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10.2217/14796694.4.1.61 © 2008 Future Medicine Ltd ISSN 1479-6694

REVIEW

Future Oncol. (2008) 4(1), 61–70 61

Molecular oncology: current trends in diagnosticsJoel A Lefferts, Claudine L Bartels & Gregory J Tsongalis†

†Author for correspondenceDartmouth Medical School, Department of Pathology, Dartmouth–Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USATel.: +1 603 650 5498;Fax: +1 603 650 4845;[email protected]

Keywords: BCR–ABL, breast cancer, chronic myelogenous leukemia, real-time PCR, sentinel lymph node

Applications of molecular diagnostics to oncology have been slow to make their way to the clinical laboratory. While numerous genes and mutation spectra have been found to be involved in tumorigenesis, it is only recently that these findings begin to become useful in a clinical setting. Building on the technical knowledge obtained from molecular infectious disease testing, new instruments and assays have been developed to answer similar questions regarding qualitative, quantitative and genotyping issues. In this manuscript we describe two current examples of clinical molecular diagnostic applications, the assessment of BCR–ABL in chronic myelogenous leukemia patients and the detection of tumor cells in the sentinel lymph nodes of breast cancer patients, to demonstrate the role of molecular techniques in a routine clinical setting.

A new paradigm in molecular diagnosticsWhile initial efforts in molecular diagnosticsbegan with applications for diagnosing thehematopoietic malignancies by identifying novelgene rearrangements in the immunoglobulinheavy-chain gene or the T-cell receptor genefamilies, applications for direct qualitative infec-tious disease testing far outpaced the oncologytests being performed. In part this was owing tothe polygenic, multifactorial complexity of thedisease. It became evident early on that mole-cular oncology testing would not be a ‘one-tar-get-fits-all’ type of algorithm as in theidentification of microbial pathogens.

Interestingly, as infectious disease applica-tions continued to be developed, the need forquantitative testing and resistance genotypingsoon became the norm for specific applica-tions, such as HIV-1 and hepatitis C virus. Asthis paradigm of being able to perform qualita-tive, quantitative and genotype infectious dis-ease testing became standard practice, similarapproaches to oncology were beginning to sur-face. The need for viral-load testing emergedfrom the development of antiretroviral thera-peutics that warranted monitoring of viralcopy numbers. Soon thereafter, genotypingefforts emerged from the need to determineviral resistance or subtypes that would betterrespond to therapeutics.

Paralleling the applications for infectious dis-ease testing, molecular oncology is now at a sim-ilar crossroads where applications for qualitative,quantitative and genotype testing are warrantedbased on novel biomarkers and therapeutics.Thus, a new testing paradigm has emerged. In

this manuscript, we discuss such applicationsthat have recently come to the forefront ofmolecular oncology testing.

Real-time PCR for patient management Real-time PCR has become a method of choicefor most molecular diagnostics laboratories. Thismodification of the traditional PCR allows forthe simultaneous amplification and detection ofamplified nucleic acid targets as it occurs. Thus,there is no need for post-amplification manipu-lation of the products. Because of this, real-timePCR platforms are closed systems that limit thepotential for amplicon contamination. In rou-tine clinical practice, the main advantages ofreal-time PCR are the speed with which samplescan be analyzed, as there are no post-PCR pro-cessing steps required, and the ‘closed-tube’nature of the technology. The analysis of resultsvia amplification-curve and melt-curve analysisis very simple and contributes to it being a muchfaster method for analyzing PCR results.

