Utilization of Ancillary Studies in the Cytologic Diagnosis of Respiratory

Page 1of30 between thisversion andtheVersion record. Please citethisarticle asdoi:10.1002/dc.23549. through thecopyediting, typesetting, pagination andproofreading process,whichmay lead todifferences This istheauthor manuscriptaccepted forpublicationandhas undergonefullpeerreview buthasnotbeen Pathology, FoxChasePathology, Cancer Center,Temple Universi Pathology andPathologyLaboratory Medicine,University ofPe University University of SchoolMichigan ofMedicine,Ann Arbo Medical Center, Medical New York, NY; 1 Respiratory Cytology Papanicolaou SocietyThe of Cytopathology Consensus of Ancillary Utilization the in Cytologic Studies D Fax: (573) Fax: 884-4612 (573) Phone: 882-1201 11 1 5

Roh, PhD, Roh, MD, York, NY; York, of Theof Pathology, University ofTexas AndersonMD C University University Jackson,of Mississippi, MS; Email: Email: Columbia,MO65212 USA Hospital Drive,One Sciences Medical M263 Building University of Missouri ofDepartmentPathology and Sciences Anatomical andChair Professor J.Layfield, Lester MD Address correspondenceto:

Department of Department Pathology Sciences,and Anatomical Un Geisinger, Kim MD, J. Layfield, Lester MD, Department ofPathology Department andLaboratory Medicine, Th [email protected] 8 Department of Pathology, HarvardDepartmentPathology, of Medical Bo School,

Accepted Article10 Fernando MD, Schmitt, PhD,

Diagnostic Cytopathology 6 Department ofandPathologyCell Biology, Columbia

11 7 OscarLin, PhD, MD, Nikoletta Sidiropoulos, Nikoletta MD nnsylvania, nnsylvania, Philadelphia, PA; r,MI; ancerCenter,Houston, TX; ty, Philadelphia,ty, PA; iversity iversity of Columbia,Missouri, MO; e University e Vermont, of VTBurlington, 10 3 ZubairBaloch, MD, PhD, Laboratoire National Laboratoire Sante, de Luxembourg; iagnosis iagnosis of Respiratory Lesions: ston,MA; 8 Neal I.Neal Lindeman, MD, Recommendations for 5 DepartmentPathology, of 9 DepartmentPathology, of 3 4 Department ofDepartment Department of Department 4 Hormoz Ehya, Hormoz MD, ancer Center,New ancer 9 2 Michael Department University

title: Running

Accepted Article Consensus Recommendations forAncillary Testing of

2 Page 2of30

Page 3of30 cytology Keywords: includingsequencing next-generation discussed. are testing Ancillary including immunocy microbiologic, present document The summarizes recommendations for selectively documents present the results ofthese andfeedback review, from at presentations national recommendationdocuments are based the expertiseon andclassificationcriteria, ancillary testing and ultrasound endobronchial guidedneedlefine aspirat includingforsputum indications examination, bronc Papanicolaou The Society ofCytopathology devel has

Acceptedimmunocytochemistry, cytometry,flow dia molecular Article

This article isprotected by copyright. All rights reserved. Diagnostic Cytopathology John Wiley& Sons post-cytologicdiagnosis management andfollow-up. Abstract discussions. tochemical,cytometric,flow andmolecular testing, hialandwashingsbrushings, CT-guidedFNA and

and international conferences.The guideline ion(EBUS-FNA), as as recommendationswell for opedsetof a guidelines for respiratory cytology ofcommittee extensiveanliteraturemembers, ancillary testing cytologic of samples. gnostics,next-generation sequencing, All

of a a sampleforcytologicof flow cytometry testing. into RPMImaterial (Roswell InstituteMemorialPark block preparations block all can for usedimmunohistoc be andparaffin embedded(FFPE) block cell is material immunocytochemistry(ICC) betoappears formalin-fi its need ownvalidationprocess becan which cumber specimen this typemostclosely approximates howsm conventionally is material convenient from most a m

lower respiratory lower tracthas greater obtained import the adventWith targeted forlung of therapy cancer evaluation with evaluation identification ofthe pathologicpr performance.test for optimal Rapid evaluaon-site the Some ancillary of testing require methods dedic carcinoma’s a indicate susceptibility specificto d nucleotidesingle variants,insertion/deletions, co documentto testing, presence the orabsence cli of Targeted therapeutic have expandedoptions need the immunohistochemical andhistochemical stains aid to techniques culture for microbiologic floorganisms, testing including testing microbiologic culture, flow cytom media requirements media for microbiologic haveb culture

