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Molecular Discoveries Aim at Advancing Early Diagnosis of

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By: American Association for Cancer Research on Apr 03 2006 16:28:16

Early Cancer Diagnosis

Molecular messages and signals circulating in blood or contained in cells lining the airway can identify early stage cancer, according to scientists presenting today at the 97th Annual Meeting of the American Association for Cancer Research in Washington, D.C. Researchers have discovered molecular signposts pointing to the presence of cancer, and those signs can provide physicians with early and, in some cases, more specific cancer detection opportunities. The goal of screening and early detection is to identify primary tumors at initial stages of development when they can be successfully controlled or cured with local therapy. Most cancer deaths are caused by metastatic disease, later stage tumors that spread to other sites in the patient. Clinical monitoring of molecular markers of primary tumors and metastasis allows for early response strategies in the treatment to control or cure the disease.

Airway Gene Expression; A Novel Diagnostic Test for Lung Cancer in Smokers: Abstract No. 2420

By piggy-backing a genetic expression assay along with the best available procedure to detect lung cancer, a team of researchers has dramatically improved the sensitivity of diagnosing lung cancer. Moreover, the RNA expression signature from normal epithelial cells of the upper airways in smokers identified three times as many early stage – and more treatable – cancers as did conventional bronchoscopy, according to Avrum Spira, M.D., assistant professor from Boston University Medical Center, and lead author of the study that included scientists from the Lahey Clinic in Burlingham, Mass., and Trinity College in Dublin, Ireland.

The genetic signature of cells from the 'field of injury' epithelial cells lining the upper bronchi complemented the clinical bronchoscopy results to identify 95 percent of patients with early or late stage lung cancers. By itself, the genetic signature identified 80 percent of 60 patients who developed lung cancer. Bronchoscopy diagnosed only 53 percent of those patients.

"We are not instituting a new test," Spira said. "These high-risk patients were suspect for having cancer, and they were already undergoing the bronchoscopy. As part of the bronchoscopy procedure, we took additional samples from a more accessible airway.

"We are adding value to the bronchoscopy exam," he said. "In the event where the bronchoscopy misses the diagnosis, our test often detects the cancer. Together, the two tests have a very high sensitivity for lung cancer."

Approximately 300,000 bronchoscopies are performed each year in the United States to diagnose lung cancer. The procedure detects from 30 to 70 percent of all lung cancers, depending on the stage of cancer development.

Where bronchoscopy is weakest, the genetic profiling is strongest, Spira said. Conventional bronchoscopy detected only 30 percent of early, stage one lung cancer in the study's patient cohort, he reported. The genetic signature picked up 90 percent of those early cancers.

"The genetic signature from the field of injury epithelial cells does really well at the stage of disease that bronchoscopy is doing a poor job," Spira observed. "Those are the early tumors, and that is the group of patients that if you make the diagnosis at that stage, you are more likely to have a cure with surgery and treatment than if you wait three months and the cancer has a chance to spread."

The five-year survival rate for people diagnosed with Stage 4 lung cancer is only five percent, Spira noted. Stage 3 survival remains low, averaging less than 20 percent survival after five years.

But with earlier detection while the cancer is still in Stage 1, five-year survival exceeds 60 percent, Spira said.

"The difference is considerable," he added. "Adding the upper airway harvest of normal epithelial cells to the bronchoscopy procedure enables clinicians to make earlier, more complete diagnosis, and improves the opportunity for successfully treating more lung cancer patients."

Detection of Blood-Borne Cancer Using Polymerase Chain Reaction for tNOX Messenger RNA: Abstract No. 5684

Cancer cells that circulate in the blood during metastasis carry a cancer-specific protein called tNOX that is generated from an alternate form of tNOX messenger RNA. Scientists now report they can detect this cancer-specific message with cutting-edge molecular technology, researchers from Purdue University reported at the 97th Annual Meeting of the American Association for Cancer Research today.

"Metastatic cancer cells produce the cancer-specific form of the tNOX messenger RNA, and the presence of the alternate message for tNOX in the blood signals the presence of circulating cancer cells in the blood," said D. James Morre, Ph.D., the Dow Distinguished Professor of Medicinal Chemistry, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.

