Xplore_9_Her2 & EGFR FISH

Embed Size (px)

Citation preview

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    1/24

    Xplore Newsletter Abbott Molecular

    Use of Fluorescentin-situ hybridisation(FISH) for selection ofpatients for targetedtherapies

    PART 1: HER2 Testing inBreast Cancer Patients

    PART 2: EGFR Testing inLung Cancer andColorectal Cancer

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    2/242

    Welcome to the latest issue of the Abbott Molecular Xplore Newsletter.

    This edition is split into two main subject areas, Part 1 covers some topical issues on HER2 testing in breast cancer, Part

    2 focuses on the rapidly developing field of EGFR testing for therapy selection in lung and colon cancer.

    Part 1. HER2 testing articles

    The first article from Dr. Jrme Couturier, Institute Pasteur, Paris, gives a very clear overview of the use of dual colorFISH for selection of breast cancer patients for trastuzumab therapy. This article will be interesting if you are new to, orunfamiliar with this application, and it highlights the current practice in most laboratories around the world, highlighting thecurrent guidelines for this testing and addressing some of the theories behind the development of this testing algorithm.

    Although current testing for HER2 status in the majority of laboratories follows the algorithm described in the Couturierarticle earlier, guidelines do not exclude use of FISH as the primary test, and there is growing evidence to support use of primary FISH testing as the most clinically accurate and cost effective protocol to select patients for trastuzumab therapy.

    This subject is thoroughly reviewed in the paper by

    Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. J Clin Oncol. 2009 Mar 10;27(8):1323-33, Guidelines for humanepidermal growth factor receptor 2 testing: biologic and methodologic considerations.

    As an example of two laboratories that have already followed the primary FISH protocol for some time we present the twoarticles from the Italian groups of Prof. Flora Bassano from Reggio Emilia and Prof. Luigi Serra from Forli. In these articles,the decision to follow FISH as the front line protocol is discussed in context of the clinicians specific request for bestquality data and how the FISH protocol (Forli are running in the region of 1000 HER2 FISH assays per annum) fits into theworkflow of the laboratory.

    The next article is from the laboratory of Prof. Sauter in Hamburg, and discusses how to interpret unusual signal patternsin HER2 testing of breast cancer. The article clarifies in very clear terms some of the reasons for unusual signal patternsand how these should be reported. This is an issue which continues to cause some confusion and we are very pleasedto be able to present such a clear guide to the interpretation of these unusual signal patterns.

    Finally, continuing the discussion on borderline HER2 score cases of breast cancer, the paper from the lab of Prof. Terrac-ciano in Basel, presents a very interesting comparison of the Abbott PathVysion probe for HER2 testing with a new Dualcolor SISH system. The article investigates concordance between these two alternative technologies specifically lookingat borderline cases and shows quite clearly that the two techniques are far from concordant for these problematic cases.

    Although some early publication have shown that different ISH techniques are highly concordant for clear cut cases, it isperhaps these borderline cases which cause most controversy and this article does, for the first time, raise some issuesover the direct comparability of FISH with SISH for HER2 testing.

    Dear Reader,

    In this Issue

    4 Dual-color FISH with directly labelled probes:optimal solution for testing the gene status of breast carcinoma with equivocal HER2 expression

    6 HER2 assessment on breast cancer in FISH frontline our 10 years experience.

    8Determination of HER2 by FISH and IHC: Experi-ence at Forli Department of Anatomical Pathology

    10 HER2 testing in breast cancer: reporting of unusualsignal patterns.1311

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    3/243

    Part 2. EGFR testing for therapy selection.

    We present an article from Prof. Pauwels from Antwerp, Belgium who presents a very interesting discussion on some of

    the issues surrounding how best to select non small cell lung cancer patients who might respond to the new anti-EGFRtargeted therapies. Anti-EGFR therapies present one of the most exciting area for targeted therapies. However, asdiscussed in this article, the case for EGFR is far more complex then it is for HER2 testing in breast cancer, with multipledrugs and multiple mechanisms of sensitivity and indeed resistance to these therapies. We are very pleased to presentthis concise, but non the less, comprehensive discussion of some of the issues surrounding anti-EGFR targeted therapies.Prof. Pauwels also discusses some other markers for resistance in NSCL which we anticipate will begin to make animpact in this field in the next few years.

    In the next article from Prof. Pelosi, Milan, data is presented on his findings of the use of FISH and EGFR mutation analysisin the selection of lung cancer patients for the targeted tyrosine kinase inhibitor drugs. Data is presented and some inter-esting conclusions drawn on how perhaps FISH and mutation analysis will be required for patient selection for these drugsin the future.

    The next two article expand on the EGFR therapy discussions with more specific examples of one of the main issues forpatient selection in NSCL and colorectal cancer, being how to standardise and achieve consistent cut of values for FISHselection of patients.

    The first of these two articles from the lab of Prof. Bubendorf in Basel, Switzerland discusses use of FISH for NSCL inbiopsies and cytology specimens, and proposes are new scoring system to allow for the two different tissue sampletypes.

    The second article from Dr. Martin, Locarno, Switzerland, goes on to discuss use of EGFR FISH in colorectal cancer,once again discussing the most appropriate cut off values for selecting patients for anti EGFR therapies.

    We are very pleased to be able to present to you these very topical articles and hope that you find them informative and

    interesting. They certainly address some issues at the forefront of molecular pathology and demonstrate how excitingthe future of molecular pathology is and some of the challenges which lay ahead.

    We hope you enjoy them.

    Yours sincerely

    Abbott Molecular Marketing Team

    12 Comparison of in situ hybridization (ISH) techniquesfor the validation of equivocal (borderline) statusof HER2 in invasive breast carcinoma: fluorescencein situ hybridization (FISH) versus automated silver-enhanced ISH (SISH)

    14 The contribution of FISH in non small cell lung cancer(NSCLC).16 FISH analysis of EGFR in metastatic adenocarcinomaof the lung: a clue of therapy18 The mean EGFR gene copy number: a different kindof scoring system with promising clinical implications

    for patients with non-small cell lung cancer

    22 Utility and advances of EGFR FISH in metastaticcolorectal cancer 18

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    4/244

    The HER2 gene (official but less used name: ERBB2) isone of the emblematic markers in Oncology, whose statusdetermines the eligibility to a targeted therapy in breastcarcinoma. This gene, located in the long arm of chromo-some 17, in 17q12, encodes the human Epidermal Growth

    Factor receptor 2 (HER2), a transmembrane receptor withtyrosine kinase activity. This protein is expressed at a lowlevel in normal cells, but is overexpressed in 18 20% of breast cancers. This has lead to the development of a mono -clonal antibody, the trastuzumab (Herceptin ) that targetsHER2 and has proved a remarkable therapeutic efficacy inpatients with a tumour overexpressing the protein, in bothmetastatic and adjuvant settings. The genetic mechanismleading to overexpression of the HER2 protein is the ampli-fication of the gene, an increase in its copy number in eachtumour cell. Thus, there is a strong correlation between theexpression of HER2 detected by immunohistochemistry (IHC)and the status of the gene defined by in situ hybridization.

    The HER2 testing algorithm

    Trastuzumab is active only in patients whose tumour over-expresses the HER2 protein. Furthermore, this treatmentmay have side-effects, especially cardiac, and is of highcost. Consequently, eligibility for the treatment requires areliable assessment of the expression or of the status of the gene in the tumours. The evaluation must be done oninvasive tumour cells, and not on the eventual in situ com-

    ponent that may be HER2 positive without clinical signifi-cation. This imposes the use of in situ techniques. Theconsensus testing algorithm involves IHC as primary screen-ing technique (Wolff et al., 2007), the results of which arescored, on the basis of the intensity of membrane stainingof invasive tumour cells, as 0/1+ (negative expression), 3+(positive expression), and 2+ (equivocal expression). Cases2+ must be checked by in situ hybridization to determinewhether the HER2 gene is amplified or not. Patients with2+ expressing and HER2 amplified tumours are consideredeligible for the treatment.