Accepted Article

This article isprotected by copyright. All rights reserved. Ancillary Ancillary TestingRespiratoryfor Cytology Diagnostic Cytopathology John Wiley& Sons Committee IV 2 rugtherapies (personalized medicine). While smears, liquid preparations,based and cell py number py andstructural variations, variantsthat ocess andappropriate ocess triage of forspecia material etricevaluation, andmolecular testing. Transport w w cytometryforproliferations,lymphoid and tion intraprocedural(ROSE) allows cytologic preferred by somelaboratories. ance.Traditionally, ancillary testing was confine nicallyrelevant genetic alterations include: that atedtransfer media special or preparation methods , , testing ancillary specimens of derivedfrom the ) ) a is medium standardapproach forthe submission hemistry and hemistry most moleculartesting, fixedformalin olecular pathology olecular validationstandpoint because eenpublished. xed,paraffin-embedded tissue recent(FFPE).A some. best preparationThe for inclassificationpulmonary of neoplasms. all biopsiesall are processed doesnot and hence for ancillary testingfor and in particular molecular 1 Direct placement of aspirated Direct 3-8 FFPE cell blockFFPE

dto lized

Page 4of30 Page 5of30 bronchoalveolar lavagebronchoalveolarand needlefine aspiration ( ofpulmonary Culture represents specimens importan are agents best definitively byidentified a variet sent be forshould culture. FNApulmonary specimens. ROSE examination be may h paraffin-embedded specimens.Thesefindings bring validation. types Other ofspecimens should individually be val andICC probable for the need forrevalidation a of demonstrated study problems with validationinnear cell blocksystem cell (fixed inPreservCyt). The lungfavored a is The site for nulocalization a of have been identifiedhave forlymph node aspirates number a culture of of requiresorganisms specializ is andthebacteria often most sensitive technique theirfacilitating recognition. While viruses are may be responsible be may fordisease.pulmonary Viralin numbergranuloma. large A infectious of agents inc producecan changesdiffuse (lobar pneumonia) ormo

Accepted Article

This article isprotected by copyright. All rights reserved. 9 Diagnostic Cytopathology The antibodies The previouslyhad been validatedforf Microbiologic Microbiologic Culture John Wiley& Sons 1 andsimilar techniques and used be mediacan for rarely byinvestigated culture methods,infec other mber mber ofinfectiousprocesses. Theseinfectious age y of culture y techniques.of ntibodies when used.methanol-fixed is material for specificestablishing a diagnosis. Successful fections fections haveoftencharacteristic cytopathic effec edand techniques media.Optimal transport media idatedandrequire libraries for of controls optimu FNA)Culture specimens. for bacilli, fast acid fun ludingfungi, bacteria, viruses, andmycobacteria

intoquestion utility the of methanol-fixedmateria lyonehalf antibodies of using tested the Cellient ant component ant of examinationfor sputum,many re localized re includingchanges andabscesses elpfulinselectingand ifin what media material ormalin-fixed., tious nts nts gi ts m m

l 5

adenocarcinomas fromadenocarcinomas squamous cell carcinoma behas the developmentWith oftargeted therapy forpulmon immunocytochemistry. by identificationofsmall and large neuroendocri cell disease.metastatic separation The primary of aden Immunocytochemistryan is diagnostic important tech appropriate techniques. culture Organization guidelinesOrganizationrequire evaluation of Ki-67 isImmunochemistry important inthe evaluation ofn andidentifyinhibitorsto targets forimmunotherap Finally, neoplasm. immunocytochemistry can used be differentiation can particularlymarkers be helpful siteprimaryoffor origin disease.metastatic The been foundhave foruseful distinction. this Immun Enumeration Enumeration stainingof Ki-67 performed be can c on sent forculture sent inappropriate transport media.R Recommendation 1: adenocarcinomafrom squamous cell carcinoma. TTF-1,napsinCK5/6, p63,A, andp40 havebeen iden

Antibodies Useful Antibodies SeparatingPulmonary In Adenocarc Accepted Article

Cytologicspecimens from lesions suspectedbe to of

This article isprotected by copyright. All rights reserved. Diagnostic Cytopathology Immunocytochemistry John Wiley& Sons 10, 14-24 use differential cytokeratinof staining anddirec necarcinomas andcarcinoid facilitattumors be may inestablishing site a the metastatiof for origin apidevaluationon-site be may helpfulinselecting index forclassification ofthese neoplasms. ohistochemistryalso is aid in of determining the y such PD-L1.y as ocarcinomafrom squamous cellcarcinomas andthe

tifiedhelpful as forseparating pulmonary ell blockmaterial. ell euroendocrinetumors. The Health World nique for the identificationnique for andclassification of for prediction response for of tyrosineto kinase ary adenocarcinomas, ary the separation of Theseantibodies can used be liquidon smears, comecriticallyimportant. inomasSquamous from Cell Carcinomas 12,13 infectious etiology be should

10 Panels of of antibodies Panels 11

c c tionof the the ed ed 6 Page 6of30 Page 7of30 sensitivity and sensitivity specificity separaof TTF-1for the carcinomas are approximatelycarcinomas 85% primarily theprimarily within ovary andsites. other replacingp63. appears p40 specific squamous more for cell carcino Core biopsy beCore should encouraged forback-up, espec it becarcinoma should kept in mind adequate that m performing p63. When immunohistochemistry fordist include,(i)panels TTF-1,napsin CK p63,A, ( 5/6; pulmonary adenocarcinomaspulmonary squamous from cell carcin of squamous of cell carincoma. squamouscarcinomas withsensitivity a and specific carcinomas carcinomas lung. of the authors have Other alsobefoundto superior p40 to TTF-1 reacts TTF-1 with cells differentiating towards fol distinction of pulmonary distinction of adenocarcinoma from squamo authors number haveA of panels recommended two of Napsin A has been shownNapsin been hasA reactive be to with pulmona adenocarcinomas, is reactive is inup tothird pulmonary one of adenoca p63 is a p63 helpful indistinguishingmarker squamous reliability questioned the use ofthe of immunocyto preparations,based andformalin-fixed paraffin emb 10

