Molecular Oncology: Current Trends in Diagnostics

For reprint orders, please contact: REVIEW [email protected]

Molecular oncology: current trends in diagnostics

Joel A Lefferts, Applications of molecular diagnostics to oncology have been slow to make their way to the Claudine L Bartels & clinical laboratory. While numerous genes and mutation spectra have been found to be Gregory J Tsongalis† involved in tumorigenesis, it is only recently that these findings begin to become useful in a †Author for correspondence clinical setting. Building on the technical knowledge obtained from molecular infectious Dartmouth Medical School, disease testing, new instruments and assays have been developed to answer similar Department of Pathology, Dartmouth–Hitchcock questions regarding qualitative, quantitative and genotyping issues. In this manuscript we Medical Center, One Medical describe two current examples of clinical molecular diagnostic applications, the Center Drive, Lebanon, assessment of BCR–ABL in chronic myelogenous leukemia patients and the detection of NH 03756, USA tumor cells in the sentinel lymph nodes of breast cancer patients, to demonstrate the role of Tel.: +1 603 650 5498; molecular techniques in a routine clinical setting. Fax: +1 603 650 4845; gregory.j.tsongalis@ hitchcock.org A new paradigm in this manuscript, we discuss such applications molecular diagnostics that have recently come to the forefront of While initial efforts in molecular diagnostics molecular oncology testing. began with applications for diagnosing the hematopoietic malignancies by identifying novel Real-time PCR for patient management gene rearrangements in the immunoglobulin Real-time PCR has become a method of choice heavy-chain gene or the T-cell receptor gene for most molecular diagnostics laboratories. This families, applications for direct qualitative infec- modification of the traditional PCR allows for tious disease testing far outpaced the oncology the simultaneous amplification and detection of tests being performed. In part this was owing to amplified nucleic acid targets as it occurs. Thus, the polygenic, multifactorial complexity of the there is no need for post-amplification manipu- disease. It became evident early on that mole- lation of the products. Because of this, real-time cular oncology testing would not be a ‘one-tar- PCR platforms are closed systems that limit the get-fits-all’ type of algorithm as in the potential for amplicon contamination. In rou- identification of microbial pathogens. tine clinical practice, the main advantages of Interestingly, as infectious disease applica- real-time PCR are the speed with which samples tions continued to be developed, the need for can be analyzed, as there are no post-PCR pro- quantitative testing and resistance genotyping cessing steps required, and the ‘closed-tube’ soon became the norm for specific applica- nature of the technology. The analysis of results tions, such as HIV-1 and hepatitis C virus. As via amplification-curve and melt-curve analysis this paradigm of being able to perform qualita- is very simple and contributes to it being a much tive, quantitative and genotype infectious dis- faster method for analyzing PCR results. ease testing became standard practice, similar During a real-time PCR assay, the amplified approaches to oncology were beginning to sur- product is directly monitored within the reaction face. The need for viral-load testing emerged tube. The exponential phase of PCR is monitored from the development of antiretroviral thera- as it occurs using fluorescently labeled molecules peutics that warranted monitoring of viral (Figure 1) [1]. The amount of PCR product present copy numbers. Soon thereafter, genotyping in the reaction tube is directly proportional to the efforts emerged from the need to determine amount of emitted fluorescence and the amount viral resistance or subtypes that would better of initial target sequence [2,3]. Thus, these reac- respond to therapeutics. tions can also be quantitative. There are two types Keywords: BCR–ABL, breast cancer, chronic myelogenous Paralleling the applications for infectious dis- of detection chemistries for real-time PCR: ease testing, molecular oncology is now at a sim- leukemia, real-time PCR, • Those that use intercalating DNA-binding sentinel lymph node ilar crossroads where applications for qualitative, dyes such as SYBR® green I; quantitative and genotype testing are warranted part of based on novel biomarkers and therapeutics. • Those that use various types of fluorescently Thus, a new testing paradigm has emerged. In labeled probes

Figure 1. Real-time PCR amplification curve.