    The HER2 amplification

    Gene amplification is a specific kind of genetic rearrange-ment involving the selective duplication of a gene or, morefrequently, of adjacent genes forming an amplicon, in a givenregion of the genome. Typically, HER2 amplifications occurunder the form of homogeneously staining regions (hsr)visible in metaphase chromosomes of tumour cells and pre-senting as clusters of signals in interphase nuclei after in situ hybridization. These clusters may be fragmented throughfurther rearrangements. A difficulty in the assessment of thestatus of the HER2 gene is that its copy number increasemay be related not to an amplification but to the aneuploidyof the tumour, a common feature of breast carcinoma (DalLago et al., 2006; Rosenberg et al., 2008; Vanden Bempt etal., 2008). This aneuploidy leads, among other chromosomecopy number increases, to polysomy 17. IHC 2+ expression

    has been shown to be relatively common in tumours withpolysomy 17 but no HER2 amplification. However, IHC andmRNA expression analyses have shown that these tumoursdo not overexpress HER2 mRNA and do not express theprotein at the high level required for an efficacy of the trastu-zumab treatment (Downs-Kelly et al., 2005; Dal Lago et al.,2006; Vanden Bempt et al., 2008). Consequently, these tu-mours should be considered as functionally HER2 negativetumours. Thus, it appears of paramount importance to dif-ferentiate tumours with true HER2 amplification from thosehaving a copy number increase of the gene simply relatedto a polysomy 17 (Rosenberg et al., 2008).

    The FISH technique for the HER2 testing

    FISH (Fluorescent In situ Hybridization) relies on probes vis-ualised by fluorochromes. It is the most widely used in situ hybridization technique for the determination of the status of HER2 because of its high sensitivity and specificity, and itsunrivalled resolution due, in particular, to the development of directly labelled probes. This has also greatly simplified theprocedure by deleting the signal amplification step. As seenbefore, the necessity to differentiate true amplification from

    polysomy 17 supports the use of a dual-probe kit, includinga probe of centromere of chromosome 17 (Dal Lago et al.,

    Dual-color FISH with directly labelled probes:optimal solution for testing the gene status of breast

    carcinoma with equivocal HER2 expression

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    5/245

    Dr. Jrme Couturier

    Head of Unit of CytogeneticsDpt. of GeneticsInstitut Curie, Paris

    References:Dal Lago L, Durbecq V, Desmedt C, Salgado R, Verjat T, Lespagnard L, Ma Y,

    Veys I, Di Leo A, Sotiriou C, Piccart M, Larsimont D. Correction for chromo-some-17 is critical for the determination of true HER2/neu gene amplificationstatus in breast cancer. Mol Cancer Ther. 2006;5:2572 9.

    Downs-Kelly E, Yoder BJ, Stoler M, Tubbs RR, Skacel M, Grogan T, Roche P,Hicks DG: The influence of polysomy 17 on HER2 gene and protein expressionin adenocarcinoma of the breast. a fluorescent in situ hybridization, immuno-histochemical, and isotopic mRNA in situ hybridization study. Am J Surg Pathol.2005;29:1221 7.

    Rosenberg CL. Polysomy 17 and HER2 amplification: true, true, and unrelated.J Clin Oncol. 2008;26:4856 8.

    Vanden Bempt I, Van Loo P, Drijkoningen M, Neven P, Smeets A, Christiaens MR,Paridaens R, De Wolf-Peeters C. Polysomy 17 in breast cancer: clinicopathologicsignificance and impact on HER2 testing. J Clin Oncol. 2008;26:4869 74.

    Walker RA, Bartlett JM, Dowsett M, Ellis IO, Hanby AM, Jasani B, Miller K, PinderSE: HER2 testing in the UK: further update to recommendations. J Clin Pathol.2008;61:818 24.

    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, DowsettM, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD,Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH,

    van de Vijver M, Wheeler TM, Hayes DF; American Society of Clinical Oncology;College of American Pathologists. American Society of Clinical Oncology/Collegeof American Pathologists guideline recommendations for human epidermalgrowth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118 45.

    2006; Rosenberg et al., 2008). It is strongly recommendedin the guidelines for HER2 testing in the UK (Walker et al.,2008). Using this control probe reduces equivocal results.FISH is particularly suitable for dual-colour in situ hybridiza-tion because of the high contrast between the green and

    the red fluorescent signals. It should be pointed out that cir-cumstances in which the HER2 in situ hybridization test isprescribed, namely cases IHC 2+, require a high resolutiontechnique to accurately enumerate HER2 and centromere17 signals, because it is particularly among these cases thatpolysomy 17 may be observed. The PathVysion test fulfilsthese requirements.

    FISH scoring

    The American Society of Clinical Oncology and the Collegeof American Pathologists have defined the rules presently

    recommended for scoring in situ hybridization results (Wolff et al., 2007).Signals should be counted in at least 20 nuclei of twoseparate areas of invasive carcinoma.

    A positive HER FISH test shows a ratio HER2 signals / centromere 17 signals >2.2.

    A negative test shows a ratio

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    6/246

    Our surgical pathology unit receives all breast cancer sam-ples from the province of Reggio Emilia, Italy. They accountfor about 550 new cases per year.

    Although HER2 status determined by immunohistochemis-try (IHC) and the status determined by fluorescent in situhybridization (FISH) are significantly correlated, now weknow (J Clin Oncol 27:1323-1333.2009) that standard con-siderations of laboratory testing, including test accuracy, re-producibility, and precision, as well as the current datafavour FISH over IHC assay methods for determining HER2status.

    These considerations are clearly important in clinical practicebecause HER2 amplification is directly linked to protein ex-pression levels in breast cancer. However, this protein isnot consistently analyzed in formalin-fixed tissues as a result

    of variability in fixation methods and times and the impactof fixation on HER2 protein antigenicity. Conversely, geneamplification and FISH are significantly less dependent ontissue fixation methods, making this assay more repro-ducible between central and peripheral laboratories thanIHC. Moreover, review of the existing data demonstratethat FISH is more strongly correlated with responsivenessto either trastuzumab or lapatinib treatment.

    Despite all these considerations and the ASCO-CAP guide-lines in J Clin Oncol 27:1323-1333.2009, only a few labora-tories apply the front-line FISH strategy to all breast cancers

    at the moment of first diagnosis and metastatic relapse.

    Our extensive practical experience in HER2 FISH testingstarted in 2000 when Pathvysion probes became availablein Italy.

    Our oncologists stressed that FISH testing should be usedfor HER2 determination, because at that time, IHC testaccuracy, reproducibility, and precision was not satisfactory,especially on the Cytological FNA samples which we re-ceived.

    As FISH needed to be carried out on the FNA samples, itwas easier from an organizational standpoint, to carry out aFISH front line testing algorithm on all samples, rather thanorganizing an algorithm where we would perform first theIHC test on all samples except cytological ones from FNAS,followed by FISH testing of only the 2+ cases.

    Now we run a weekly session for FISH, strictly following ASCO/CAP guidelines, JCO 27,8 MARCH 2009 1323-1333for every step. Our results are summarized in Table 1.

    We run all types of samples: cytological from FNAs, histo-logical from tru-cut biopsies and from surgical specimensdepending on the oncologists therapeutic decision andcare strategy, were they apply the St. Gallen guidelines (A.Goldhirsch et al. Annals of Oncology 20: 1319-1329, 2009)

    Ultimately, HER2 FRONT LINE FISH testing permits themost accurate determination of whether or not an individualtumour specimen contains HER2 alteration, this potentiallygreatly enhances therapeutic efficacy and significantly in-creases oncologists confidence in their therapeutic decisions.

    HER2 assessment on breast cancer in FISHfront line our 10 years experience.

    Figure 1: Breast Cancer Reggio Emilia Incidence and Mortality Trend Figure 2: Breast Cancer Reggio Emilia Overall Survival

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    7/247

    Dr.ssa M. Flora, Dr. C. Bassano;

    Prof. G. Gardini, Dr. G. Bisagni, Dr. L. Magone

    Department of Oncology, Arcispedale S. Maria Nuova,Reggio Emilia Italy

    The latest data from our Regional Emilia Romagna TumourRegistry, show very encouraging results in terms of overallsurvival for breast cancer patients in the province: 92 %versus 85 % in the National Registry (Fig 1).