By monitoring the levels of a cancer-specific message in blood from 32 patients and 22 healthy volunteers, Morre and colleagues determined that all cancer patients in the study were positive for the presence of an alternate form of tNOX RNA message. None of the cancer-free volunteers or patients with non-cancer disease were detected with the marker.

"Despite the relative small sample size of this study, the assay seems highly correlated with metastatic disease and/or the potential for metastatic disease," Morre said. He explained that tNOX appears essential to maintain unregulated growth in cancer cells, which is one of the hallmarks of cancer and bolsters the rationale for its use as diagnostic marker.

"tNOX and its constitutive counterpart, CNOX, fulfill an essential role in cell enlargement," Morre said. "tNOX is translated from a cancer-specific splice variant messenger RNA that is missing exon 4. Both the exon-4-minus mRNA and the mature expressed tNOX protein are specific to cancer and remain undetected in cells or sera from healthy volunteers or patients with diseases other than cancer."

By harvesting messenger RNA from circulating cells in the blood, Morre and his colleagues were able to amplify the message for tNOX using the reverse transcription polymerase chain reaction. The message was elevated in patients with cancer, but not in healthy individuals or those with non-cancerous diseases.

"Because cells containing the variant tNOX message are shed into the serum, it is a useful pan-cancer marker for clinical metastatic disease," Morre said. "The test detects circulating cancer cells, and it does have the potential to become a standard screening assay especially for post-operative breast cancer patients to monitor the potential for metastatic spread." Morre noted that if there was no evidence of metastatic spread post-surgery, patients and their physicians may have the opportunity to circumvent or at least delay the onset of certain chemotherapy strategies until the time that circulating cells appear.

Morre noted that cancer patients participating in the study included breast, lung and ovarian cancers.

"This assay is a promising tool for detecting circulating cancer cells in patients," Morre said. "It has implications for monitoring tumor progression and therapy in clinical use."

A Real-Time PCR Test for Septin 9 Gene Methylation Identifies Early Stage Colorectal Cancer in Plasma: Abstract No. LB-224

Free-floating DNA gleaned from blood is a key to early detection of colorectal cancer, according to researchers from the Epigenomics, a molecular diagnostics company.

The researchers found that presence of the methylated form of DNA encoding the so-called Septin 9 gene is found in plasma of up to 57 percent of patients with all stages of colorectal cancer at high levels of specificity (95 percent).

"The presence of methylated Septin 9 DNA in blood is a marker suited as an excellent population screening tool to identify a large number of asymptomatic cancers, and our study is the first of this magnitude and with this level of clinical performance to describe a DNA methylation-based blood test for the early detection of colorectal cancer." said Catherine Lofton-Day, Ph.D., vice-president, Molecular Biology at Epigenomics.

Lofton-Day noted that a blood-based screening assay will be more patient-friendly than the conventional colorectal cancer screening tool, which requires patients to collect and submit fecal samples for fecal occult blood testing – a considerable obstacle to patient compliance.

While the test to identify methylated Septin 9 DNA in the blood at present is not intended to replace periodic colonoscopy or sigmoidoscopy screening for colon cancer, it can be used more frequently as part of routine yearly physical check-ups to increase the chances of detecting cancer at early stages.

"The test is already better than existing non-invasive early detection methods such as fecal occult blood testing," Lofton-Day said.

Lofton-Day's findings come from two independent studies that determined Septin 9 levels in a total of 1,500 patients in three distinct categories. One group was known to be free from colon cancer after examination by colonoscopy. A second group included individuals with non-colorectal cancers and other non-cancerous conditions, and a third group consisted of patients with a confirmed diagnosis of colorectal cancer.

In an initial study of 501 samples, 57 percent of colorectal cancers were detected. After validating the Septin 9 assay among the three known-status groups in a set of several hundred samples, Lofton-Day and her colleagues predicted the diagnosis of colorectal cancer in an independent set of 790 patients. In this study the detection rate for all colorectal cancers was 50 percent (104 detected in 209 cases). The false positive rate for both studies was between 4 and 6 percent.