Plateau

Log-Linear

Baseline

Intercalating DNA-binding dyes allow for the the probe and away from the quencher, gener- simple determination of the presence or absence ating a fluorescent signal. Molecular beacons of an amplicon. SYBR Green I, like ethidium are self-complementary single-stranded oligo- bromide, is a dye that emits fluorescence when it nucleotides that form a hairpin-loop structure is bound to dsDNA. During the PCR, there is and consist of a probe homologous to the target an increase in the copy number of the amplicon, sequence, flanked by sequences that are homo- as well as a simultaneous increase in the amount logous to each other. Attached to one end is a of intercalated SYBR Green I. This will then reporter dye (e.g., FAM and TAMRA) and to increase the level of emitted fluorescence in the other, a quencher (DABCYL). When the direct proportion to the copy number [4]. One beacon binds to the target sequence, the disadvantage to these types of dyes is that they quencher and reporter are separated and fluores- are nonspecific and will bind to any dsDNA, cence is emitted. Fluorescence resonance energy including nonspecific PCR amplicons and transfer probes are two separate fluorescently primer-dimers. labeled oligonucleotides, one with a 5´ donor Detection of real-time PCR products can also molecule and the other with a 3´ acceptor mole- be accomplished using fluorescently labeled cule attached. When these probes hybridize very probes of various types. There are three main close to one another, energy can be transferred detection chemistries for these probes: cleavage- from the donor to the acceptor, which then based (5´ exonuclease), molecular beacons and emits fluorescence. fluorescence resonance energy transfer probes. Real-time PCR is quickly becoming the Cleavage-based probes are the most commonly method of choice for most molecular diagnos- used and depend upon the 5´ to 3´ exonuclease tics laboratories because of its increased sensi- activity of Taq DNA polymerase, also known as tivity/specificity and decreased turn-around the Taqman® assay (Figure 2). During the ampli- times. This technology can be used for both fication process, the probe hybridizes to the tar- qualitative and quantitative assessment of target get sequence. The exonuclease activity of the sequences, as well as distinguish mutant from polymerase then cleaves the reporter dye off of wild-type sequences. For SNP genotyping and

62 Future Oncol. (2008) 4(1) futurefuture sciencescience groupgroup Molecular oncology: current trends in diagnostics – REVIEW

Monitoring disease: chronic Figure 2. TaqMan® real-time PCR assay. myelogenous leukemia Chronic myelogenous leukemia (CML) belongs to a group of diseases referred to as the myeloproliferative disorders. These are clonal hematopoietic malignancies characterized by the proliferation and survival of one or more of the myeloid cell lineages [5,6]. CML has an estimated 5000 newly diagnosed cases per year, and it accounts for approximately 20% of all adult cases of leukemia [7,8]. Typically, the disease progresses through three clinical phases: a chronic phase (which may be clinically asymp- tomatic), an accelerated phase and a blast phase or blast crisis. Most patients are diagnosed with the disease during the chronic phase, often through the results of a complete blood count performed for unrelated reasons. A conclusive diagnosis can be made and/or confirmed through cytogenetics and molecular testing. More than 95% of patients with CML have the distinctive Philadelphia chromosome (Ph) that results from a reciprocal translocation of the long arms of chromosomes 9 and 22 (q34;q11), and involves the transfer of the ABL gene on chromosome 9 to the BCR on chromosome 22 (Figure 3) [9]. The fused BCR–ABL gene produces a protein (p210) with deregulated tyrosine kinase activity that affects multiple signal trans- small mutation testing, two different labeled duction pathways, leading to uncontrolled cell probes are designed, one for the wild-type allele proliferation and reduced apoptosis [10,11]. and one for the mutant allele. The mismatch The recognition of the role of BCR–ABL between the wild-type allele and the mutant kinase activity in CML has provided an attractive probe facilitate competitive hybridization. target for the development of therapeutics. Therefore, fluorescence will only be detected Although interferon therapy and stem cell trans- when the correct probe binds the target plantation were previously the first-line treat- sequence. If binding-dye chemistries are being ments of choice, tyrosine kinase inhibitors such used, another powerful feature of most real- as imatinib mesylate (Gleevec®; Novartis, Basel, time PCR instruments is the ability to perform Switzerland) and dasatinib (SPRYCEL®; Bris- melting-curve analyses [4]. The melting temper- tol–Myers Squibb, NY, USA) have now become ature of a specific amplicon can be identified by first- and second-line therapies. Imatinib binds to an additional thermal step on the PCR product, the inactive form of the wild-type BCR–ABL in the same tube. protein, preventing its activation. However, Real-time PCR can also be used to determine acquired mutations in the BCR–ABL kinase the copy number of specific target sequences for domains prevent imatinib binding. Dasatinib both infectious disease and oncology applica- binds to both the inactive and active forms of the tions. By multiplexing the primers and probes protein, and also binds to all imatinib-resistant for the target sequence with the primers and mutants except the threonine to isoleucine muta- probes for a control sequence, accurate assess- tion at amino acid residue 315 (T315I). Trials are ment of the target copy can be made. This currently underway comparing dasatinib with describes the basis of a relative quantification imatinib for initial CML therapy. reaction. By contrast, absolute quantification can CML has traditionally been diagnosed and be performed by using external standards of monitored by cytogenetics, FISH and Southern known concentration to create a standard curve blot analysis (Figure 4). Gene-expression profiling and determine the target copy number. has also been recently utilized as a discovery tool