    Also encouraging is the decreasing mortality trend despitethe increasing incidence for breast cancer Fig 2.

    Such encouraging results, are achieved by cooperation of specialist groups in the breast unit, and, in our opinion, thefront-line FISH testing strategy is an intergral part of achiev-ing this success.

    Last, but not least, questions about FISH costs:

    Many studies in the past have shown (Lidgren M. ActaOncologica, 2008; 47: 1018-1028) That trastuzumab andchemotherapy treatment for FISH positive patients is a

    cost-effective treatment option from a societal perspective.

    Moreover ASCO /CAP guidelines (JCO 27,8 March 20091323-1333) clearly stated that costs are not increasing byusing FISH testing.

    Our Unit of Surgical Pathology belongs to the same depart-ment as the Oncology Unit and the global budget is thesame. In this environment it is clear that if you perform aninappropriate but very expensive treatment due to a unrelia-ble test (IHC), you not only waste the cost of the treatmentbut will also not reach the primary goal of best treatment forthe patient.

    (See G.Sauter et al. Journal of Clinical Oncology, Vol 28,2010)

    HER2 STATUS RESULTS as DETERMINED by FISH PATHVYSION at SURGICAL PATHOLOGY UNIT Reggio Emilia HOSPITALand year

    Data from January 2004 to September 2009: Patient numbers Percentage

    TOTAL FISH TEST 2906

    HER2 AMPLIFICATION 383 13 %

    HER2 NORMAL 2523

    Chromosome 17 Polysomy 319 11 %

    These percentages are broadly in agreement with published rates, Sauter et. al, JCO 2009.Please note that we do not have borderline cases, as we continue to count more cells until we reach a positive or negative category. Moreover, the standardizationof preanalytic steps has completely eliminated unsuccessful FISH tests.

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    8/24

    The evaluation of HER2/neu oncoprotein expression isessential for the management of breast cancer patients.Studies in a large number of cases have found hyperex-pression of the oncoprotein to be associated with anunfavourable prognosis and correlated with response to

    a number of chemotherapeutic treatments; it is also thetarget of the anti-HER2/neu antibody (trastuzumab).Demonstration of HER2 protein hyperexpression and/orHER2 gene amplification is in fact essential for adminis-tration of this drug.

    The molecular basis for overproduction of the oncoproteinis amplification of the HER2 gene. There are currently 2investigation methods:

    a) measuring HER2 protein levels with a semiquantitativeimmunohistochemistry (IHC) method (HercepTest: score

    0, 1+, 2+, 3+); (Fig. 1)b) evaluating gene amplification, expressed as the ratio of

    HER2/CEP17, using in situ hybridisation methods (FISH,CISH, SISH).

    Before deciding which method to use in our department,we compared the results of IHC and FISH conducted inparallel on the same cases from a routine caseload.

    Samples were examined from 418 cases of infiltratingbreast cancer surgically treated at the AUSL Forl BreastUnit between 01/06/2004 and 31/01/2006 and fixed informalin, embedded in paraffin and diagnosed by the ForlDepartment of Anatomical Pathology.

    The samples were classified by histological diagnosis asfollows: 291 ductal carcinomas, 34 lobular carcinomas,17 mucinous carcinomas and 76 different histotypes.

    Histological sections from the paraffin blocks were cut toa thickness of 4 5 m, processed and evaluated by IHCusing the Dako HercepTest and by FISH using the VysisPathVysion kit.

    IHC scores of 3+ were considered positive, 0 or 1+ negativeand 2+ doubtful.

    With FISH, cases were considered as non-amplified whenthe signal ratio of the HER2 gene / chromosome 17 centro-mere (CEP 17) (counted in at least 60 nuclei) was < 2. (Fig.2b)

    They were considered amplified when the LSI HER2/CEP

    17 ratio was 2. (Fig. 2a)

    Determination of HER2 by FISH and IHC:Experience at Forl Department of Anatomical Pathology

    8

    Figure 1a: Score IHC 0

    HERCEPTEST / FISH CORRELATION

    Total cases: 418

    FISH HercepTest

    0 1+ 2+ 3+ Totals % Amp. 7 12 15 39 73 17.5

    Non-Amp. 165 149 31 0 345 82.5

    Total%

    17241.1

    16138.5 4611.0 399.3 418 Totalcases

    Figure 1b: Score IHC 1+ Figure 1c: Score IHC 2+ Figure 1d: Score IHC 3+

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    9/24

    Of the 418 cases examined in parallel by both immuno-histochemical testing of HER2 hyperexpression and theFISH test for HER2 gene amplification, 17.5 % testedpositive with FISH and 9.3 % were assigned a score of 3+with the HercepTest.

    As shown in the table, 7 (4.1%) and 12 (7.5 %) casesrespectively scored 0 and 1+ for HER2 with IHC (and werethus considered negative) but were amplified with FISH.

    There was a significant correlation between cases not am-plified using FISH and those scoring 0 or 1+ using IHC(95.9 % and 92.5 % respectively). 32.6 % (15/46) of thecases assigned a score of 2+ presented gene amplification.

    Comparison of the results obtained with the two methodsrevealed a strong correlation (100%) between positive caseson FISH testing and those hyperexpressing the receptor

    (3+); in fact none of the cases classified as 3+ was not am-plified according to FISH.

    All this might suggest that IHC alone could be sufficient toselect patients for treatment with trastuzumab and thatFISH does not provide any further information.

    Unfortunately, the same concordance was not achievedwith negative results: in fact, 7 cases (4.1 %) with a scoreof 0 and 12 cases (7.5 %) with a score of 1+ (equivalent to26 % of the amplified cases) underwent gene amplificationwith FISH. This discrepancy would result in the exclusion

    of all patients considered negative on the basis of IHCalone, and would thus deprive them of the benefits of suchspecific treatment.

    Given these results, the decision was taken some time agoto use FISH as the screening test for HER2 expressionsince, as our cases have shown, cancers found negative(0 and 1+) with IHC as well as those scoring 2+ would alsoneed to be confirmed by FISH: this would have both pro-

    longed the response time and been more costly, as bothtests would have had to be conducted in around 90 % of cases.

    Another point in favour of this solution was the opportunityto use dedicated staff handling all aspects of this sector,from technical setup to microscopic evaluation. This staff has now gained considerable experience through examina-tion of around 1500 cases per year, given that all cases of breast cancer seen in the Romagna hospitals are referredto our service.

    Last but not least, the issue of reproducibility of resultsshould be considered; this is a problem we regularly facewhen checking the blind readings taken by differentworkers.

    A multicentre prospective study carried out by BCIRG(Breast Cancer International Research Group) on 2600breast cancer patients found 92 % agreement betweenFISH evaluations obtained at the reference laboratory andat external laboratories, but only 79 % for IHC. The Collegeof American Pathologists (CAP) also found a higher agree-ment between laboratories using FISH (100 %) compared

    with those using IHC (72 %) (Press and Slamon, 2005).

    9

    Dr.ssa Laura Medri, Dr. Luigi Serra

    Department of Pathology, Hospital Morgagni-Pierantoni,Forl, Italy

    Figure 2 a: Amplified case Figure 2 b: Non-amplified case

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    10/2410

    Figure 1: Schematic diagrams of unusual signal patterns in HER2 FISH.

    The accepted measure of HER2 amplification is a HER2-to-control chromosome-17-centromere (CEP17)-ratio greaterthan 2.0. This approach results in an unequivocal classifica-tion of the HER2 status in the vast majority of cases 1. Thereis, however, a small fraction of tumors with unusual FISH-

    signal patterns that require a deviation of this standard rulein order not to report an inadequate HER2 amplificationstatus. This article will describe these cases.

    Loss of Centromere 17

    Due to loss of centromere 17 the HER2-to-CEP17 ratiocan be greater or equal 2.0. Of course, amplification bydefinition requires an increase in HER2 gene copy numberper tumor cell nucleus. Therefore a loss of centromere 17without high HER2 copy number does not qualify for HER2positivity. The ASCO/CAP recommendations suggest a

    minimum of 4 HER2 signals per nucleus 2. Cases with lessHER2 signals are not amplified because HER2 gene copynumber is not increased above the level observed in non-amplified breast cancer cases. In our own experiencea centromere 17 signal loss is seen with a frequency of approximately 2 5%.

