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Molecular diagnostics of tissue samples are guiding the use of therapeutics for personalized—and successful—cancer treatments.

Breast cancer and colorectal cancer together affect nearly 3.5 million people in the US.^1,2 It had been estimated that, in 2007, over 178,000 women would be diagnosed with breast cancer and 40,000 would die, while over 153,000 men and women would be diagnosed with colorectal cancer and 52,000 would die.^1,2 A number of factors are prognostic for a favorable five- or ten-year survival, including lymph nodes that are histopathologically-determined to be free of disease.

However, despite this, 15% of estrogen receptor-positive (ER+), node-negative breast cancer patients end up with recurrent disease within ten years.³ Up to 30% of stage I and II colorectal cancer patients recur within five years. Better diagnosing, staging, and identifying individual patients likely to recur and respond to therapy is critical in increasing survival and reducing morbidity.

Molecular testing of tissue specimens, including primary tumor and localized lymph nodes, shows great promise in stratifying cancer patients for staging, prognosis, treatment selection and drug development. Molecular diagnostics to guide the use of therapeutics is becoming increasingly important as the demand for individualized therapies grows.   

In breast cancer, several companies including Agendia, Amsterdam, The Netherlands; AviaraDx, San Diego, Calif.; and Genomic Health, Redwood City, Calif., have developed molecular tests that analyze multiple genes in a patient’s tumor to create a prognostic assay for recurrence and/or predictive of therapeutic response. Veridex, Warren, N.J., markets a test that interrogates two genes in sentinel lymph nodes to help guide the intra- or post-operative decision to excise additional nodes. These assays utilize various molecular technologies to measure the expression of genes in patients who are candidates for the testing. These assays are showing great promise in individualizing therapy and are having an impact on the way oncologists are treating patients.

Agendia’s MammaPrint interrogates 70 genes to assess risk of recurrence in patients with invasive breast cancer who are stage I (ER+ or ER-) or stage II lymph node-negative (ER+ or ER-).⁴ MammaPrint is the first IVDMIA (In Vitro Diagnostic Multivariate Index Assay) test to receive FDA clearance. The assay looks at expression of 70 genes involved in the regulation of cancer including cell cycle, invasion, angiogenesis and metastasis and generates a patient-specific gene signature. The signature is prognostic for development of distant metastases in patients determined, histologically, to be node-negative and was validated on more than 1,000 patients. The assay is performed on fresh primary breast tumor tissue. Messenger RNA is extracted from the tissue and labeled with a fluorescent dye. It is then hybridized to a DNA microarray and scanned to determine the gene expression pattern. The pattern is then processed through an algorithm to calculate a risk of recurrence. MammaPrint identifies about 40% of patients as low risk, with a good prognosis of metastasis-free survival. 

AviaraDx’s H/I test measures the ratio of expression of two genes to predict clinical outcome of ER-positive, node-negative patients.⁵  AviaraDx’s test is performed on formalin-fixed paraffin-embedded tumor tissue from slides, with tissue sections at least 7µ thick. The test interrogates the expression of the HOXB13 and IL17BR genes, both regulated by estrogen. The HOXB13 gene is over-expressed in subsets of pre-invasive and invasive breast cancers and appears to predict poor benefit from endocrine therapy. In contrast, low expression of the IL17BR gene correlates with loss of tumor suppressor genes. The ratio of expression of the two genes can be used to stratify ER+ patients into low- or high- risk of recurrence as well as good versus poor response to endocrine therapy.

Veridex’s two-gene assay, marketed under the name GeneSearch Breast Lymph Node Assay, rapidly identifies metastases in lymph nodes to help guide surgical decisions.⁶ The test is unique in that it is an intra-operative assay performed on sentinel lymph nodes during surgery. It is the first FDA-approved molecular diagnostics test for breast lymph node testing, and is designed to detect metastases greater than 0.2mm. The sentinel lymph node is removed during surgery and sections of the tissue are interrogated on-site by real-time PCR using the Cepheid SmartCycler for expression of Mammaglobin and Cytokeratin-19 genes. These genes are over-expressed in breast tissue, but not in the lymph nodes. Results of the test are rapidly available, allowing surgical decisions to be made while the patient is still in the operating room. Results of the test can be helpful in deciding if more-extensive, axillary lymph node surgery is required. 