futurefuture sciencescience groupgroup www.futuremedicine.com 63 REVIEW – Lefferts, Bartels & Tsongalis

Figure 3. The Philadelphia chromosome: t(9;22) translocation. patients that are FISH-negative can be positive by RT-PCR, demonstrating the high sensitivity of the RT-PCR method for the detection of 9 9+ minimal residual disease [13]. With more CML patients achieving remission with the new therapeutics, there is a need for highly sensitive molecular assays that can detect small numbers of Ph+ cells upon relapse. Molecular assays such as real-time reverse Ph transcriptase quantitative (RQ)-PCR can achieve a 3-log improvement in sensitivity compared 22 with cytogenetics and FISH, making them the preferred method of monitoring patients treated Bcr with imatinib [14]. Although RQ-PCR methods Bcr–abl are not as labor-intensive as cytogenetics or FISH, RNA purification and reaction setup steps abl can be relatively time-consuming. There are numerous laboratory-developed assays and assays using analyte-specific reagents that can be performed using various real-time PCR instruments. The newest innovation on this front is the GeneXpert® BCR–ABL Assay (Cepheid, CA, USA). This assay is designed to detect the BCR–ABL fusion transcript and the ABL endog- to further characterize molecular phenotypes enous control sequence in peripheral blood or within the already-existing clinical phenotypes of bone marrow samples. The test is run on the CML [12]. Conventional cytogenetics, such as GeneXpert Dx System (Figure 5) using single-use bone marrow karyotyping that is performed on disposable cartridges. All extraction and purifica- 30–50 cells, can be used to visualize the trans- tion of RNA is performed within the cartridge, location by G-banding analysis. This method followed by reverse transcription and nested real- can also provide valuable information with time PCR [14,15]. Quantitative results are regards to other chromosomal aberrations, such achieved by calculating the percentage ratio as trisomy 8, isochromosome 17 and duplicate BCR–ABL:ABL (Figure 6). Ph chromosomes. FISH, typically performed on As with infectious disease applications, novel 100–1000 cells in interphase or metaphase, therapeutics in oncology have made it manda- allows the visualization of the translocation tory for clinical laboratories to provide highly through the use of fluorescently labeled probes. sensitive and quantitative molecular assays such The probes specific for the BCR and ABL genes as this to monitor the effectiveness of treatment. are differentially labeled, so that a normal cell CML now represents a molecular oncology para- will have two green signals and two red signals. digm similar to HIV-1 and hepatitis C virus When the Ph chromosome is present, the colo- whereby qualitative, quantitative and resistant calization of the green and red signals causes the genotyping can be performed routinely. signal to appear yellow. More recently, reverse transcriptase PCR (RT-PCR) has been imple- Molecular evaluation of the sentinel mented as a more sensitive method of detecting lymph node the fusion transcript that results from the trans- Increased medical and biological knowledge of location [13]. human diseases has resulted in technological The introduction of more efficacious thera- advances that continue to revolutionize the field peutics, such as imatinib and dasatinib, has rede- of medicine. This is especially evident in the fined the therapeutic responses that are field of breast oncology. Higher-resolution achievable in the CML patient (Table 1). It has imaging, targeted drugs and improved surgical been shown that residual disease can be detected techniques have all contributed to improved by FISH in more than 50% of patients who have patient care by detecting, diagnosing and treat- a complete cytogenetic response as determined ing breast cancer more effectively. Despite these by conventional cytogenetics. In addition, most advances, patients whose breast cancer has