    Polysomy of Chromosome 17

    Aneuploidy, gains and losses of chromosomes, is frequentlyseen in tumors. Additional chromosomes 17, of course re-sult in an increased copy number of HER2 genes. This extragene copies lead to a somewhat increased expression of the HER2 protein and can be the reason for an equivocalHER2 result by immunohistochemistry (1+, 2+). In contrastto classical HER2 amplification, polysomy leads to an in-creased expression of a very large number of different genesthat randomly includes HER2. It seems possible that chro-mosome 17 polysomic tumor cells are not as dependent onHER2 expression are HER2 amplified tumor cells. This mayexplain the lack of reports showing response to trastuzum-ab in chromosome 17 polysomic tumors. In HER2 FISHtesting, polysomic cases show a parallel increase of HER2and centromere 17 copies with a random, non-clustered

    distribution of both signals (Fig. 1C, Fig. 2). The HER2/ CEP17 ratio is not significantly increased in these casesand allows the distinction between a polysomy 17 andtrue HER2 amplification.

    HER2 Testing in Breast Cancer:Reporting of unusual signal patterns.

    FISH Pattern

    A) normal

    CEP 17

    HER2

    17p

    17q21

    B) Amplification C) Polysomy 17 D) Coamplification

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    11/2411

    Coamplification of Centromere 17

    The centromere 17 is sufficiently distant from the HER2locus, so it is normally not included in the HER2 amplicon.Nevertheless, HER2 amplification is sometimes accompa-nied by amplification of parts of the centromere 17 regionresulting in a greatly increased number of HER2 and CEP17signals which are always clustered. It seems that a complextranslocation is neighboring the HER2 locus to the centro-mere followed by amplification of the common locus 3. Sig-nal numbers of HER2 and CEP17 are comparable or evenidentical resulting in a ratio of 1. Nonetheless this patternequates a true HER2 amplification which is reflected by aconsistent strong HER2 overexpression 4. Polysomy can beeasily distinguished from coamplification on the basis of thecharacteristic signal pattern. Coamplification results in theformation of HER2 signal clusters overlaid with clusters of

    CEP17 signals due to the close proximity of the ampli conson one chromosome (Fig. 1D, Fig. 3). In contrast, poly somyshows randomly distributed HER2 and CEP17 signals with-in the nucleus of a tumor cell. In our experience coamplifica-tion is a rare observation, corresponding to approximately1% of cases.

    References:1. Sauter G, Lee J, Bartlett JM, et al: Guidelines for human epidermal growth

    factor receptor 2 testing: biologic and methodologic considerations.J Clin Oncol 27:1323 33, 2009

    2. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of ClinicalOncology/College of American Pathologists guideline recommendations forhuman epidermal growth factor receptor 2 testing in breast cancer.J Clin Oncol 25:118 45, 2007

    3. Marchio C, Lambros MB, Gugliotta P, et al: Does chromosome 17 centromerecopy number predict polysomy in breast cancer? A fluorescence in situ hybridi-zation and microarray-based CGH analysis. J Pathol 219:16 24, 2009

    4. Troxell ML, Bangs CD, Lawce HJ, et al: Evaluation of HER2/neu status incarcinomas with amplified chromosome 17 centromere locus.

    Am J Clin Pathol 126:709 16, 2006

    Dr. Tobias Grob, MD

    Institute for PathologyUniversity HospitalHamburg-Eppendorf

    Figure 3: HER2 FISH analysis of a breast cancer case with coamplification of HER2 (A)and centromere 17 (B).

    Figure 2: HER2 FISH analysis of a breast cancercase with polysomy 17.

    (A) (B)

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    12/2412

    Probe/Ventana) and the dual color SISH (dSISH-ultraViewSISH Detection Kit, ultraView AP Red ISH Detection Kit,INFORM HER2 DNA Probe, INFORM Chromosome 17Probe/Ventana) technique. HER2 gene and CEP17 signalswere counted separately in at least 60 invasive nonoverlap-

    ping tumor cells in at least 2 different areas of the tumor.

    The strength of linear correlation of HER2/CEP17 measure-ments between FISH and sSISH as well as between sSISHand dSISH was evaluated and agreement assessed usingBland-Altman plots, a method which describes the averageHER2/CEP17 of the two methods versus the differencebetween both methods. Concordance of HER2/CEP17 as-sessed by FISH and dSISH for ratios of 1.5 1.8, 1.8 2.0,2.0 2.2 and 2.2 2.5 (called categories) were determinedusing the weighted kappa statistic.

    ResultsFor FISH and sSISH, the correlation of HER2/CEP17 be-tween both methods was 0.61 and considerably improvedwhen comparing sSISH versus dSISH (correlation coeffi-cient: 0.79). Compared to FISH, dSISH tended to over-estimate HER2/CEN17 by 0.42 signals on average, withdifferences reaching 2.4 signals. In tumors with equivocalFISH-HER2 status (ratio 1.8 2.0 and 2.0 2.2) dSISHwas highly discrepant with concordance only of 11.1%and 42.9%, respectively. In 22.2% and 28.6% of thesecases, dSISH resulted in HER2 amplification. Concordancebetween FISH and dSISH was greatest for HER2/CEP17ratios of 1.5 1.8 and 2.2 2.5, yet kappa values still dem-onstrated only fair agreement (k=0.30 and =k0.33).

    Conclusions

    Trastuzumab is effective only in patients whose tumoursare positive for HER2 gene amplification and/or proteinoverexpression. Therefore, accurate HER2 testing is essen-tial for selecting patients eligible for trastuzumab- and/orlapatinib-based therapies. Although for this testing agold standard technique is not indicated from ASCO/CAPguidelines, in situ hybridization techniques such as FISHand SISH seems to be more accurate to determine HER2amplification status.

    Background

    Overexpression of human epidermal growth factor receptortype 2 (HER2), a 185-kD receptor first described more thantwo decades ago, occurs in 20% to 25% of invasive breastcarcinomas. In general, patients with breast cancers over-expressing this receptor have a high copy number of itsgene. Moreover they have decreased overall survival andmay have differential responses to a variety of chemothera-peutic and hormonal agents. In particular HER2 amplifica-tion and overexpression are the molecular targets for specif-ic therapies, such as trastuzumab (Herceptin; Genetech,South San Francisco, CA) and lapatinib (GlaxoSmithKline,London, United Kingdom). Therefore, accurate determina-tion of HER2 gene amplification is necessary for the selec-tion of patients for trastuzumab therapy. FISH is regardedas the gold standard method for detecting HER2 gene am-

    plification. In comparison to FISH, SISH has the advantagesof being a method evaluated by bright-field microscopy andgenerating stable signals. Several studies have shown anexcellent concordance between different ISH methods indetection of clearly HER2 amplified and non-amplified breastcarcinomas. Aim of this study was to compare the perform-ance of dual color SISH (dSISH) versus conventional FISHanalysis in breast carcinoma with equivocal HER2 status.Equivocal (or borderline) HER2 gene amplification was de-fined as HER2/CEP17 (chromosome 17 centromere control)ratio between 1.8 and 2.2 in an average of 60 cells, as indi-cated by American Society of Clinical Oncology/College of

    American Pathologists (ASCO/CAP) guidelines.

    Material & Methods

    62 invasive breast carcinoma biopsies with equivocal HER2status as assessed by FISH assay (HER2/CEP17 ratiosfrom 1.8 to 2.2) were retrieved from the archives of theInstitutes of Pathology of the University Hospital, Basel,Switzerland and the University of Insubria, Varese, Italy.

    They were investigated independently for HER2 usingFISH (PathVysion HER2 DNA Probe Kit, Abbott/Vysis),

    single color SISH (sSISH-ultraView SISH Detection Kit,INFORM HER2 DNA Probe, INFORM Chromosome 17

    Comparison of in situ hybridization (ISH) techniques forthe validation of equivocal (borderline) status of HER2 in

    invasive breast carcinoma: fluorescence in situ hybridiza-tion (FISH) versus automated silver-enhanced ISH (SISH)

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    13/2413

    Patients with equivocal HER2 test as assessed by FISH orSISH represent a poorly studied small subgroup (3 to 5%of breast cancer samples) with uncertain association of testscores to benefit from HER2-directed therapy.

