchpinofcancerdiagnosisfor
over 100 years (Lakhani & Ashworth, 2001). This knowledge provides a
vitalservice;thepathologyreportisintendedtoguidethephysicianinfind-
ingtheoptimaltreatment foraparticular patientandbuildstheframework
for future selection of new markers and new therapies for a given patient.
In the era of individualized medicine, standard clinical care is continu-
ously becoming more complex, resulting in more complex and extensive
diagnosticpathology workup. Thisbecomes obviousby comparingguide-
lines for tissuepreparationof lungcancer surgical specimens,wheresimple
histochemistrystainssuchashematoxylinandeosinandperiodicacid–Schiff
stain were sufficient in the 1960s. In contrast, today’s pathology workup
requires a plethora of additional information, including subtyping based
on a number of immunohistochemical stains, semiquantitative assessment
of growth pattern in the case of a pulmonary adenocarcinoma, and often
additional molecular analyses. The increasing complexity in pathology
workupisalsoreflectedbyamorethan60%increaseinthenumberoftissue
blockspercaseandslidespertissueblockoverthelastdecade(e.g.,immu-
nohistochemistry and PCR) (Warth et al., 2016). Especially in the field of
oncology, cell- and tissue-related molecular diagnostics results from tech-
niquesincludingimmunohistochemistry,fluorescenceinsituhybridization,
and extraction-based molecular diagnostics such as PCR and sequencing
today determine therapy decisions. And although extraction-based molec-
ular diagnostics such as PCR and sequencing are molecular tests, they still
strongly rely on an initial pathological examination of tissue samples to
ensure sample adequacy, correct annotation of tumor areas for possible
tumorcellenrichment,togetherwithestimationofthetumorcellpercent-
age.Inparticular,thepercentageoftumorcellswithinagivenareaselected
forextractionwillultimatelydeterminetheyieldandpossible“dilution”of
tumorDNAbyothercelltypeswhichisessentialfortheinterpretationand
accuracy of molecular test results. Consequently, inadequate sampling can
result in a false-negative test result due to insufficient tumor DNA yield
andultimatelyinthewrongtherapy(Thunnissenetal.,2012).Thus,correct
aprioriinterpretationandannotationoftissuemorphologybypathologistsis
key for the correct outcome of molecular testing.
Alongthesameline,pathologicalevaluationisalsoofutmostimportance
in MALDI imaging. This is obvious in all histology-directed approaches
4 K.Schwamborn
where only small predefined areas of a sample are analyzed (Cornett et al.,
2006).However,histologycoannotationisalsoofparamountimportancein
allotherMSIapproaches.Possiblepitfallscausedbyinexperience,explana-
tions, and solutions will be given within the following paragraphs.
2. POSSIBLE ERRORS CAUSED BY TISSUE INHERENT
FACTORS—WHY HISTOLOGY IS IMPORTANT FOR
SUPERVISED ANALYSIS
With the advent and rise of molecular biology predictions on
substituting histological assessment with molecular analyses were made.
However, until now traditional microscopic morphological tissue analysis
isstillthegoldstandardandtheconditiosinequanonforsubsequentmolec-
ular testing. The rationale behind that fact is the need for high-quality
microscopic tissue analysis prior to any other molecular test to establish
adequacy of the sample (Szasz, Gyorffy, & Marko-Varga, 2016).
Thereisanequivalentneedforhigh-qualityhistologicalexaminationin
mass spectrometry imaging, in particular in studies with supervised data
analysis.Thesetypesofstudiestypicallyaimtounraveldifferencesbetween
two or more different sample sets, e.g., normal and diseased tissue or two
different subtypes of disease. Thus, in order to ensure, sample adequacy
severalfactorshavetobeconsidered.Firstofall,ithastobeestablishedthat
thesamplerepresentstherighthistologicaltypefortheintendedanalysisand
is representative of the underlying disease. Second, the number of disease-
defining cells within a given area has to be sufficient and above the detec-
tion/sensitivitylimitoftheanalysis.Additionally,thereshouldbenomajor
interfering cell component (e.g., inflammatory cells or red blood cells)
withinthetissueareaofinterestandnostrongmorphologicalheterogeneity.
To exclude samples with inadequate preanalytical conditions, features
suggestingthesamplehasbeencompromised(e.g.,autolysis)havetoberec-
ognized.Theseandmanyotheraspectsofhistomorphologicalinterpretation
oftissuesamplesrepresentanessentialfirststeptoensuretheinterpretability
ofthesubsequentimagingmassspectrometryexperiment.Onlycorrectand
precise annotation can lead to valid, robust, and meaningful data. In the
following paragraphs, these aspects will be discussed in detail using the
example of cancer.
Whencomparingdifferenttumortypes,itisobviousthatthesamplehas
to contain tumor cells. On the other hand, when comparing tumor and