64 Future Oncol. (2008) 4(1) futurefuture sciencescience groupgroup Molecular oncology: current trends in diagnostics – REVIEW

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

11 ‘Ph’ 12 22 34 13 9 22 9 der(9)

9 22 9 t(9;22) 22

-23

-9.4

-6.5

-4.4

become metastatic still face high mortality rates. Tumor cells in the breast are shed and are cap- Early and accurate detection of metastatic tured in the axillary lymph nodes (ALNs), where spread is essential for successful management of they can develop into metastatic lesions and then this advanced form of breast cancer. spread to other body sites. Therefore, pathologic One of the primary channels of metastatic examination of the ALNs has become an essen- spread of breast cancer from the primary tumor tial and routine component in the care of breast site to distant sites is the lymphatic system. cancer patients.

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.

futurefuture sciencescience groupgroup www.futuremedicine.com 65 REVIEW – Lefferts, Bartels & Tsongalis

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

Complete ALN dissection (ALND), in which breast cancer patients help in determining what a large number of lymph nodes are surgically type of therapy is appropriate and how aggressive removed from the axillary fat pad, is regularly that treatment should be. Although ALND is a performed, especially when clinical or pathologic valuable tool in treating breast cancer patients, evidence suggests metastasis to one or more the surgical procedure carries with it common lymph nodes. A primary goal of ALND is to stop risks, including pain, lymphedema, numbness the spread of tumor cells beyond the lymph and restricted shoulder movement [17]. nodes. Evaluation of ALNs also allows for proper In the past decade, sentinel lymph node (SLN) staging of the disease. This analysis is one of the biopsy has replaced ALND in patients with no most valuable prognostic factors for breast cancer clinical indications of or high-risk factors for [16]. The presence or absence and histological lymph node metastasis [18]. The term ‘sentinel’ is appearance of metastatic tumor cells in ALNs of used to describe the lymph node that is the

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

Positive for BCR–ABL Positive result 600 500 400 300 200 Internal control BCR–ABL Flourescence 100 0 01020304050 Cycles

66 Future Oncol. (2008) 4(1) futurefuture sciencescience groupgroup Molecular oncology: current trends in diagnostics – REVIEW

Figure 7. Intraoperative photograph of a breast sentinel using a γ-probe or by the blue color seen by the lymph node. naked eye; it can then be removed for pathologic examination [19]. In patients with metastasis-free SLNs, it can be assumed with some degree of cer- tainty that the remaining ALNs also lack metastases. This technique spares a large population of breast cancer patients from the more extensive ALND surgical procedure and its associated mor- bidities. Additionally, reducing the number of nodes biopsied allows the pathologists to devote more time to each case in the search for metastatic tumor cells. The exact procedure for histological examination of lymph nodes varies from institution to institution. Some institutions only obtain tissue sections from paraffin-embedded samples while others use frozen sections or touch preparation cytology, which can be less accurate but require much less processing time, allowing for intra- Courtesy of Kenneth A Kern, Surgical Oncology, Hartford Hospital, Hartford, operative evaluation of SLNs. The number of CT, USA. sections examined also varies from a single sec- tion to numerous serial sections taken from vari- primary site of lymph drainage from the tumor. ous regions throughout the lymph node. This lymph node is also the most likely to contain Standard hematoxylin and eosin staining can be shed tumor cells. SLNs are localized by injecting performed alone or in combination with immu- the tumor site with a radioisotope before surgery nohistochemical staining for cytologic makers and/or a blue dye during surgery (Figure 7). These such as cytokeratin (Figures 8 & 9). tracers travel through the lymphatic system and Newer molecular techniques have been devel- collect in the first lymph node encountered. The oped by several investigators for detecting the SLN can then be identified by its radioactivity presence of tumor cells in lymph nodes. Publica- tions from as early as 1994 describe procedures Figure 8. Hematoxylin and eosin stain of a breast sentinel using RT-PCR to detect mRNA of breast can- lymph node showing metastatic tumor cells. cer-specific markers [20–22]. The success of these early attempts was often limited owing to the