    In comparison to FISH, ISH methods like SISH have theadvantage of generating stable signals and can be evalu-ated by bright-field microscopy. However, only fair agree-ment was reached with dSISH when HER2/CEP17 wascompared to FISH for ratios between 1.5 1.8 and 2.2 2.5,respectively. Breast carcinoma with equivocal status forHER2 in FISH analysis was poorly reproducible with dSISHwith concordance of only 54% of the cases. In such equiv-ocal cases, therefore FISH analysis seems to represent amore reliable technique to more effectively score HER2 sta-tus. Furthermore this study of equivocal cases underlinesthe importance of using an internal control, such as CEP17

    to avoid a false positive diagnosis in cases of increasedCEP17 copy number without true HER2 gene amplification.

    Prof. Luigi Terracciano, MD

    Sandra Schneider, techn. Assistant

    Institute of PathologyDivision of Molecular PathologyUniversity of Basel

    References:Elkin EB, Weinstein MC, Winer EP, Kuntz KM, Schnitt SJ, Weeks JC.HER2 testing and trastuzumab therapy for metastatic breast cancer:a cost-effectiveness analysis. J Clin Oncol. 2004; 22:854 63

    Hicks DG, Tubbs RR. Assessment of the HER2 status in breast cancer byfluorescence in situ hybridization: a technical review with interpretive guidelines.Hum Pathol. 2005; 36:250 61.

    Press MF, Sauter G, Bernstein L, Villalobos IE, Mirlacher M, Zhou JY, Wardeh R,Li YT, Guzman R, Ma Y, Sullivan-Halley J, Santiago A, Park JM, Riva A, SlamonDJ. Diagnostic evaluation of HER2 as a molecular target: an assessment of accu-racy and reproducibility of laboratory testing in large, prospective, randomizedclinical trials. Clin Cancer Res. 2005; 11:6598 607

    Press MF. How is HER2/neu status established when HER2/neu and chromo-some 17 centromere are both amplified? Am J Clin Pathol. 2006; 126:673 4

    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, DowsettM, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD,Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH,van de Vijver M, Wheeler TM, Hayes DF; American Society of Clinical Oncology/ College of American Pathologists. American Guideline recommendations forhuman epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol.2007; 25:118 45

    Dietel M, Ellis IO, Hfler H, Kreipe H, Moch H, Dankof A, Klble K, Kristiansen G.Comparison of automated silver enhanced in situ hybridisation (SISH) and fluo-rescence ISH (FISH) for the validation of HER2 gene status in breast carcinomaaccording to the guidelines of the American Society of Clinical Oncology andthe College of American Pathologists. Virchows Arch. 2007; 451:19 25

    Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for humanepidermal growth factor receptor 2 testing: biologic and methodologic considera-tions. J Clin Oncol. 2009; 27:1323 33

    Figure 1a: Same case as detected by double color silver enhanced insitu (dSISH). Clusters of HER2 signals (black) and CEP17signals (red) in tumor cells. (original magnification x630)

    The red signals appear to be somewhat blurred makingmore difficult a precise assessment of CEP17 copies.

    Figure 1b: Coamplification of HER2 (red signals) and CEP17 (green signals)as detected by fluorescent in situ hybridization (FISH) (FITC,DAPI counterstain, original magnification x630). This caseyielded a HER/CEP17 ratio of 1.9

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    14/2414

    Much of cancer therapy is based on the assumption thatpatients with specific types and stages of cancer should betreated according to standardized predetermined protocols.However, recent scientific advances have made individual-ised selection of treatment possible: a treatment based on

    the characteristics of the patient and the tumor. Targetedtherapies attack cancer cell-specific attributes that are es-sential for growth or survival (oncogenic addiction).

    Successful examples of targeted therapy include the use of trastusumab in breast cancer, where HER2 FISH analysis isthe best predictor for success of therapy. More recentlyseveral new compounds have become available for treat-ment of non small cell lung carcinoma (NSCLC). Howevercompared to the case for breast cancer, the selection of ap-propriate patients who will respond to these agents is farmore complex. This complexity is due to different modes of

    actions of the compounds, either Tyrosine Kinase Inhibitors(TKI) or monoclonal antibodies, and the fact that the targetof these compounds, the Epidermal Growth Factor Recep-tor (EGFR) protein, can be either overexpressed due togene amplification (as in the HER2 protein in breast cancer)or constitutively activated by gene mutations, or indeedboth! A further layer of complexity is added by alternativemolecular abberations in other genes which confer resist-ance to the targeted therapies or by specific mutation in theEGFR gene which confer resistance rather than sensitivity tothese compounds. A brief overview of some of these issues

    is discussed below.

    Predictors of response to targeted therapyin NSCLC

    i) Tyrosine Kinase Inhibitors in NSCLC

    Recently, several trials with EGFR inhibitors in NSCLC of-fered promising results. Some of these inhibitors were smallmolecules, targeting the tyrosine kinase activity of the EGFR.Well known examples are erlotinib and gefitinib.

    However, these tyrosine kinase inhibitors (TKIs) are only as-

    sociated with significant clinical responses in 10-20% of Caucasian patients with heavily pre-treated metastaticNSCLC. This contrasts with East Asian patients, where aresponse rate of 40-50% is seen.

    Initially, the basis for the selective response to TKIs in pa-tients with NSCLC was unknown. Subsequently, the identifi-cation of mutations in the tyrosine kinase domain of EGFRthat resulted in ligand-independent gene activation provideda seemingly rational basis for the response.

    However, the association between mutations and respon-siveness to TKIs was much more complex than had beenenvisioned. Although mutations correlated well with respon-siveness and tumor free survival, overall survival appeared tocorrelate better with increased EGFR gene copy numbers.

    So, what is the best predictor of responseto TKIs: EGFR mutation analysis or lookingfor EGFR amplification?

    The debate is still going on. Several recent findings con-

    fused the issue. For example, mutation and amplification of EGFR gene are not independent factors: most amplifiedcases are also mutated. Also, several studies have reportedthat non-TKI-treated patients with NSCLC and EGFR muta-tions have a more favourable prognosis than do patientswith wild type EGFR. In contrast, EGFR gene amplificationportends a poorer prognosis in this group of patients. Fur-thermore, the current adoption of EGFR mutation as a se-lection marker would exclude EGFR TKI therapy in 80 90%of non-East Asian patients, whereas 50-60% would be ex-cluded when using EGFR gene copy numbers (FISH positiv-ity) as a marker.

    ii) Anti EGFR Monoclonal antibodies in NSCLC

    Not only TKIs have been used in NSCLC, but also an anti-EGFR monoclonal antibody, cetuximab. Studies with pani-tumumab have also been initiated.

    In the S0342 clinical trial, 229 chemotherapy-nave patientswith advanced stage NSCLC were enrolled onto a phase IIsection trial evaluating sequential or concurrent chemother-apy (paclitaxel plus carboplatin) with cetuximab. EGFR FISHwas assessable in 76 patients and classified as positive

    (four or more gene copies per cell in 40% of the cells ongene amplification) in 59. The study demonstrated improvedresponse, progression free survival and overall survival inFISH-positive patients. The median survival time of 15

    The contribution of FISH in non smallcell lung cancer (NSCLC).

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    15/2415

    months in FISH-positive patients is by far the longest surviv-al time achieved in a SWOG trial in this clinical setting and islonger than the median survival time reported with chemo-therapy and bevacuzimab, which in the United States isconsidered the current standard of care for patients with

    advanced NSCLC. So, in this setting, EGFR FISH is a pre-dictive factor for selection of NSCLC patients for cetuximabplus chemotherapy. Furthermore, preclinical studies alreadysuggested that response to cetuximab is independent of EGFR mutation status.

    Predictors of resistance to EGFR targetedtherapies

    But what are the predictors of resistance to EGFR directedtherapies? Several have been described.

    A T790M EGFR kinase mutation inhibits TKI binding to theactive site of the enzyme. This mutation typically developsas a secondary resistance, but has also been described intumor samples before TKI therapy.