Acceptedap40has andsensitivity a of 94% specificity of Article

17,29 Diagnostic Cytopathology and97% 30 John Wiley& Sons The sensitivity The andspecificity fornapsin inse A 29 ii)TTF-1and p40; napsin (iii) andA or(iv)TT p40; tion ofadenocarcinomapulmonary from squamous cell licularthyroidepithelium, respectively. respectively. celladenocarcinoma.carcinomas from rcinomas, chemistry on methanol-fixed block cell material. ity respectivelyof 100%and 85% for the recognitio p63for identification the of squamouscell mas than p63masthanand some authors haveadvocated its eddedblock cell A material. studyrecent has aterialbe preserved must formolecular testing. ially when ially ROSE thatshows sample the cellularity ry epithelium ry inction inction of adenocarcinomasquamous from cell omas are 85% andare 85% omas respectively. 94% uscell carcinoma ofthe lung. to four immunohistochemical four to markers forthe 10 it remains useful forremains separating it 96% forsquamous carincoma. cell 20 and some neoplasms arisingand some 25 arcinomas. somegastric 29,31-36 27,28 29,31 The The Potential Potential parating 17 While p63 p63 While

F-1and 29 9

n 7

positive, specimenpositive, the can atoreflexed be molecul predictive some Immunocytochemical markers. techni molecular methods (FISHorPCR-based techniques), i predictive While testing forsusceptibility tyroto directed against directed rearranged detecting specific detecting mutationsbase (15 pairdeletion TTF-1 andp40 TTF-1 appears forseparation optimal ofade preservationof cellular forrequested material or squamouscell carcinomashould selected. be Excess tissue forpreserve subsequent molecular testing, p recommended recommended significant when differentiati cellular panelsimmunohistochemical including somecombinati Pulmonaryadenocarcinomas beshould distinguished f Recommendation 2: utility of this utility approach of mayAntibodielimited. be Because diagnostic Because material savedfor must be molec immunocytochemistryinthe sub-classification no of adenocarcinomas. Johnsonadenocarcinomas. al et considered be should when using immunocytochemistry be low might too foranalysis.

Accepted Article

Immunocytochemistryfor Predictive Molecular Marker This article isprotected by copyright. All rights reserved. ALK 34 Kimbrell etal Kimbrell , 38 39-41 has describedhas double a staining method forTTF-1 a

ROS1 Diagnostic Cytopathology , and , mutated John Wiley& Sons 37 addressed the issues concerningthe addressed utilization the of sine sine kinaseinhibitors generally is by performed s directed s against mutated anticipated testing. molecular combination The of sin and19p.L858R exon inexon mutation andf 21) referably adenocarcinoma one marker of and one of ar laboratoryfor ar confirmatoryAntibodies testing. ondistinct as such keratinization notseen. is To n-small cell lungn-small carcinomas. nocarcinomasform squamous carcinoma. cell ive immunostainingive should toavoided be ensure mmunocytochemical methodsforexist testing of ularusetesting, the double stainingof techniques rom squamous rom carcinomas. cell Use of ques have been usedhave been ques initial as tests whenwhich on of TTF-1,napsin on A, of p40p63,and CK 5/6 is fordistinguishing squamous from EGFR 42,43 are three such However, three markers. theare EGFR s are limited are to ndNapsin A.

ail

8 Page 8of30 Page 9of30 carcinomas carcinomas lung. of the the expression endogenousprotein has of shown been against aberrations that aberrationsthat low occurat frequency such as immunohistochemistry Currently, a be reduc may cost have developed clones been inclinical andare use. antibody detects antibody expressedendogenouslevels oftot D5F3 antibodythe (Cell Signaling) testing per with with subsequent with reflexpositive results of FISHto Immunocytochemicaltesting for mutated Recommendation 3: technique fortechnique Food and AdministrationFood Drug (FDA) approvedimmunoh results. detect to other Immunocytochemicaltesting rearranged for ALK may b limited volumesample whenmolecular testing cannot susceptibility tumor theto tyrosine associated kin overall cost theoverall as percentage ofadenocapulmonary 43-46 ROS1 Immunohistochemistry appears Immunohistochemistry beto useful a scree anddetect the inappropriately expressedendogenou ROS1

Accepted Article EGFR

rearrangementswith subsequent reflexpositive of

mutations;while the 47 Antibodies raised Antibodies against This article isprotected by copyright. All rights reserved. PD-L1 ImmunocytochemicalPD-L1 Testing Diagnostic Cytopathology EGFR John Wiley& Sons ALK antibody havefalse may positive negativ andfalse is not is preferred testingthe fordeterminat method ROS1 analysis.approach This using reflextesting reduc ase inhibitors, ase but utilized be may inthe setting formed the BenchMarkon XT This instrument.