During a real-time PCR assay, the amplifiedproduct is directly monitored within the reactiontube. The exponential phase of PCR is monitoredas it occurs using fluorescently labeled molecules(Figure 1) [1]. The amount of PCR product presentin the reaction tube is directly proportional to theamount of emitted fluorescence and the amountof initial target sequence [2,3]. Thus, these reac-tions can also be quantitative. There are two typesof detection chemistries for real-time PCR:

• Those that use intercalating DNA-bindingdyes such as SYBR® green I;

• Those that use various types of fluorescentlylabeled probes

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Intercalating DNA-binding dyes allow for thesimple determination of the presence or absenceof an amplicon. SYBR Green I, like ethidiumbromide, is a dye that emits fluorescence when itis bound to dsDNA. During the PCR, there isan increase in the copy number of the amplicon,as well as a simultaneous increase in the amountof intercalated SYBR Green I. This will thenincrease the level of emitted fluorescence indirect proportion to the copy number [4]. Onedisadvantage to these types of dyes is that theyare nonspecific and will bind to any dsDNA,including nonspecific PCR amplicons andprimer-dimers.

Detection of real-time PCR products can alsobe accomplished using fluorescently labeledprobes of various types. There are three maindetection chemistries for these probes: cleavage-based (5´ exonuclease), molecular beacons andfluorescence resonance energy transfer probes.Cleavage-based probes are the most commonlyused and depend upon the 5´ to 3´ exonucleaseactivity of Taq DNA polymerase, also known asthe Taqman® assay (Figure 2). During the ampli-fication process, the probe hybridizes to the tar-get sequence. The exonuclease activity of thepolymerase then cleaves the reporter dye off of

the probe and away from the quencher, gener-ating a fluorescent signal. Molecular beaconsare self-complementary single-stranded oligo-nucleotides that form a hairpin-loop structureand consist of a probe homologous to the targetsequence, flanked by sequences that are homo-logous to each other. Attached to one end is areporter dye (e.g., FAM and TAMRA) and tothe other, a quencher (DABCYL). When thebeacon binds to the target sequence, thequencher and reporter are separated and fluores-cence is emitted. Fluorescence resonance energytransfer probes are two separate fluorescentlylabeled oligonucleotides, one with a 5´ donormolecule and the other with a 3´ acceptor mole-cule attached. When these probes hybridize veryclose to one another, energy can be transferredfrom the donor to the acceptor, which thenemits fluorescence.

Real-time PCR is quickly becoming themethod of choice for most molecular diagnos-tics laboratories because of its increased sensi-tivity/specificity and decreased turn-aroundtimes. This technology can be used for bothqualitative and quantitative assessment of targetsequences, as well as distinguish mutant fromwild-type sequences. For SNP genotyping and

Figure 1. Real-time PCR amplification curve.

Baseline

Log-Linear

Plateau

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small mutation testing, two different labeledprobes are designed, one for the wild-type alleleand one for the mutant allele. The mismatchbetween the wild-type allele and the mutantprobe facilitate competitive hybridization.Therefore, fluorescence will only be detectedwhen the correct probe binds the targetsequence. If binding-dye chemistries are beingused, another powerful feature of most real-time PCR instruments is the ability to performmelting-curve analyses [4]. The melting temper-ature of a specific amplicon can be identified byan additional thermal step on the PCR product,in the same tube.

Real-time PCR can also be used to determinethe copy number of specific target sequences forboth infectious disease and oncology applica-tions. By multiplexing the primers and probesfor the target sequence with the primers andprobes for a control sequence, accurate assess-ment of the target copy can be made. Thisdescribes the basis of a relative quantificationreaction. By contrast, absolute quantification canbe performed by using external standards ofknown concentration to create a standard curveand determine the target copy number.

Monitoring disease: chronic myelogenous leukemiaChronic myelogenous leukemia (CML) belongsto a group of diseases referred to as the myelo-proliferative disorders. These are clonal hemato-poietic malignancies characterized by theproliferation and survival of one or more of themyeloid cell lineages [5,6]. CML has an estimated5000 newly diagnosed cases per year, and itaccounts for approximately 20% of all adultcases of leukemia [7,8]. Typically, the diseaseprogresses through three clinical phases: achronic phase (which may be clinically asymp-tomatic), an accelerated phase and a blast phaseor blast crisis. Most patients are diagnosed withthe disease during the chronic phase, oftenthrough the results of a complete blood countperformed for unrelated reasons. A conclusivediagnosis can be made and/or confirmedthrough cytogenetics and molecular testing.