    In addition, amplification of MET, with subsequent recruit-ment of ErbB3 signaling has frequently been identified. To-gether, T790M and MET amplification account for 60-70%of tumors with secondary TKI resistance.

    In 2007, Soda et al identified another potential driver muta-tion in NSCLC: a fusion of the N-terminal portion of the pro-

    tein encoded by the echinoderm microtubule-associatedprotein-like 4(EML4) gene with the intracellular portion of thereceptor tyrosine kinase encoded by the anaplastic lympho-ma kinase (ALK) gene.

    These fusions are the result of inversions within the shortarm of chromosome 2 (involving 2p21 and 2p23, approxim-atively 12Mb apart).

    ALK rearrangements are already well known in anaplasticlarge cell lymphomas, inflammatory myofibroblastic tumorsand neuroblastomas.

    Multiple EML4-ALK variants have already been identified inlung cancer, but all these variants demonstrate gain of func-tion properties.

    In an EML4-ALK mouse lung tumor model, treatment withan ALK inhibitor resulted in reduced tumor burden com-pared with controls.

    Several ALK kinase inhibitors are currently in early clinicaltesting. Up to 7% of the lung tumors harbour EML4-ALK fu-sions. Most of them are adenocarcinomas. It seems thatEML4-ALK fusions are associated with a lack of EGFR orKRAS mutations.

    Importantly, EML4-ALK positivity is associated with resist-ance to EGFR TKIs. So, a new NSCLC patient subpopula-tion, caracterized by ALK gene rearrangements, isemerging.

    In conclusion, in NSCL, selection of the patients which willrespond to the various compounds is far from straitforwardand will require a thorough understanding of the underlyingmolecular profiles of these patients or the oncogenic ad-diction of the tumour. As we progress towards this goal it isrevealing a fascinating new role for applying personalisedmedicine and the lessons learnt will pave the way for newtargeted therapies for other carcinomas.

    FISH is a valuable predictive marker when considering anti-EGFR therapy in NSCLC, particularly when anti-EGFR anti-bodies (cetuximab) are considered. MET amplification is anegative predictor for response to therapy. The ALK generearrangement status has to be determined when consider-

    ing ALK kinase inhibitors. Up to know, tumors with EML4- ALK positivity seem to be resistant to anti-EGFR therapy.

    Selected readings1. Gazdar A.F. Personalized medecine and inhibition of EGFR signalling in

    lung cancer. N Engl J Med. 2009; 361: 1018-1020.

    2. John T., Liu G., Tsao M-S. Overview of molecular testing in NSCLC:mutational analysis, gene copy number, protein expression and otherbiomarkers of EGFR for the prediction of response to tyrosine kinaseinhibitors. Oncogene 2009; 28: 514-523.

    3. Hirsch F.R., Herbst R.S., Olsen C., Chamsky K., Crowley J. et al. In-creased EGFR gene copy number detected by FISH predicts outcomein NSCLC patients treated with cetuximab and chemotherapy. J ClinOncol 2008; 26; 3351-3357

    4. Shaw A.T., Yeap B.Y., Mino-Kenudson M., Digumarthy S.R., Costa D.B.et al. Clinical features and outcome of patients with NSCLC who harborEML4-ALK. J Clin Oncol 2009; 27: 4247-4253.

    Prof. Patrick Pauwels,MD

    Head of the DiagnosticPathological AnatomyUniversity Hospital Antwerp.

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    16/2416

    Lung cancer is one of the leading causes of morbidity anddeath all over the world and a significant burden on healthcare resources of most countries. The inherent biologicalaggressiveness of lung cancer makes its detection too latein most instances (accounting for 70 80 % of patients),

    when the disease is locally advanced or metastatic and theresponse rate to multimodality therapy disappointing. This isparticularly worrisome for adenocarcinoma, which is be-coming the main subtype of lung cancer in most countriesin both males and females. Recently, tyrosine kinase inhibi-tor (TKI) therapy targeting EGFR has been demonstrated tobe effective in the treatment of pulmonary adenocarcinomaswhen specific EGFR gene mutations or cytogenetic altera-tions involving EGFR gene and/or chromosome 7 arepresent. In this setting, fluorescence in situ hybridization(FISH) has been purported as a powerful and valuable tech-nique allowing the precise gene copy number assessment(amplification) or the refinement of chromosomal alterations,translocations or inversion to be consistently evaluated.

    An increased EGFR copy number has been correlated withimproved survival after TKI therapy in a subset of adeno-carcinoma patients, inasmuch as most EGFR FISH-positivepatients (i.e. patients showing amplification or high polys-omy according to previously refined criteria) have demon-strated control of disease. Moreover, EGFR increased copynumber and mutations are not mutually exclusive events,but 56% to 67% of EGFR mutation patients have been re-ported to harbor increased EGFR copy number, leading to

    the contention that a combined evaluation in the same ade-nocarcinoma patient for EGFR gene status by two-colorFISH analysis and EGFR mutations in exon 18 to 21 couldmaximize our efforts to identify subsets of adenocarcinomapatients amenable of TKI therapy.

    Pre-requirement for obtaining optimal and reproducible re-sults, however, remains adopting rigorous and accuratetechnical procedures when accomplishing FISH analysis byusing commercially available reagents. Briefly, in our labora-tory, two to 4 micron-thick formalin-fixed paraffin-embeddedtissue sections (fixation time should not be over 24 hours)are freshly cut and placed on positively charged glassslides, leaving them to dry for at least 3 hours at 37 C.Sections are thoroughly dewaxed in xylene, rehydrated ingraded ethanol and washed in distilled water, before un-masking them with TRIS/EDTA buffer at pH 9, for 15 min-

    utes at 80C. After washing in 2 x saline buffer (SSC), slidesare transferred into a protease solution (pepsin 0.4 % in HCl0.01N) for 15 minutes at 37 C and then washed in 2 x SSC.Subsequently, sections are dehydrated in graded ethanol,left to dry, incubated with 10 ml probe solutions (Abbott

    Molecular/Vysis), covered with a 22 x 22 mm coverslip andthen sealed with rubber cement. After co-denaturation of target DNA and the probe by heating at 80 C in the Hybrytechamber for 10 minutes, an overnight hybridization is per-formed at 37 C by means of a moist chamber. Coverslip isthen peeled off and the sections are washed in stringencysolution (0.1 x SSC) for 3 min at 73 C, then in 2 x SSC for2 minutes at room temperature and in PBD for 10 seconds.Finally, the slides are counterstained with DAPI solution,covered with a glass coverslip and sealed with fingernailpolish.

    For the microscopic evaluation, tumor cells are identifiedaccording to their position in parallel hematoxylin andeosin-stained sections, taking care to avoid necrotic areas.

    Analysis of DAPI stained nuclei allows not overlapping andappropriately digested nuclei to be precisely localized andevaluated for gene copy number assessment. Sixty nucleiare counted in 6 8 randomly selected different tumor areasby means of 100 x oil objective. Digital images are obtainedusing a Leica DM6000 epifluorescence microscopeequipped with a Leica digital camera DC250 (Leica ImagingSystems Ltd, Cambridge, United Kingdom). Green, orange,

    and DAPI fluorescent signals are detected using specificfilters. The images are then recorded, pseudocolored andmerged using the CW4000 software (Leica).

    The orange and green signals of the individual nuclei arerecorded in a dedicated score sheet, summed to obtainthe mean copy number value of the EGFR gene per cell andthe ratio between the number of gene copies and chrmo-mosome 7 centromere (CEP7) signals. For EGFR geneevaluation, we apply previously refined criteria for whichgene amplification is defined as an EGFR/CEP7 ratio being 2 in tumor cells or the occurrence of at least 15 orange

    specific signals in 10 % or more tumor cells. High polysomyor high trisomy is considered when 40 % neoplastic cellsshow 4 or 3 green specific signals per cell, respectively.Low polysomy and trisomy categories corresponded totumors accounting for less than 40% polysomic or trisomic

    Fluorescence in situ hybridization (FISH) analysis of epidermal growth factor receptor (EGFR) in metastatic

    adenocarcinoma of the lung: a clue for therapy

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    17/2417

    neoplastic cells, respectively. Representative images of EGFR FISH analysis of pulmonary adenocarcinoma showingamplification, polysomy or eusomy are presented in Figure 1

    A D.