ROS1

al rcinomas with rcinomas . number A of antibodieshavebeen developed tion technique tion foridentification of molecular be performed. be to occurto inapproximately non-small 1.6% of cell ALK e used inplaceused ofe FISH testing. istochemical assessing method protein represent may screeninguseful a protein (when present). Other antibody ningfortechnique s protein.Such s inappropriate ALK resultsfor FISHtesting. alterations low. is Currently,the ALK rearrangements ALK status uses 48 48 e of a of ion ion of es es 9

of response of therapyto depends drugon selectedthe squamouscell carcinomaand adenocarcinoma.Sectio havepembrolizumabbeen approvedfortreatment of n treatment for ofpatients when standardchemotherap surveillanceandimmune destruction. is sometimes PD-L1 expressed amountsin large ca on ofExpressionProgrammed Ligand-1Death is (PD-L1) have activity against have some including malignancies n with Nivolumab with therapy. Detection of expressingPD-L1 cells carcinoma i may non-squamous,non-small pulmonary cell carcinomas m depends testing specificon anti the druPD-1/PD-L1 become have refractory to standardchemotherapy reg Immunohistochemical anti testingfor thePD-1/PD-L1 Recommendation 4: made at this made time. published inthe literature recommenda andspecific

Accepted Article 53 PD-L1 testingPD-L1 of cytology specimens nothasunder This article isprotected by copyright. All rights reserved. 49,50 49,50 Diagnostic Cytopathology A new class of drugs class target of new andA are PD-1 r or PD-L1 John Wiley& Sons on-small cellon-small lung cancers. g Immunocytochemicalused. for testing inPD-L1 ndicate improved ndicate survival patients are when treated tionsfoits for cytology use cannotbe material .protocols Testing havebeen published. apredictive foranti-PD-1/PD-L1 marker therapies. ncer cells andncer allows from their immune escape rapyappropriateappears forsome patients who nofthe appropriate antibody cloneforprediction y has becomehas y ineffective. andNivolumab iments. Selection iments. antibody usedof the for on-small lung cancer,cell on-small including both ayaidinthe selection of targeted therapy. 51,52 goneextensive validation Thesedrugs are useful eported to eported 52 52 10

Page 10of30 Page 11of30 patients for useful non-responsive tyrosineto kina testingPD-1/PD-L1performed is at the discretion o and some andsome chemotherapeutic used agents fortreatment

discretion the local of the team.oncology Currently, testingMET cannotbe recommended forro Recommendation 5: for amplificationMET is and performedFISH. is by testing testing havepotential may for predicting value pro occurs inup non-small occurs 20% to of cell carcinomas re elucidated, but elucidated, therapyc-MET based immunohistoc on roleimmunohistochemistry The of for prediction the Mesenchymal-epidermal transition( overexpression may beoverexpression may important in development the studies havesuggested studies utility forthe combined use carcinoma Nivolumabcarcinoma with done be withoutcan PD-L1 target for treatmenttarget the of non-small cell Immunocytochemistryfor andtheIdentification Clas

Accepted Article

This article isprotected by copyright. All rights reserved. Immunocytochemical for Testing c-MET MET Diagnostic Cytopathology ) receptor ) tyrosine has identifiedkinase been a as 54,55 John Wiley& Sons and some drug resistantsmalland cell some lung cancers. seinhibitor therapies. Treatment ofsquamous cell gression targeted on therapy ( fractoryto f thelocaloncology and team be may especially ofdrugs inresulting of response of inhibitorsofc-MET has beennotyet utine use,utine butsuch performedtesting be atcan sification ofMetastatic sification Malignancies thetoLung ofresistance EGFR-tyrosineto kinase inhibitors hemical staininghemicalshown has promise. testing. of lungof small cancer.cell EGFR -tyrosinekinase therapy andsome MET and 56 EGFR EGFR EGFR

MET ) butsuch ) testing co-inhibition. amplification potential 57 56 MET c-MET 54

54 11

squamouscell carcinomas. noThus, immunocy single lung.GATA-3, usually which supports urothelial a consideredsupportto gastrointestinal a origin may (a findingpositive consideredoften supportinga n primariesanddisease.metastatic instance, For so fororiginmetastases a is complexprocess with ove neoplasms.metastatic Immunocytochemical testing o Table neoplasm.lists Iselection a ofimmunocytoc andCD117 (SMA) among becan others extremely helpf (LCA),antigenCDX-2,p40, desmin,GATA-3, PAX-8, m lineage specific such markers HMB-45,as p melan-A, diagnose neoplasms.metastatic Differential cytoke combination of cytomorphologicfeaturesand immunoh stainingfor these markers not does excludelung a assuch markers napsin andA suggest TTF-1 may no a Metastaticdiseaseresponsible is a significantfor Selected panelsSelectedantibodies of beshould used est to Recommendation 6: disease.metastatic a specific for buttypeof neoplasm, patterns ofst suspected suspected diseasemetastatic theto lung. The prec