More than 95% of patients with CML havethe distinctive Philadelphia chromosome (Ph)that results from a reciprocal translocation of thelong arms of chromosomes 9 and 22 (q34;q11),and involves the transfer of the ABL gene onchromosome 9 to the BCR on chromosome 22(Figure 3) [9]. The fused BCR–ABL gene producesa protein (p210) with deregulated tyrosinekinase activity that affects multiple signal trans-duction pathways, leading to uncontrolled cellproliferation and reduced apoptosis [10,11].

The recognition of the role of BCR–ABLkinase activity in CML has provided an attractivetarget for the development of therapeutics.Although interferon therapy and stem cell trans-plantation were previously the first-line treat-ments of choice, tyrosine kinase inhibitors suchas imatinib mesylate (Gleevec®; Novartis, Basel,Switzerland) and dasatinib (SPRYCEL®; Bris-tol–Myers Squibb, NY, USA) have now becomefirst- and second-line therapies. Imatinib binds tothe inactive form of the wild-type BCR–ABLprotein, preventing its activation. However,acquired mutations in the BCR–ABL kinasedomains prevent imatinib binding. Dasatinibbinds to both the inactive and active forms of theprotein, and also binds to all imatinib-resistantmutants except the threonine to isoleucine muta-tion at amino acid residue 315 (T315I). Trials arecurrently underway comparing dasatinib withimatinib for initial CML therapy.

CML has traditionally been diagnosed andmonitored by cytogenetics, FISH and Southernblot analysis (Figure 4). Gene-expression profilinghas also been recently utilized as a discovery tool

Figure 2. TaqMan® real-time PCR assay.

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to further characterize molecular phenotypeswithin the already-existing clinical phenotypes ofCML [12]. Conventional cytogenetics, such asbone marrow karyotyping that is performed on30–50 cells, can be used to visualize the trans-location by G-banding analysis. This methodcan also provide valuable information withregards to other chromosomal aberrations, suchas trisomy 8, isochromosome 17 and duplicatePh chromosomes. FISH, typically performed on100–1000 cells in interphase or metaphase,allows the visualization of the translocationthrough the use of fluorescently labeled probes.The probes specific for the BCR and ABL genesare differentially labeled, so that a normal cellwill have two green signals and two red signals.When the Ph chromosome is present, the colo-calization of the green and red signals causes thesignal to appear yellow. More recently, reversetranscriptase PCR (RT-PCR) has been imple-mented as a more sensitive method of detectingthe fusion transcript that results from the trans-location [13].

The introduction of more efficacious thera-peutics, such as imatinib and dasatinib, has rede-fined the therapeutic responses that areachievable in the CML patient (Table 1). It hasbeen shown that residual disease can be detectedby FISH in more than 50% of patients who havea complete cytogenetic response as determinedby conventional cytogenetics. In addition, most

patients that are FISH-negative can be positiveby RT-PCR, demonstrating the high sensitivityof the RT-PCR method for the detection ofminimal residual disease [13]. With more CMLpatients achieving remission with the new thera-peutics, there is a need for highly sensitivemolecular assays that can detect small numbersof Ph+ cells upon relapse.

Molecular assays such as real-time reversetranscriptase quantitative (RQ)-PCR can achievea 3-log improvement in sensitivity comparedwith cytogenetics and FISH, making them thepreferred method of monitoring patients treatedwith imatinib [14]. Although RQ-PCR methodsare not as labor-intensive as cytogenetics orFISH, RNA purification and reaction setup stepscan be relatively time-consuming. There arenumerous laboratory-developed assays andassays using analyte-specific reagents that can beperformed using various real-time PCR instru-ments. The newest innovation on this front isthe GeneXpert® BCR–ABL Assay (Cepheid,CA, USA). This assay is designed to detect theBCR–ABL fusion transcript and the ABL endog-enous control sequence in peripheral blood orbone marrow samples. The test is run on theGeneXpert Dx System (Figure 5) using single-usedisposable cartridges. All extraction and purifica-tion of RNA is performed within the cartridge,followed by reverse transcription and nested real-time PCR [14,15]. Quantitative results areachieved by calculating the percentage ratioBCR–ABL:ABL (Figure 6).