high sensitivity of RT-PCR in combination with the low-level expression of breast-specific or cancer-specific markers in normal nodal tissue. Although the mRNA markers used in these stud- ies code for proteins found in breast cancer cells and not normal lymph nodes, the RT-PCR methods were not quantitative and, therefore, could not effectively distinguish between very low levels of expression in normal lymph nodes and the higher levels of mRNA expression in lymph nodes harboring metastatic tumor cells. More recently, researchers have revised some of these methods to include more modern real- time RT-PCR techniques that can easily distinguish between low and high levels of expression [23–28]. Analysis of a large number of potential targets for the identification of positive nodes in a real-time RT-PCR test revealed two targets, mammaglobin and cytokeratin 19, as ideal markers for this diagnostic application. Arrow points to metastatic tumor cells. These markers were used to evaluate SLNs from

futurefuture sciencescience groupgroup www.futuremedicine.com 67 REVIEW – Lefferts, Bartels & Tsongalis

Figure 9. Immunohistochemical stain for cytokeratin in a may be missed and other histological features. breast sentinel lymph node showing a cluster of metastatic Post-operatively, this assay would supplement tumor cells. standard histological techniques by confirming these findings and minimizing the numbers of sections requiring review by the pathologist. A technological advancement that could possibly improve this assay would be the incorporation of this real-time RT-PCR into the GeneXpert platform. Thus, the entire procedure, from RNA isolation to real-time RT-PCR, could be performed in a single cartridge [28].

Conclusion The two molecular oncology applications described in this manuscript represent examples of qualitative and quantitative molecular diagnostic assays that can be applied to routine management of CML and breast cancer patients. In some cases, these types of assays will replace more traditional Arrow points to a cluster of metastatic tumor cells. methods of analysis and in others they will be highly complementary to existing analytical tools. over 250 breast cancer patients. When compared Building on these applications with the advances with histology, their RT-PCR assay was 90% in technology that are occurring will surely result sensitive and 94% specific [27]. in better management of the oncology patient. Recently, the US FDA approved a molecular diagnostic assay for breast lymph node (BLN) Future perspective testing (Veridex LLC, NJ, USA). This assay Clearly, the advantages of molecular diagnostics in includes a standardized protocol and reagents for the diagnosis and treatment of the cancer patient homogenization of lymph node tissue, isolation are only now becoming evident. A better under- of RNA from the homogenized tissue and the standing of tumor cell biology and the pathways GeneSearch™ BLN test kit for setting up a involved in carcinogenesis have led to novel biom- multiplexed real-time RT-PCR assay using the arkers and therapeutics for human cancers. The SmartCycler real-time platform (Cepheid) diagnostic algorithms set in place for qualitative, (Figure 10). The entire procedure can be com- quantitative and genotype infectious disease test- pleted in less than 1 h. The Intended Use state- ing have shed light on these similar applications ment provided by Veridex recommends this test for oncology. for intraoperative or postoperative detection of In the context of patient management, not nodal metastases greater than 0.2 mm. When only will molecular biomarkers be responsible for Veridex performed a pivotal study of 423 patients the reclassification of many of these tumor types, from 11 sites, the sensitivity and specificity of the but they will also be responsible for directing BLN assay, with respect to overall histological therapy. Novel small-molecule therapeutics will findings, were found to be 95.6 and 94.3%, require companion diagnostics to assess the feasi- respectively. When compared with the sensitivity bility and eligibility of a patient for a particular of frozen sections and touch preparation cytology targeted therapy. As molecular technologies con- (85.6 and 45.5%), the GeneSearch BLN assay tinue to improve, so will management practices. identified more histologically positive cases. The GeneSearch BLN assay appears to be ade- Financial & competing interests disclosure quate for intraoperative analysis and represents a The authors have no relevant affiliations or financial involve- reasonable replacement for frozen sectioning and ment with any organization or entity with a financial interest touch preparations to determine whether or not in or financial conflict with the subject matter or materials additional lymph nodes should be removed dur- discussed in the manuscript. This includes employment, ing the same surgery required for the SLN biopsy. consultancies, honoraria, stock ownership or options, expert If possible, SLNs negative for metastasis by the testimony, grants or patents received or pending, or royalties. BLN assay should still be examined histologically No writing assistance was utilized in the production of to allow for proper staging of micrometastases that this manuscript.