"Different stages of colorectal cancers were detected with similar sensitivity, and detection was not affected by location of the tumor in the colon," Lofton-Day said. "This is a reliable, non-invasive method to detect many patients with colorectal cancer, but who show little or no symptoms. "

Expression Profiling of Peripheral Blood Cells for the Early Detection of Breast Cancer: Abstract No. 125

A cryptic message may be found in cells circulating in the blood of women – and that cipher may be useful in early diagnosis of breast cancer, according to results from research conducted at the University Clinic at the Norwegian Radium Hospital in collaboration with DiaGenic ASA.

The blood-borne message is read with a genetic expression test to interpret the possible reaction of peripheral blood cells in response to environmental changes in the body due to breast cancer, said Anne-Lise Børresen-Dale, Ph.D., head of the Department of Genetics, Inst. for Cancer Research, DNR, Norway.

"Early detection of breast cancer is the key to optimizing the success of treatment and patient survival," Børresen-Dale said. "We have identified a gene signature that can discriminate between breast cancer and non-breast cancer in 75 percent of the cases."

Børresen-Dale and colleagues in partnership with DiaGeneic ASA defined a set of 58 genes that were differentially expressed by peripheral blood cells in the presence or absence of breast cancer. An array platform with 22,000 probes interpreted the blood-borne message from samples of 64 women with breast cancer and 76 women with no sign of the disease. The gene signature correctly predicted the diagnosis of breast cancer in approximately 75 percent of the cases after triple cross validation. The gene signature included genes belonging to similar gene families as the 37 genes identified in the first pilot study performed on filter macroarrays.

In this pilot, published in Breast Cancer Research last year, which included 24 women with breast cancer and 32 controls, the researchers failed to classify cancer in 26 percent of the samples from women diagnosed with breast cancer. The assay also errantly suggested the presence of cancer in 12 percent of samples from cancer-free patients; however, three of the four false positive predictions were from samples donated by healthy women who were pregnant.

"This suggests a possible hormonal effect," Børresen-Dale commented.

"To be used as a diagnostic tool, we have to increase substantially both sensitivity and specificity, so we indeed have more work to do before any clinical use," she said. "First we need to sort out if this is specific for breast cancer. And we must analyze whether there is any impact of hormone replacement therapy on the gene signature.

"Furthermore, we aim to determine how early in the development of cancer that this signature indicates the presence of a tumor.

"The first gene signature set that was upregulated in cancer belongs to defense-related genes; genes involved with oxygen stress and immune response. Those genes that were down-regulated in cancer samples are involved with ribosome production and translation and control."

While the signature was expressed in a woman with ductal carcinoma in situ, indicating that it may be useful in very early stage tumors, it was not evident in a Stage 4 cancer.

"That is probably a systemic disease that has altered the expression profile in the peripheral blood cells very differently," she said.

Early detection of breast cancer currently relies primarily on mammography, Børresen-Dale noted.

"Mammographic screening has really helped us achieve earlier diagnosis," Børresen-Dale said. "But it is still not early enough."

The sensitivity of imaging technology is highly dependent on individual characteristics of the patient's breast. In a study of more than 11,000 women with no clinical symptoms of breast cancer, mammography detected tumors in 78 percent of all women, but in just 48 percent of women with extremely dense breast tissue. Mammography typically falters at detecting tumors less than 5 millimeters in size, which can be a relatively late stage of tumor development.

"It could really help if a blood sample was taken for gene expression screening at the time the woman had the mammogram done," Børresen-Dale said. "That may answer discrepancies in the reading and analyzing of the mammogram."

The use of array technology in the clinical setting generally requires blood sampling and can be performed as part of routine normal health physicals.

"An assay to detect the presence of early stage breast cancer by reading the message made by blood cells in response to cancer can be less demanding on women that need repeated mammography because of uncertain findings, and offers the hope of much earlier detection," Børresen-Dale said. "That is when the patient has the greatest opportunity for successful treatment options and long term survival."

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