As with infectious disease applications, noveltherapeutics in oncology have made it manda-tory for clinical laboratories to provide highlysensitive and quantitative molecular assays suchas this to monitor the effectiveness of treatment.CML now represents a molecular oncology para-digm similar to HIV-1 and hepatitis C viruswhereby qualitative, quantitative and resistantgenotyping can be performed routinely.

Molecular evaluation of the sentinel lymph nodeIncreased medical and biological knowledge ofhuman diseases has resulted in technologicaladvances that continue to revolutionize the fieldof medicine. This is especially evident in thefield of breast oncology. Higher-resolutionimaging, targeted drugs and improved surgicaltechniques have all contributed to improvedpatient care by detecting, diagnosing and treat-ing breast cancer more effectively. Despite theseadvances, patients whose breast cancer has

Figure 3. The Philadelphia chromosome: t(9;22) translocation.

Bcr–abl

Ph

9+9

22

Bcr

abl

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become metastatic still face high mortality rates.Early and accurate detection of metastaticspread is essential for successful management ofthis advanced form of breast cancer.

One of the primary channels of metastaticspread of breast cancer from the primary tumorsite to distant sites is the lymphatic system.

Tumor cells in the breast are shed and are cap-tured in the axillary lymph nodes (ALNs), wherethey can develop into metastatic lesions and thenspread to other body sites. Therefore, pathologicexamination of the ALNs has become an essen-tial and routine component in the care of breastcancer patients.

Figure 4. Methods for detecting the BCR–ABL translocation include traditional cytogenetic karyotyping (A), FISH (B) and Southern blot analysis (C).

-23

-9.4

-6.5

-4.4

9 22 9 t(9;22) 22

111213

22

22

9

34

‘Ph’

der(9)9

Table 1. Clinical response of chronic myelogenous leukemia patients to current therapies.

Therapeutic goal Therapeutic response

Hematologic response Normal peripheral blood values, normal spleen size

Cytogenetic response Reduction of Ph+ cells in blood or bone marrow

Complete 0% Ph+ cells

Partial 1–35% Ph+ cells

Minor 36–95% Ph+ cells

Molecular response Reduction or elimination of BCR–ABL mRNA in peripheral blood or bone marrow

Ph+: Philadelphia chromosome positive.

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Complete ALN dissection (ALND), in whicha large number of lymph nodes are surgicallyremoved from the axillary fat pad, is regularlyperformed, especially when clinical or pathologicevidence suggests metastasis to one or morelymph nodes. A primary goal of ALND is to stopthe spread of tumor cells beyond the lymphnodes. Evaluation of ALNs also allows for properstaging of the disease. This analysis is one of themost valuable prognostic factors for breast cancer[16]. The presence or absence and histologicalappearance of metastatic tumor cells in ALNs of

breast cancer patients help in determining whattype of therapy is appropriate and how aggressivethat treatment should be. Although ALND is avaluable tool in treating breast cancer patients,the surgical procedure carries with it commonrisks, including pain, lymphedema, numbnessand restricted shoulder movement [17].

In the past decade, sentinel lymph node (SLN)biopsy has replaced ALND in patients with noclinical indications of or high-risk factors forlymph node metastasis [18]. The term ‘sentinel’ isused to describe the lymph node that is the

Figure 5. The GeneXpert® system and schematic of the single-use cartridge.

Figure 6. GeneXpert® amplification curve for a BCR–ABL-positive result and spreadsheet used to calculate ratio.