68 Future Oncol. (2008) 4(1) futurefuture sciencescience groupgroup Molecular oncology: current trends in diagnostics – REVIEW

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

Surgical removal of SLN

SLN sectioned in Department of Pathology

Molecular laboratory Histology

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

Real time RT-PCR Sectioning, staining and immunohistochemistry set-up (~5 min)

Running GeneSearch assay Pathologist’s review of slides on SmartCycler® (~30 min)

Total time: <1 h Total time: 1–3 days

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.

futurefuture sciencescience groupgroup www.futuremedicine.com 69 REVIEW – Lefferts, Bartels & Tsongalis

Bibliography 13. Schoch C, Schnittger S, Bursch S et al.: a real-time quantitative RT-PCR assay for 1. Wilhelm J, Pingoud A: Real-time Comparison of chromosome banding mammaglobin 1 mRNA. Breast Cancer Res. polymerase chain reaction. Chembiochem 4, analysis, interphase- and hypermetaphase- Treat. 98, 185–190 (2006). 1120–1128 (2003). FISH, qualitative and quantitative PCR for 24. Gillanders WE, Mikhitarian K, Hebert R 2. Wittwer CT, Herrmann MG, Moss AA, diagnosis and for follow-up in chronic et al.: Molecular detection of Rasmussen RP: Continuous fluorescence myeloid leukemia: a study on 350 cases. micrometastatic breast cancer in monitoring of rapid cycle DNA amplification. Leukemia 16, 53–59 (2002). histopathology-negative axillary lymph Biotechniques 22, 130–138 (1997). 14. Dufresne SD, Belloni DR, Levy NB, nodes correlates with traditional predictors 3. Parks SB, Popovich BW, Press RD: Tsongalis GJ: Quantitative assessment of the of prognosis: an interim analysis of a Real time polymerase chain reaction with BCR–ABL transcript using the Cepheid prospective multi-institutional cohort study. fluorescent hybridization probes for Xpert BCR–ABL Monitor assay. Arch. Ann. Surg. 239, 828–837 (2004). detection of prevalent mutations causing Pathol. Lab. Med. 131, 947–950 (2007). 25. Gimbergues P, Dauplat MM, Cayre A et al.: common thrombophilic and iron overload 15. Winn-Deen ES, Helton B, van Atta R et al.: Correlation between molecular metastases in phenotypes. Am. J. Clin. Pathol. 115, Development of an integrated assay for sentinel lymph nodes of breast cancer 439–447 (2001). detection of BCR–ABL RNA. Clin. Chem. patients and St Gallen risk category. Eur. 4. Wittwer CT, Reed GH, Gundry CN, 53(9), 1593–1600 (2007). J. Surg. Oncol. 33, 16–22 (2007). Vandersteen JG, Pryor RJ: High resolution 16. Lacroix M: Significance, detection and 26. Weigelt B, Verduijn P, Bosma AJ et al.: genotyping by amplicon melting analysis markers of disseminated breast cancer cells. Detection of metastases in sentinel lymph using LC Green. Clin. Chem. 49, 853–860 Endocr. Relat. Cancer 13, 1033–1067 (2006). nodes of breast cancer patients by multiple (2003). 