    In this frame of mind, studying 75 metastatic (stage IV) lungadenocarcinomas (from 38 females and 37 males, 34 of whom non smokers, 18 former smokers, 18 current smok-ers and 5 unknown), we have found that seven tumorsshowed EGFR amplification, 23 high polysomy, 6 high tri-somy, 22 low polysomy and 17 low trisomy of chromosome7, respectively. Overall, the percentage of EGFR amplifi-cation/chromosome 7 high polysomy accounted for 40 %.EGFR amplification or chromosome 7 high polysomy corre-lated significantly with the occurrence EGFR gene mutationsin exon 19 or 21, this relationship being also confirmed bythe close association between a gene copy number thresh-

    old > 4 and the presence of mutations. More interestingly,

    patients with EGFR amplification/chromosome 7 high polys-omy exhibited a complete or partial response to tyrosinekinase inhibitor (TKI) therapy, whereas patients complainingof stable or progressing disease were mostly associatedwith the occurrence of low polysomy or trisomy.

    In conclusion, these results are in keeping with the literaturedata suggesting that formalin-fixed paraffin-embeddedmaterial is suitable for predicting the status of EGFR geneand chromosome 7 by means of a two-color FISH analysisapproach. The correlation between these cytogeneticalterations and EGFR mutations, two non random andmutually not exclusive phenomena, supports once againthe view that both assays should be carried out togetherwhen dealing with pulmonary adenocarcinoma for thebest selection of patients subsets to be effectively treatedwith TKI therapy.

    Giuseppe Pelosi, MD, Patrizia DellOrto, DSc,Simona Pessina, DSc, Viviana Stufano, DSc

    Division of Pathology and Laboratory Medicine,European Institute of Oncology and University of MilanSchool of Medicine, Milan

    Figure 1 A D: FISH analysis of pulmonary adenocarcinoma showing amplification of EGFR gene (A), also with the presence of clustered signals (B), polysomyof chromosome 7 (C) or eusomy condition (D).

    A B

    DC

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    18/2418

    Background

    The analysis of Epidermal Growth Factor Receptor (EGFR)by FISH has been identified as a predictor of outcome inpatients treated with anti-EGFR-based therapies in differenttumor types. Evidence from clinical trials using tyrosinekinase (TK)-inhibitors for the treatment of patients withnon-small cell lung cancer (NSCLC) suggests a significantimprovement in overall and progression-free survival ac-companied by a higher response rate in patients with EGFRFISH-positive compared to EGFR FISH-negative tumors. AFISH-positive result in NSCLC is currently defined, accord-ing to the Colorado criteria, as presence of high polysomy(4 EGFR gene copies per nucleus in 40% of the analyzedcancer cells) or amplification (a ratio of EGFR gene to chro-mosome of 2, or 15 EGFR gene copies per nucleus in10% of the analyzed cancer cells) [1]. These criteria are

    established for defining EGFR-positive NSCLC from biopsy(histological) material and continue to be applied in clinicaltrials based on analysis of histological specimens.

    The problem

    However, nearly 40% of all diagnoses of NSCLC are madeusing cytological, rather than histological specimens, sug-gesting that the Colorado criteria applied to cytology may besuboptimal for defining EGFR-positivity. In fact, the numberof signals detected in cells from cytological specimens re-presents the true number of signals, since the nuclei remainintact while in histological specimens, a fraction of the nu-clear DNA is missing due to the effect of nuclear truncationwhich occurs during section cutting of histological tissueblocks. Thus, to effectively select patients for EGFR TK-in-hibitor therapy by EGFR FISH irrespective of specimen type,it is mandatory to establish a FISH scoring system whichcan be applied to both cytological and histological speci-mens in a reproducible manner.

    A possible solution

    An alternative evaluation system for EGFR FISH signalsin NSCLC and possibly other tumor types could be toquantify the mean number of EGFR gene copies per case

    (MCN: mean gene copy number). To this end, we evaluatedthe inter-observer reproducibility of the MCN in both cyto-logical and histological cancer specimens from patients withdifferent tumor types, including 50% with NSCLC andcompared its performance to the Colorado criteria in both

    specimen types [2].

    Analysis

    For FISH analyses the LSI EGFR SpectrumOrange/CEP 7SpectrumGreen dual color probe set (Vysis Inc., AbbottLaboratories). Details have been described elsewhere [2].Four scorers, two for histology, and two for cytology, ex-perienced in FISH analysis independently performed theevaluation according to first, the Colorado criteria, andsecond using the MCN.

    Reproducibility of scoring systemOur findings on more than 150 histological specimensunderline a 25% inter-observer variability with the Coloradocriteria. On cytological smears, agreement was over 90%and clearly biased by the overall increased number of EGFRgene copies which can be observed with cytological com-pared to histological specimens due to the effect of nucleartruncation (Figure 1). Together, these findings suggest thatthe Colorado criteria are sub-optimal for the evaluation of EGFR gene copy number by FISH in cytological specimensand may lead to substantial inter-observer variability onhistological specimens as well. The MCN was on the otherhand highly reproducible between observers regardless of specimen and tumor type (whether histological, cytological,from pulmonary or non-pulmonary origin) (Figure 2).

    Additional advantages of MCN

    The evaluation of MCN may be advantageous for severalreasons. First, it may better account for the heterogeneityin EGFR patterns which often renders evaluation challeng-ing using standard criteria. Second, in conjunction withcut-point determination methods such as receiver operat-ing characteristic (ROC) curve analysis, the optimal cut-off score for classifying patients according to a specific end-

    The mean EGFR gene copy number: a different kindof scoring system with promising clinical implications

    for patients with non-small cell lung cancer

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    19/2419

    Dr. Inti Zlobec, PhD

    Prof. LukasBubendorf, MD

    Institute for PathologyUniversity Hospital Basel

    CytologyFISH+

    HistologyFISH

    EGFR Centromer 7

    Figure 1: Difference in EGFR gene copy numbers between histological and cytological specimens from the same pulmonary adenocarcinoma. Nucleartruncation leads to loss of signals in histological specimens, while all signals are preseved in whole cells of cytological specimens. A & C: Cytologicalspecimen with 4 EGFR gene signals per tumor cell. B & D: Histological specimen with 4 EGFR gene signals per tumor cell.

    A

    C

    B

    D

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    20/2420

    Figure 2: Scatter plots of observers scores showing strong correlation of mean EGFR gene copy number (MCN)independent of specimen (histology or cytology) and tissue (lung or other) type.

    point of interest such as response or non-response toanti-EGFR therapy can be made. Changes in sensitivity andspecificity at different potential MCN cut-off values can beevaluated. Analysis of the ROC curve can help to determinethe trade-off between sensitivity and specificity that is rea-

    sonable and clinically justifiable for selecting patients fortherapies. The use of MCN with ROC curve analysis has

    recently been applied for patients with metastatic colorectalcancer (mCRC) treated with cetuximab-based therapyto identify the most appropriate cut-off score to classifypatients into responsive and non-responsive treatmentgroups. To our knowledge this method has not yet been

    applied to identify possible threshold values for responsein patients with NSCLC.

    A) Lung, histology B) Lung, Cytology

    D) Other, CytologyC) Other, histology

    The mean EGFR gene copy number: a different kind of scoring system withpromising clinical implications for patients with non-small cell lung cancer(continued)

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    21/2421

    Figure 3: A potential application of mean EGFR gene copy number (MCN) in clinical practice. The scale demonstrates that for each MCN a different probability of outcome can be predicted.

    References:1. Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, Haney J,

    Witta S, Danenberg K, Domenichini I, Ludovini V, Magrini E, Gregorc V, DoglioniC, Sidoni A, Tonato M, Franklin WA, Crino L, Bunn PA, Jr., Varella-Garcia M.Epidermal growth factor receptor gene and protein and gefitinib sensitivity innon-small-cell lung cancer. J Natl Cancer Inst 2005; 97: 643 655.