Positive for BCR–ABL

0

100

200

300

400

500

600

Cycles

Flo

ure

scen

ce

Internal controlBCR–ABL

0 10 20 30 40 50

Positive result

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primary site of lymph drainage from the tumor.This lymph node is also the most likely to containshed tumor cells. SLNs are localized by injectingthe tumor site with a radioisotope before surgeryand/or a blue dye during surgery (Figure 7). Thesetracers travel through the lymphatic system andcollect in the first lymph node encountered. TheSLN can then be identified by its radioactivity

using a γ-probe or by the blue color seen by thenaked eye; it can then be removed for pathologicexamination [19]. In patients with metastasis-freeSLNs, it can be assumed with some degree of cer-tainty that the remaining ALNs also lack metas-tases. This technique spares a large population ofbreast cancer patients from the more extensiveALND surgical procedure and its associated mor-bidities. Additionally, reducing the number ofnodes biopsied allows the pathologists to devotemore time to each case in the search for metastatictumor cells.

The exact procedure for histological examina-tion of lymph nodes varies from institution toinstitution. Some institutions only obtain tissuesections from paraffin-embedded samples whileothers use frozen sections or touch preparationcytology, which can be less accurate but requiremuch less processing time, allowing for intra-operative evaluation of SLNs. The number ofsections examined also varies from a single sec-tion to numerous serial sections taken from vari-ous regions throughout the lymph node.Standard hematoxylin and eosin staining can beperformed alone or in combination with immu-nohistochemical staining for cytologic makerssuch as cytokeratin (Figures 8 & 9).

Newer molecular techniques have been devel-oped by several investigators for detecting thepresence of tumor cells in lymph nodes. Publica-tions from as early as 1994 describe proceduresusing RT-PCR to detect mRNA of breast can-cer-specific markers [20–22]. The success of theseearly attempts was often limited owing to thehigh sensitivity of RT-PCR in combination withthe low-level expression of breast-specific or can-cer-specific markers in normal nodal tissue.Although the mRNA markers used in these stud-ies code for proteins found in breast cancer cellsand not normal lymph nodes, the RT-PCRmethods were not quantitative and, therefore,could not effectively distinguish between verylow levels of expression in normal lymph nodesand the higher levels of mRNA expression inlymph nodes harboring metastatic tumor cells.

More recently, researchers have revised someof these methods to include more modern real-time RT-PCR techniques that can easily dis-tinguish between low and high levels ofexpression [23–28]. Analysis of a large number ofpotential targets for the identification of positivenodes in a real-time RT-PCR test revealed twotargets, mammaglobin and cytokeratin 19, asideal markers for this diagnostic application.These markers were used to evaluate SLNs from

Figure 7. Intraoperative photograph of a breast sentinel lymph node.

Courtesy of Kenneth A Kern, Surgical Oncology, Hartford Hospital, Hartford, CT, USA.

Figure 8. Hematoxylin and eosin stain of a breast sentinel lymph node showing metastatic tumor cells.

Arrow points to metastatic tumor cells.

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over 250 breast cancer patients. When comparedwith histology, their RT-PCR assay was 90%sensitive and 94% specific [27].

Recently, the US FDA approved a moleculardiagnostic assay for breast lymph node (BLN)testing (Veridex LLC, NJ, USA). This assayincludes a standardized protocol and reagents forhomogenization of lymph node tissue, isolationof RNA from the homogenized tissue and theGeneSearch™ BLN test kit for setting up amultiplexed real-time RT-PCR assay using theSmartCycler real-time platform (Cepheid)(Figure 10). The entire procedure can be com-pleted in less than 1 h. The Intended Use state-ment provided by Veridex recommends this testfor intraoperative or postoperative detection ofnodal metastases greater than 0.2 mm. WhenVeridex performed a pivotal study of 423 patientsfrom 11 sites, the sensitivity and specificity of theBLN assay, with respect to overall histologicalfindings, were found to be 95.6 and 94.3%,respectively. When compared with the sensitivityof frozen sections and touch preparation cytology(85.6 and 45.5%), the GeneSearch BLN assayidentified more histologically positive cases.