17. Mansel RE, Fallowfield L, Kissin M et al.: mRNA markers. Br. J. Cancer 90, 5. Tefferi A, Gilliland DG: Oncogenes in Randomized multicenter trial of sentinel 1531–1537 (2004). myeloproliferative disorders. Cell Cycle 6, node biopsy versus standard axillary 27. Backus J, Laughlin T, Wang Y et al.: 550–566 (2007). treatment in operable breast cancer: Identification and characterization of 6. Delhommeau F, Pisani DF, James C, the ALMANAC Trial. J. Natl Cancer Inst. optimal gene expression markers for Casadevall N, Constantinescu S, 98, 599–609 (2006). detection of breast cancer metastasis. J. Mol. Vainchenker W: Oncogenic mechanisms in 18. Singletary SE: Breast cancer surgery for the Diagn. 7, 327–336 (2005). myeloproliferative disorders. Cell. Mol. Life 21st century: the continuing evolution of 28. Hughes SJ, Xi L, Raja S et al.: A rapid, fully Sci. 63, 2939–2953 (2006). minimally invasive treatments. Minerva automated, molecular-based assay accurately 7. Schiffer CA: BCR–ABL tyrosine kinase Chir. 61, 333–352 (2006). analyzes sentinel lymph nodes for the inhibitors for chronic myelogenous 19. Clark SE, Bowen RL: Positive and negative presence of metastatic breast cancer. Ann. leukemia. N. Engl. J. Med. 357, 258–265 aspects of sentinel lymph node biopsy in breast Surg. 243, 389–398 (2006). (2007). cancer. Future Oncol. 3, 65–72 (2007). 8. Quintas-Cardama A, Cortes JE: Chronic 20. Manzotti M, Dell’Orto P, Maisonneuve P Affiliations myeloid leukemia: diagnosis and treatment. et al.: Reverse transcription-polymerase •Joel A Lefferts Mayo Clin. Proc. 81, 973–988 (2006). chain reaction assay for multiple mRNA Dartmouth Medical School, Department of 9. Jabbour E, Cortes JE, Giles FJ et al.: markers in the detection of breast cancer Pathology, Dartmouth–Hitchcock Medical Current and emerging treatment options in metastases in sentinel lymph nodes. Int. Center, One Medical Center Drive, Lebanon, chronic myeloid leukemia. Cancer 109, J. Cancer 95, 307–312 (2001). NH 03756, USA 2171–2181 (2007). 21. Marchetti A, Buttitta F, Bertacca G et al.: • Claudine L Bartels 10. Sessions J: Monitoring your patients with mRNA markers of breast cancer nodal Dartmouth Medical School, Department of chronic myeloid leukemia. Am. J. Health metastases: comparison between Pathology, Dartmouth–Hitchcock Medical Syst. Pharm. 63(23 Suppl. 8), S5–S9 (2006). mammaglobin and carcinoembryonic Center, One Medical Center Drive, Lebanon, 11. Daley GQ, van Etten RA, Baltimore D: antigen in 248 patients. J. Pathol. 195, NH 03756, USA Induction of chronic myelogenous leukemia 186–190 (2001). • Gregory J Tsongalis in mice by the P210bcr/abl gene of the 22. Schoenfeld A, Luqmani Y, Smith D et al.: Dartmouth Medical School, Department of Philadelphia chromosome. Science 247, Detection of breast cancer micrometastases Pathology, Dartmouth–Hitchcock Medical 824–830 (1990). in axillary lymph nodes by using polymerase Center, One Medical Center Drive, Lebanon, 12. Melo JV, Barnes DJ: Chronic myeloid chain reaction. Cancer Res. 54, 2986–2990 NH 03756, USA leukaemia as a model of disease evolution in (1994). Tel.: +1 603 650 5498 human cancer. Nat. Rev. Cancer 7, 441–453 23. Dell’Orto P, Biasi MO, del Curto B et al.: Fax: +1 603 650 4845 (2007). Assessing the status of axillary sentinel [email protected] lymph nodes of breast carcinoma patients by

70 Future Oncol. (2008) 4(1) futurefuture sciencescience groupgroup