Accepted Article

This article isprotected by copyright. All rights reserved. Diagnostic Cytopathology John Wiley& Sons percentage lung nodules. of While result negative aininghelpful inassessingare the organ of origin origin, be may byexpressed somepulmonary me adenocarcinomaspulmonary be may CK20 on-pulmonaryorigin). Similarly,CDX-2 a marker hemical helpful markers inthe evaluation of origin,in particularly mucinousadenocarcinoma. A be in positive somemucinous carcinomas ofthe iseantibodies of panel should determined be by ablish the andorigindirection differentiation of rlappingofstaining betweenpatterns pulmonary ratinstaining patterns (CK7, and CK5/6) CK20, and rostaticspecific antigen (PSA),leukocyte common uscle specific actinuscle (MSA),smooth actinmuscle n-primary pulmonary n-primary adenocarcinoma, lack of tochemical stainingresult is diagnost definitively f samplesf determineto tumor typesite or of ul in the specific uldiagnosisinathe of metastatic istochemical reactionistochemical patterns is toused for in s s of 12 ic ic Page 12of30 Page 13of30 satisfactory substratesatisfactory for molecular testing haveStudiesdemonstrated formalin parafthat fixed guidance.Since performed is samplingunder endobronchial ultrasoun CBs beingacellular CBs orof borderline cellularityfo findings. morphologicanalysis ofcytologic specimen andrevi pathologists to performto pathologists molecular used studies i to adventnumber a The of of targeted therapiesforlu radiologist andradiologistpathologist. tissue acquisitionProper and g management requires the utility formalin-sixed, the of paraffin-embedded su but a number buta haveaddressed of studies use the ofc embedded cell block embeddedcell material, the use use direct the of smears,CBs, andliquid prebased block. testing. testing. Presently,the ofmajoritytargetedsuch is responsible he/she ensureto that adequate andr is pathologist Not the only foridentif responsible pathologist everyattempt must make possible obt to and ROS1 63 The choicethe appropriateThe of specimen typefor mol ), butnew therapies), targeting squamouscell carcin

Accepted Article Molecular Techniquesfor TestingPredictive Associa EGFR,ROS1, KRAS,

This article isprotected by copyright. All rights reserved. 10 ROSE can be be canhelpful very ROSE inassuring adequate tis 10,60-63 and ROSE can be be can very ROSE inusefultriaging material forp Diagnostic Cytopathology ALK John Wiley& Sons 64 testing can reliably be performed on fixe formalin butcellularity CBs problemremains a of with up t r moleculartesting. therapies are directedtherapies are adenocarcinomas at ( ying carcinomaswhich should testing,undergo but parations. data Current indicates that a variety o rgicalresection specimens or small biopsyspecimen dentifypatients likely respondto therapiesuchto ng cancer has placedhas ngcancer new responsibilities on epresentativematerial present is for ancillary ew of the patient’s ewofthe medical andimaging history ytologicpreparations. finblock embeddedcell (CB)is material a aintissue foradequate moleculartesting. ood communicationood between pulmonologist, d (EBUS) or computerized d(EBUS) or tomography(CT) ecular testing ecular remains controversial. omas are underomas are development. The ted Targetedwith Therapy 65 Most studieshaveMost reported on 66,67 These studies reviewed These suecollected is when rocessing cell to EGFR dparaffin o 57% of o 58,59

, , f ALK, s. 13 s, s,

having clinical utility.clinicalhaving Potentialtargetgenes in testing Molecular forpulmonary squamouscell carci rearranged tumors,rearranged immunocytochemical FISH testi or performed following performed resultsnegative for significant number significant number haveshownof that studies smear samples. preparationscytologic yield resumolecular testing related amplification related in performanceof prioritizationofthese beenhave markers publi yet erythroblasticleukemia homolog2viral oncogene ( the the impact of impact theselattertwo With thegenes. recent cancer. for for The of majority organizationsrecommend andauthors based techniquesbased alldetection were for useful ofc rates detection similarnotifsuperior histolog to cytologic that using techniques fixatio array an of 75 higherthosethan histologicobtained by sampling m comparable to comparable biopsies core insomestudies fordet ROS1, BRAF, ROS1, RET before In an analysis of 181 of181 In analysis anarticles focusing a on EGFR EGFR KRAS ALK 66 67-70 and geneproduct present is in control pathwaythe befo . 63,75 In some cases, Insome samples cytologic havebeen associ ALK Additional that be markers tested may inpulmonar

AcceptedKRAS Article

, MET, . 75,76

analysis. In general, when tissuelimited, is Ingeneral, neurotrophic tyrosine kinase receptor type ( 1 FGFR2,FGFR3 This article isprotected by copyright. All rights reserved. KRAS appear mutations excludeto in mutations , and , Diagnostic Cytopathology EGFR John Wiley& Sons FGFR4 and clude: fibroblast growthclude: receptorfactor ( 1 ic Moreover, blocks,material. cell smears, andli nand processing techniques yielded mutation . 78 nalysisinlung cancer,Cunha da Santos et al. lts similar to lts achievedsimilar those with histologic linicallyimportant in mutations lun non-smallcell FDAapprovalof crizotinib for oftreatment shed, some recommendauthorities early ERBB2 ALK ectionclinically importantof mutations. Other potential Other targetsare phosphatase andtensi ethods. nomaremainsinits infancy few with markers yet sareand CBs equivalent for molecular testing. ng for ngfor molecular molecular pulmonary testing of adenocarcinoma testing. EGFR ). 76,77 70 ROS1 Cell blocks haveblocksbeen Cell shown be to re re testingprioritized is andperformed While no recommendations no forWhile ated with detectionated with for rates mutations EGFR rearrangements nowbeshould y adenocarcinomas y include and NTRK1 EML4-ALK ) and ) v-erb-b2 EGFR , thus , negatingthe and FGFR1 EML4-ALK 71,72 ROS1 66 A KRAS, ) andthe showed quid