click to enlarge Cross-section of colorectal lining with cancer cells and guanylyl cyclase C protein marker. (Source, DiagnoCure, © 2008. Illustration by Linda Nye)

Genomic Health’s OncotypeDX test analyzes 21 genes found to be associated with tumor aggressiveness and response to treatment in ER+, node-negative patients.^3,7,8  The test utilizes formalin-fixed paraffin-embedded, primary tumor tissue submitted on slides. RNA is extracted and then reverse transcriptase PCR (RT-PCR) is performed to interrogate the expression of the 21 genes. Results of the gene expression analysis are run through an algorithm to calculate a Recurrence Score for the patient. The score can then be used to estimate risk of recurrence and aid in the selection of treatment. Introduced in 2004, OncotypeDx has been used on more than 33,000 patients. Both American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) have recently adopted the OncotypeDx assay into their breast cancer treatment guidelines, signaling a major milestone for the use of molecular markers in therapeutic decision making in the treatment of breast cancer. Although all these molecular assays examine different gene profiles, they clinically give similar results, suggesting that recurrence and response to therapy is a complex and multivariate process. 

In colorectal cancer (CRC), research assays for prognostic molecular markers, such as loss of heterozygosity (LOH) at chromosome 18q21 locus and Microsatellite Instability (MSI), are being used in to stratify patients enrolling in clinical trials. In the Eastern Cooperative Oncology Group E5202 trial, patients’ tumor cells are analyzed for both MSI and 18q21LOH. Depending on the result, patients will either receive no therapy or will be randomized to one of two adjuvant chemotherapy arms of the trial: FOLFOX (fluorouracil plus leucovorin plus oxaliplatin) or FOLXFOX plus bevacizumab.⁹ These potentially prognostic molecular markers are expected to help guide clinicians in optimizing treatment selection.

Another key prognostic factor in CRC recurrence and survival is the spread of metastases to the local lymph nodes (LNs) of patients. Proper staging of CRC depends on accurately identifying these metastases. This single factor is the most important in predicting recurrence,^10,11 yet traditional methods of detection are severely limited by both inadequate sampling of the LNs and limits of optical detection. Often, a single 10µ section of each harvested LN is examined histologically with a limit of detection of about one cancer cell in a field of 200 normal cells. Occult metastases detected by molecular methods such as RT-PCR are up to five orders of magnitude more sensitive than traditional histological examination. Molecular detection of occult metastases has shown to be clinically important in identifying cancer missed by histopathology alone.^11,12 

Staging is also critical in guiding therapeutic decisions. Current guidelines do not generally recommend adjuvant chemotherapy for stage I or II CRC patients following surgical removal of the tumor. However, stage III patients, who have demonstrated spread of metastases to the lymph nodes, are offered adjuvant chemotherapy. 

In colorectal cancer, up to 30% of stage I/II patients have recurrence of disease and die, despite having lymph nodes characterized pathologically as negative for metastases. This suggests that the cancer has spread but is not being detected by traditional methods.¹¹

A new molecular marker, Guanylyl Cyclase C (GCC), shows promise in augmenting traditional staging of CRC, and identifying those stage I and II patients with occult metastases, missed by traditional histopathology, for whom treatment may be warranted. GCC is a transmembrane receptor protein expressed exclusively on epithelial cells lining the intestine.^13,14 It is a receptor for the hormones guanylin and uroguanylin, and for the bacterial heat-stable enterotoxin, which causes diarrhea.¹⁴ GCC modulates water transport across the intestinal epithelium. Because GCC is highly specific for the gastrointestinal tract and remains expressed in colorectal cancer and metastases,¹³ it appears to be a sensitive and specific marker of metastatic disease outside of the colon. Early studies, performed by Dr. Scott Waldman at Thomas Jefferson University, on pathologically lymph node-negative patients indicated that the presence or absence of GCC mRNA expressed in the lymph nodes was associated with risk of recurrence; patients who recurred all expressed GCC mRNA whereas patients who did not express GCC mRNA did not recur.^14,15 Additionally, GCC appeared to be more specific than other markers of metastases, including carcinoembryonic antigen (CEA),¹⁵ and identified tumor cells in the blood of all metastatic colorectal cancer patients.¹⁴