    2. Zlobec I, Raineri I, Schneider S, Schoenegg R, Grilli B, Herzog M, Savic S,Bubendorf L. Assessment of mean EGFR gene copy number is a highlyreproducible method for evaluating FISH in histological and cytological cancerspecimens. Lung Cancer 2009.

    Potential for individualized predictionof outcome

    An additional advantage of using the MCN is again relatedto the quantitative nature of the scores. Although one alter-native is to categorize patients as potentially responsive andnon-responsive according to some MCN cut-off value, itis also possible, using relatively simple statistical modeling,to assign to a particular patient a probability of responsefor each MCN. A hypothetical example is given in Figure 3.

    This approach may be more applicable in the clinical settingsince it incorporates the patients individual MCN to deter-mine the degree to which response to therapy may beexpected.

    The MCN: a simple and reproduciblescoring system

    The assessment of EGFR FISH signals using the MCN isa highly reproducible and simple scoring system which canbe applied to both histological and cytological specimensfrom tissue of pulmonary and non-pulmonary origin. Thequantitative nature of the scores has several advantagesover categorical scoring systems for evaluating EGFR FISH.Most importantly, the assessment of MCN may allow fora more individualized prediction of outcome to targetedtreatment with anti-EGFR therapies.

    Mean copy number in histology

    3.01 3.13 3.26 3.39 3.53 3.67 3.82 3.98 4.14 4.31 4.49 4.67 4.86 5.06 5.26 5.48 5.70 5.94 6.18

    HYPOTHETICAL PROBABILITY OF OUTCOME

    2% 15% 40% 65% 88% 95%

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    22/2422

    Utility and advances of EGFR FISH in metastaticcolorectal cancer

    Metastatic colorectal cancer is one of the major publichealth concerns in Europe and US.

    One million new cases are diagnosed and half a milliondeaths are registered per year worldwide. During the pastfew years, new effective treatments have evolved, thusimproving the outcome of affected patients in terms of response rate, time to progression and overall survival.

    In particular, monoclonal antibodies (MoAbs) targeting theepidermal growth factor receptor (EGFR) have recentlyshowed promising results in metastatic colorectal cancerpatients (with wild-type K-Ras gene) when used as a singleagent therapy or in combination with irinotecan. However,the biological and molecular mechanisms underlyingresponse or resistance to these drugs are only partiallyknown.

    Initially, patient enrolment for anti-EGFR MoAbs therapy wasbased on EGFR protein expression, but the comprehensionthat detection of positive immunostaining does not predictclinical outcome of EGFR-targeted treatment has led to anintense search for alternative predictive markers.

    Recent evidence indicated that EGFR gene copy number,as detected by fluorescence in situ hybridization (FISH),could influence response to anti-EGFR MoAbs therapy inmetastatic colorectal cancer patients. Different independentstudies demonstrated that an increased EGFR gene copy

    number, due either to EGFR gene amplification or to chro-mosome 7 polysomy, is correlated with an improvementin response and allows the identification of patients thatare likely to benefit from cetuximab or panitumumab. Bycontrast, a disomic status of EGFR gene seems to revealpatients resistant to these drugs [1 5].

    Following these studies, the request of the assessment of EGFR gene status by FISH rapidly increased in clinical prac-tice of pathology laboratories. Its precise evaluation is there-fore fundamental, but represents today a contentious issuein tumor pathology, molecular genetics and oncology. At the

    moment, in fact, the value of EGFR gene copy number asa predictive marker is well ascertained, but the definition of a threshold to define the increased gene copy number orcopy number gain (CNG) is a crucial point that has not beencompletely addressed yet.

    During the last four years, different cut-off values have beengenerated in different cohorts of treated patients, by corre-lating the EGFR gene status to the clinical response, andby searching for the proper value of EGFR signals per cellable to discriminate responders vs. non-responder patients.

    However, no studies of cross-validation and reproducibilityhave been performed so far, and a general consensus hasnot been reached (see Table 1).

    Table 1: Cut-off values to define CNG obtained inthe principal studies dealing with metastatic colorectalcancer patients treated with anti-EGFR MoAbs.

    Authors and year Pts MoAb cut-off*

    Moroni et al 1, 2005 31 cetuximab orpanitumumab

    3

    Frattini et al 2, 2007 27 cetuximab 4

    Sartore-Bianchi et al 3, 2007 58 panitumumab 2.47

    Cappuzzo et al 4, 2008 85 cetuximab 2.92

    Scartozzi et al 5, 2009 44 cetuximab 2.6

    * copy number of EGFR signals per nucleus

    The main reasons that complicate the achievement of ageneral agreement in EGFR FISH interpretation criteriaconcern the specimen histological features and the scoringof hybridized signals.

    Histomorphological characteristics of metastatic colorectalcancer, such as closely packed cells, nuclear overlappingand presence of mucininous material, can make FISH sig-nals difficult to score and preclude cells for FISH evaluation.However in the majority of cases signals can be countedin individual cells and tumours classified according to thefollowing criteria:

    i) Two copies per cell of the EGFR specific probe (red) andof the chromosome 7 centromere (CEP7, green), indicat-ing balanced normal copies (i.e. disomy)

    ii) Multiple (more than 2) copies of both probes in balancednumber, indicating polysomy

    iii) Multiple and unbalanced copies with a ratio EGFR/ CEP7 < 2 (i.e. chromosome instability)

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    23/2423

    iv) Multiple copies of red signals (in cluster or doubleminutes) in excess with respect to green signals andwith a ratio EGFR/CEP7 2 (i.e. gene amplification).

    In addition, a combination of these patterns might beobserved within the same sample, representing differentcellular clones in the same specimen, most likely as aconsequence of tumor genetic heterogeneity (see Figure 1).

    The definition of the precise EGFR gene copy number ischallenging and requires expert and trained personnel [6].

    At the moment, it is strongly recommended to evaluatea minimum of 10 15 nuclei from at least 8 10 differentareas, in order to ensure tumor representativeness, andto record carefully all the raw data.

    Despite the technical difficulties of FISH scoring, metastaticcolorectal cancer patient in which the majority of the cellsare characterized by an increased EGFR gene copy numbercould be reasonably addressed to anti-EGFR targetedtherapy. However, important issues need to be addressedin future prospective studies. In particular, it remains unclearwhether EGFR gene copy number should be investigatedon primary tumor or on related metastatic lesions [7], andmore information are warranted on the exact definition of the relationship between EGFR gene status, cetuximab/ panitumumab response, and the role played by EGFR down-stream members, such as BRAF, PIK3CA and PTEN [8].

    In conclusion, for best patients care, the major tasks forpathologists over the next few years will be the elaborationof widely accepted evaluation criteria of EGFR FISH incolorectal cancer, similar to that which has been realizedfor HER2 assessment in breast cancer. Prospective large

    clinical trials should then address the significance of theintegration of EGFR gene status in a molecular predictivealgorithm.

    References:1. Moroni M, et al. Lancet Oncol 2005;6:279 86.

    2. Frattini M, et al. Br J Cancer 2007;97:1139 45.

    3. Sartore-Bianchi A, et al. J Clin Oncol 2007;25:3238 45.

    4. Cappuzzo F, et al. Ann Oncol 2008;19:717 23.

    5. Scartozzi M, et al. BMC Cancer 2009; 9:303.

    6. Martin V, et al. J Clin Pathol 2009;62:314 24.

    7. Molinari F, et al. Br J Cancer 2009;100:1087 94.

    8. Siena S, et al. J Natl Cancer Inst 2009;10:1308 24.

    Figure 1: EGFR FISH assay on formalin-fixed paraffin-embedded tissue samples of metastatic colorectal cancer. Different constellations of EGFR gene (in red)and CEP7 (in green) signals are illustrated. A) disomy, b) high polysomy, c) gene amplification interspersed with low polysomy, d) widespread geneamplification.

    Dr. Vittoria Martin, MD

    Institute of Pathology (ICP)in Locarno, Switzerland

  • 8/8/2019 Xplore_9_Her2 & EGFR FISH

    24/24

    Abbott GmbH & Co. KG Abbott Molecular EuropeMax-Planck-Ring 265205 Wiesbaden, Germany

    Please explore the new Website:www.abbottmolecular.com