68,73- g and 14 n Page 14of30 Page 15of30 containing receptorcontaining ( 2 based testing.based frozen, fresh, biopsy), driedair smears, or alcoho EGFR (homolog substitutionsanddeletionssmall frame in exon19. confirmed by pathologist.confirmed a While specimens with 2 analysis in analysis laboratories,many clinical features.clinical Lower stagepatientsbe may tes

alcohol-fixed cytologic alcohol-fixed smears be usedcan reliably two mostcommonly two observed studies. The testingstudies. methodable be should deteto provided sample provided the meets analytic sensitivity the have laboratories reported results usingsuccessful for recommended EGFR selectionbased of therapy. disease (stageIII disease andhigher). EGFR mutations. mutations. stage I,Patients orbeII,III may tes beshould EGFR considered new recurrenceswhen or m of testing should of available be within workingda10 testingbe can performed formalin-fixed on paraffi testing mutation should performed be at time ofdi PTEN

67-72

Accepted Article ),platelet-derived growth factoralpha receptor ( While FFPE FFPE is material While one of the most commonsou DDR2 This article isprotected by copyright. All rights reserved. ). EGFR 75 75,79 76,78 76,78 Patients notshouldbe from Patients excluded such testing EGFR recent studies have studies recent shown that material obtainedf TestingPulmonaryFor Adenocarcinoma inpulmonary mutations adenocarcinomas the p.Lare Diagnostic Cytopathology John Wiley& Sons ted at theted discretionthe local of team.oncology l fixedl forpolymerase specimens chainreaction (PC ted recurrence time ted at of progression. or Retestin 76 . significantly lower concentration ofcancer cells, oftheused platform andwith appropriate validatio ys. 62,63,76,77 ct in mutations immunohistochemistry Currently not is 0% or0%greater cancer cells are prefered, 75

etastasesare detected to fornew look n-embedded n-embedded (FFPE)block/corematerial (cell agnosis forpatients agnosis presenting high with stage Sample adequacy forSample should analysis be PDGFRA EGFR rces forof material molecular exons 19, 18, and2021 ) ) anddomain-discoidin based solely based on rom air-dried rom and 858R 63 many many Results 75 as the the as

g ofg R) R) n 15

ALK recent data indicates recent that patients upof 3% to wit predictive valuepredictive of cells suitable cells forevaluation. When in is material techniques and techniques present arewhen thought excludeto more traditional more chemotherapyjustifying someto on EGFR

Moreover, patientsMoreover, with KRAS

Recommendation 7: results. for for This finding This question brings into on FFPE material,on alsobebutcan performed on smea anddiagnosison stage lower patients at re time of EGFR ALK rearrangement testing performed be should all on h mutational helpfulanalysis be in may therapysele testing priority.has FISH methodThe appears opt therapy selection. Acceptedrearrangements performed be may immunocytochemi by Article

KRAS

ALK response testingfor anti- to KR AS K RAS Rearrangement TestingPulmonaryfor Adenocarcinoma This article isprotected by copyright. All rights reserved. KRAS codon 13, 12, and146 61, mutations be can tested mutations havepoor may responses bothto KRAS TestingPulmonaryFor Adenocarcinoma testing as a guidetesting a as forresponse anti- to Diagnostic Cytopathology John Wiley& Sons sufficientforboth h currence or progression. currence or Testing performed be may K RAS

EGFR cologists,the need forearly rs.Ingeneral,slide the should contain cancer 50 mutations in mutations ction, butitction, shouldsole the not methodbe for imalfor igh stage lung ighadenocarcinomasstage at time of mutations respond will anti- to based therapybased remains controversial. Some ALK strywith FISHreflex forpositive to EGFR EGFR rearrangement testing. and and EGFR EGFR ALK ALK based therapybased and rearrangement testing rearrangements. KRAS for by for PCR-based therapy.

testing. The EGFR 41 Screening therapy.

16 80

Page 16of30 Page 17of30 abnormalities. abnormalities. Patients with and KRAS Recommendation 8: becan and forsmears used following testing approp team. Reflex team. testingto disease. disease. I,Testing of stage IIand III patients m EGFR

discretion the local of the team.oncology ROS1 FISH testing forFISH testing Recommendation 9: 1 to of1 non-small 2.5% cell carincoma EGFR, ALK, EGFR, pulmonaryhigh stage adenocarcinomas whohavebeen ROS1 ALK remains testing controversial and performed be may testing mutation should performed be at time ofdi rearranged pulmonaryrearranged adenocarcinomas are rare, on is a receptor a is phylogeneticallytyrosine kinase rel should tested be for and KRAS ROS1 Accepted Article

. Immunocytochemical testing for rearrangements be performed atmay the discretion ALK ROS1 This article isprotected by copyright. All rights reserved. should be performed be should when ROS1 ROS1 for Testing Primary Pulmonary Adenocarcinoma rearrangments appear benefitto from crizotinib th . 76 83,84 Diagnostic Cytopathology and are mutuallyexclusive andare with tyrosineother kina John Wiley& Sons ay be performeday be at the discretionthe local of onco

riate validationspecimenof this type. ated to ated ROS1 EGFR shown shown lack to molecular/geneticchanges in agnosis forpatients agnosis presenting high with stage pulmonary on non-small-cell carcinomas at ly patients lackingly changes in can becan acceptablean alternative FISH.to analysis mutational negative. is block Cell ALK . 81,82