The GeneSearch BLN assay appears to be ade-quate for intraoperative analysis and represents areasonable replacement for frozen sectioning andtouch preparations to determine whether or notadditional lymph nodes should be removed dur-ing the same surgery required for the SLN biopsy.If possible, SLNs negative for metastasis by theBLN assay should still be examined histologicallyto allow for proper staging of micrometastases that

may be missed and other histological features.Post-operatively, this assay would supplementstandard histological techniques by confirmingthese findings and minimizing the numbers ofsections requiring review by the pathologist. Atechnological advancement that could possiblyimprove this assay would be the incorporation ofthis real-time RT-PCR into the GeneXpert plat-form. Thus, the entire procedure, from RNA iso-lation to real-time RT-PCR, could be performedin a single cartridge [28].

ConclusionThe two molecular oncology applicationsdescribed in this manuscript represent examples ofqualitative and quantitative molecular diagnosticassays that can be applied to routine managementof CML and breast cancer patients. In some cases,these types of assays will replace more traditionalmethods of analysis and in others they will behighly complementary to existing analytical tools.Building on these applications with the advancesin technology that are occurring will surely resultin better management of the oncology patient.

Future perspectiveClearly, the advantages of molecular diagnostics inthe diagnosis and treatment of the cancer patientare only now becoming evident. A better under-standing of tumor cell biology and the pathwaysinvolved in carcinogenesis have led to novel biom-arkers and therapeutics for human cancers. Thediagnostic algorithms set in place for qualitative,quantitative and genotype infectious disease test-ing have shed light on these similar applicationsfor oncology.

In the context of patient management, notonly will molecular biomarkers be responsible forthe reclassification of many of these tumor types,but they will also be responsible for directingtherapy. Novel small-molecule therapeutics willrequire companion diagnostics to assess the feasi-bility and eligibility of a patient for a particulartargeted therapy. As molecular technologies con-tinue to improve, so will management practices.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involve-ment with any organization or entity with a financial interestin or financial conflict with the subject matter or materialsdiscussed in the manuscript. This includes employment,consultancies, honoraria, stock ownership or options, experttestimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production ofthis manuscript.

Figure 9. Immunohistochemical stain for cytokeratin in a breast sentinel lymph node showing a cluster of metastatic tumor cells.

Arrow points to a cluster of metastatic tumor cells.

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Figure 10. Workflow in the analysis of the sentinel lymph node.

RT: Reverse transcriptase; SLN: Sentinel lymph node.

Executive summary

A new paradigm in molecular diagnostics

• Applications in molecular oncology are similar to what we have experienced in molecular infectious disease testing.• The introduction of new therapeutics will drive the development of diagnostic tests.

Real-time PCR for patient management

• Real-time PCR has revolutionized qualitative and quantitative testing in clinical laboratories.• The advantages of decreased theoretical arrival time, ease of use and minimal contamination potential are ideal for the clinical

laboratory setting.

Monitoring disease: chronic myelogenous leukemia

• Chronic myelogenous leukemia represents a new paradigm for molecular oncology testing that includes qualitative, quantitative and resistance genotyping.

• The introduction of targeted therapies has made it necessary to quantify tumor cell burden beyond traditional test sensitivities.• The GeneXpert instrument consolidates molecular testing to a random-access, single-use cartridge platform.

Molecular evaluation of the sentinel lymph node

• Evaluation of the sentinel lymph node is standard practice and a necessary step in managing the breast cancer patient.• Molecular detection of metastatic tumor cells in the sentinel lymph node can provide useful information to the surgeon and

pathologist with respect to patient care.

Surgical removal of SLN

SLN sectioned in Department of Pathology

Molecular laboratory Histology

Tissue homogenization

and RNA isolation (~10 min)Tissue fixation and paraffin embedding

Real time RT-PCR

set-up (~5 min)

Running GeneSearch assay

on SmartCycler® (~30 min)

Total time: <1 h

Sectioning, staining and immunohistochemistry

Pathologist’s review of slides

Total time: 1–3 days

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Affiliations• Joel A Lefferts

Dartmouth Medical School, Department of Pathology, Dartmouth–Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA

• Claudine L BartelsDartmouth Medical School, Department of Pathology, Dartmouth–Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA

• Gregory J TsongalisDartmouth Medical School, Department of Pathology, Dartmouth–Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USATel.: +1 603 650 5498Fax: +1 603 650 [email protected]

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