In a new assay (Previstage GCC Colon Cancer Staging Test) to be released in the second half of 2008 by DiagnoCure Oncology Laboratories, West Chester, Pa., formalin-fixed paraffin-embedded lymph nodes (LNs) of stage I and II patients will be examined molecularly by RT-PCR for the presence of GCC mRNA. Because RT-PCR is up to 100,000 times more sensitive than traditional histopathology, occult metastases may be detected in LNs that could be missed by conventional methods. This molecular staging of the patient may help identify those stage I and II patients who are most likely to recur and who may be candidates for adjuvant chemotherapy. A major five-year, prospective, National Cancer Institute (NCI)-sponsored study of nearly 700 CRC patients molecularly-staged by GCC is expected to be published in 2008. 

In summary, the rapidly growing number of companies offering advanced tissue testing of breast and colorectal cancer patients and the incorporation of some of these tests into treatment guidelines, illustrates the growing impact that molecular testing of tissues is making on the treatment of cancer. Properly identifying patients who are likely to have disease recurrence and respond to specific therapies will no doubt have a positive impact on treatment success. New molecular tests, which characterize genes and mRNA from primary tumors and associated lymph nodes, are showing great promise in helping to characterize patients as individuals and may better guide clinicians to make treatment decisions that will result in reduced morbidity and mortality.

References

1. http://SEER.cancer.gov/statfacts/html/breast_print.html
2. http://SEER.cancer.gov/statfacts/html/colorect_print.html
3. Paik S, Shak S, Tang G, et al.  A multigene assay to predict recurrence of tamoxifen-treated node-negative breast cancer.  N Engl J Med 2004;351:2817-26.
4. www.agendia.com January 15, 2008
5. www.aviaradx.com January 15, 2008
6. www.veridex.com January 15, 2008
7. www.genomichealth.com January 15, 2008
8. Cronin M, Sangli C, Liu M-L, et al.  Analytical validation of the OncotypeDX genomic diagnostic test for recurrence, prognosis and therapeutic response prediction in node-negative, estrogen receptor-positive breast cancer.  Clin Chem 2007;53:1084-91. 
9. Lenz HJ.  Prognostic/predictive molecular markers in colorectal cancer.  Gastrointestinal Cancer Research 2007;1:S29-32.
10. Compton CC, Fielding P, Burgart LJ, et al.  Prognostic factors in colorectal cancer: College of American Pathologists Consensus Statement 1999.  Arch Pathol Lab Med 2000;124:979-94.
11. Iddings D, Ahmad A, Elashoff D, Bilchik A.  The prognostic effect of micrometastases in previously staged lymph node negative (N0) colorectal carcinoma: a meta-analysis.  Ann Surg Oncol 2006;13:1386-92.
12. Nicastri DG, Doucette JT, Godfrey TE, Hughes SJ.  Is occult lymph node disease in colorectal cancer patients clinically significant?  J Mol Diag 2007;9:563-71.
13. Birbe R, Palazzo JP, Walters R, Weinberg D, Schulz S, Waldman SA.  Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia, and adenocarcinoma of the gastrointestinal tract.  Hum Pathol. 2005 Feb;36(2):170-9.
14. Frick GS, Pitari GM, Weinberg DS, Hyslop T, Schulz S, Waldman SA.  Guanylyl cyclase C: a molecular marker for staging and postoperative surveillance of patients with colorectal cancer.  Expert Rev Mol Diagn. 2005 Sep;5(5):701-13.
15. Cagir B, Gelmann A, Park J, Fava T, Tankelevitch A, Bittner EW, Weaver EJ, Palazzo JP, Weinberg D, Fry RD, Waldman SA.  Guanylyl cyclase C messenger RNA is a biomarker for recurrent stage II colorectal cancer.  Ann Intern Med. 1999 Dec 7;131(11):805-12.