ROS1 of localoncology of the in team rearrangements are seen in erapy. EGFR,KRAS 85 segenetic Since logy , , 17

andpleuralfluids. from extracted stained Papanicolaou low smears of t demonstratedthe high sensitivity NGS compared of t in pulmonary cytopathologyinpulmonary utilizingaspiratio both very smallamounts very DNA. recentSeveral of studies screening simultaneous genes inof amultiple massi profile for molecular cytologic specimens. Clinica is cancer, increasinganthere forcomp a need more precision In era an of with medicine new emergingb relatively low relatively tumor fraction. sensitivity analytical ofNGS (approximat platforms

dictates the quantitative dictatesthe DNAyield. theyarecomponents, frequently limited the by volu usually specimens inherentlyprovide high tumor fra cytology samples cytology andNGS rates success oflung FNAs andneedleadditional passes improve the may overal in EBUS FNA, samplesinEBUS brushing,obtained by orbody background tumor cells and/or wherethe tumor cells yield, due yield, overallto cellularity,low insuffici or common The causes most NGS failure of inlung cytol as well as exfoliativewell as as cytology (bodycavity fluids

Accepted Article SequencingNext-Generation and Molecular Analysis o

91 NGS success ratesdo notNGS success significantlyvary betwe

This article isprotected by copyright. All rights reserved. 87,91,93,94 89,93 One ofthe One utilizingearlier studies NGS cytol on Diagnostic Cytopathology Implementing measures such measures ImplementingROSE as for adequacy a John Wiley& Sons enttumor cellularity, incaseswith high numbers o andbronchoalveolar lavage) specimens. l next-generationl sequencing allowedhas (NGS) for elypermits5-10%) samples evaluation of with ncytology (CT-guidedFNA and EBUS-guided FNA) havedescribed the utility of analysNGS mutational velyparallel manner from usingsamplescytologic rehensive approach generate a to complete ctionlowertodue numbers ofstromalbackground me of me tissue aspirated (overallthatcellularity) iomarkersguiding therapeutic decisionsinlung umor volumesamplesumor oflavagebronchoalveolar cavityfluid samples. are similarto are that of sites.other ogic specimens ogic are relatedinsufficient to DNA oconventionaltesting by using methods cells l cellularitytol the input meet requirement DNA for are heterogeneously are distributed, frequently seen f Pulmonary Pulmonary f Carcinoma en aspiration en andexfoliative 88 Although cytology aspiration ogic ogic specimens 89

89,91,92 ssessment The high The f non- f 86-90 18 is is

Page 18of30 Page 19of30 Illumina NGS.Illumina block preparations block are comparableFFPE thattoh of reads, number of of mappedand reads read on-target NGSassay. the which allows forallows which evaluation of multiple biomarkers diagnostics Molecular inlung cytopathologyund has with significantly with higher DNAfrom theyield LBC ri successfullyperformed using extracted DNA from res eliminating background eliminating non-tumor cells,red thereby ordirectly section the smearon for slides macro/m (Illumina Inc, SanInc, (Illumina CA) Diego, platforms. between ranges ng10for Ion Torrent PGM Tech(Life employedforNGS. Cell blockpreparations Cell as as directwell smears an or cytological or stains/fixatives. type, roughly this type, translates approximatelyto 100 In this changingIn this landscapeprecision of t medicine, selecting suitable selecting successful material implemen for clinical trials for trials clinical targetedtherapy. mutations, have thesemay somerelevance inthe pro clinical proper contextfor subsequent clinical man populations,andvariants ofunknown ( significance but likelytherapy, will also passenger identify mu needs toin one keep mind NGS not detectsthat only 98 95

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Mar;25(3):405-15. Mar;25(3):405-15. p63to superior diagnosis forthe pulmonary of squa JA,Teruya-Feldstein Bishop Westra J, Pelosi G, WH, Accepted Article

th ed. Lyon:ed.International Agency for ResearchCan on

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Roy-Chowdhuri, S. Roy-Chowdhuri, doi:10.1371/journal.pone.0080478(2013). Kanagal-Shamanna,R. diagnostics. diagnostics. tumors usingsolid needle fine aspiration samples: multigenenextgeneration sequencingpanel. outcomes inpreviously outcomes non-small untreated cell lun registry of trials.clinical Clin Res. Cancer 2009 glioblastoma Natlcells. Proc USciAcad A.S 1987 C, Birchmeier Sharma WiglerS, M. andExpressionre development andcancer. development Biochim BiophysActa. 2009 J, Acquaviva CharestWong A. R, roThe multifaceted molecular class molecular lung cancers. of JClinOncol. 2012 EngelmanH, JA,Mino-Kenudson M, Pao AJ.W,Iafrate A, Gonzalez C, Fang R,Mark BattenJM,EJ, H,Chen W Shaw K, BergethonAT, Ou SH,KatayamaR, CM,Lovly doi:10.1002/cncy.21338(2013). Scarpa, A.Scarpa, Oncol. Oncol. Sep;24(9):2364-70.2013 rearrangement clinical on innever outcomes smokers Soo Haack RA, H,KimDR, HS,ChoJH, Shim BC. fr The HR, SM,Lim Kim HJ,KimHwang SK, Shin JK, ParkE, crizotinib inadvanced crizotinib non-smallcell lung cancer ( Camidge AT, Shaw DR,Engelman JA,Solomon BJ, Kwak aspirations of pulmonary aspirations of and neoplasms. pancreatic 2012 ASCO 2012 Meeting.AbJClin (suppl;2012Oncol 30,

AcceptedG. Young, Article et al.

et al. et et al. et Cancercytopathology Mod Pathol Mod Clinicalnext-generation sequencing successfullya Molecular oftypinglung adenocarcinomaon cytolog Effectiveassessment of egfr inbrmutation status al. et This article isprotected by copyright. All rights reserved. al. et Factors affecting the successnext-generation s of 27, 314-327,doi:10.1038/modpathol.2013.122 (2014) Next-generation Next-generation sequencing-basedmulti-genemutati Diagnostic Cytopathology 123, 659-668,doi:10.1002/cncy.21597 123, (2015). John Wiley& Sons 691-698,doi:10.1158/1078-0432.CCR-12-1958 19, Clinicalresearch canceran official : journal t of PloS onePloS Aug 15;15(16):5267-73. rgeted next-generation sequencingusing fine- Dec;84(24):9270-4. NSCLC)harboring rearrangement. ROS1gene Mar 10;30(8):863-70. promisesandchallenges forroutine clinical BaeSH,Ou MK, JewellJ,Wang SS,Kang g cancerg resultspatients: of an tumor online lesofthe receptor tyrosine ROS kinase in equencyandimpact of ROS1 abstr 7508). arrangementofthe ROS1gene inhuman Cancer cytopathology Cancer ilnerKwak KD, JW, EL,Clark JiCarbone DP, Cancercytopathology with lung with adenocarcinoma. Ann McDonald NT, McDonald Massion PP, Siwak-Tapp ROS1 ROS1 rearrangementsdefine unique a 8, 8, e80478, Jan;1795(1):37-52. EL,Clark Clinicalal. JW, et activity of ppliedfine-needleto onchoalveolarlavageand ical samples ical using a equencing incytology 122, 104-113, 121, 688-694, . on profiling on of he 28 Page 28of30 Page 29of30

100. 101. 99. 97. 98. 95. 96. 93. 94. 92.

tumor tumor profiling: mutation are weready forroutine ClinicalOncology of genomic of medicine. doi:10.1186/s12916-014-0140-3 140, (2014). British the forof Clinical Society Cytology evidence andfuture evidence directions. C., Bellevicine, Malapelle, Luca,Iaccar de C., U., doi:10.4132/jptm.2015.06.16 (2015). Dumur, I.&Dumur, C. Kraft,A. O. sequenc Next-generation cytopathology of next-generation sequencing:of cytopathologist's a Malapelle,Vigliar, E., deC.,U., Luca, Bellevicin

Histopathology Monaco, S. CytopathologyMonaco, E. ofLung Cancer: Moving Diagnostic Testing.Diagnostic H.The Roh, M. Utilization ofCytologicFine-Needle

cytopathology Cancer performedtests oncology cytology on specimens and Stence, M. Gailey, P., A.A., S.C.& Jensen, Ma,D Malignancies. AffectingFactorsSuccess the ofClinical Next-Gene H.,Luthra,Chen, Goswami, R., S.,R. Singh, R. R. sequencing: Aninstitutional sequencing: experience.

Wei, S. Wei,

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Tripathy, Harnden, Tripathy, D., Blackwell, K., K. Robson, & AcceptedJanne, A.J.A. Redig, & P. Basket and trials e the Article

Using"residual" FNAand body rinse fluid specimen 123, 69-70, 123, doi:10.1002/cncy.21515 69-70, (2015). Cancers 18, 313-320(2012). 18, 33, 975-977, doi:10.1200/JCO.2014.59.8433 975-977, 33, (2015). This article isprotected by copyright. All rights reserved. Journal pathologyof and translational medicine 123,doi:10.1002/cncy.21476 30-39, (2015). Journalclinicalof oncologyofficial ofjournal: 7, 1699-1715,doi:10.3390/cancers7030859 (2015). Diagnostic Cytopathology Diagn CytopatholDiagn John Wiley& Sons 26, 271-283, 26, doi:10.1111/cyt.12265 (2015). Cancercytopathology e,C. Troncone,Challenges G. & and opportunities ino,A. Troncone, G. & analysis: EGFR current & Roy-Chowdhuri, & S.Analysis of Pre-Analytic .Multiplatform comparison of molecular 42, 42, 984-992,doi:10.1002/dc.23142 (2014). Aspirates for of Lung Cancer Molecular rationSequencing Solid Organof use inthe clinic?oncology perspective. ingcytopathologist. andthe from Morphology from Molecular.to formalin-fixed, paraffin-embeddedformalin-fixed, tissue. volutionofclinical designin trial era an M. Next generation M.Next sequencing and , doi:10.1002/cncy.21666 , (2015). Cytopathology : officialjournal s fornext-generation s 49, 49, 300-309, the American the of Society BMCmedicine Cancer Cancer Diagnostic 12, 29

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