A detailed study of how domains within the Cas9 protein move when the molecule binds to DNA has allowed scientists to locate the protein that monitors the fidelity of binding between the Cas9 single-guide RNA and its DNA target. The researchers then tweaked this domain to boost specificity, creating the highest fidelity Cas9 protein to date.
In a new study, published online in the July 26 issue of PNAS, researchers at University of California San Diego School of Medicine, with colleagues in Xijing Hospital and Sun Yat-sen Cancer Center in China, report that DNA methylation can provide effective markers for at least four major cancers, not only correctly differentiating malignant tissues from normal, but also providing information on prognosis and survival.
“Choosing the proper cancer treatment with the best chance of recovery and survival depends greatly upon accurately diagnosing the specific type or subtype of cancer,” said Kang Zhang, MD, PhD, founding director of the Institute for Genomic Medicine and co-director of biomaterials and tissue engineering at the Institute of Engineering in Medicine, both at UC San Diego School of Medicine. “If you can do that using a minimally invasive biopsy, it has significant implications for cancer science and medicine. Using DNA methylation markers may be a new and more effective a way forward.”
DNA methylation involves methyl groups — one carbon atom bonded to three hydrogen atoms — attaching to DNA molecules. It is a fundamental epigenetic process that regulates gene function without changing the DNA sequence of a gene, essential to normal development and associated with numerous key processes, including initiation and progression of cancer.
Zhang and colleagues looked at DNA methylation for differentiating tumor tissue and normal tissue for the four most common cancers (lung, breast, colon and liver) in three different databases: a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples; a testing cohort of 791 tumor samples from The Cancer Genome Atlas and 93 matched adjacent normal tissue samples and another independent testing Chinese cohort of 394 tumor samples; and 324 matched adjacent normal tissue samples.
They found that DNA methylation analysis could predict cancer versus normal tissue with more than 95 percent accuracy in the three cohorts, comparable to typical diagnostic methods, according to Zhang.
In addition, the analysis correctly identified 97 percent colorectal cancer metastases to the liver and 94 percent colorectal cancer metastases to the lung. “Since 10 percent of cancers present as metastatic lesions or cancers of unknown primary origin, identification of tissue of origin is critical for choosing a correct therapy. This new simple method will be of great value to pinpoint the primary source of the tumor,” said Michael Karin, co-senior author of the study and Distinguished Professor of Pharmacology, also at UC San Diego School of Medicine.
Zhang suggested DNA methylation has the potential to improve outcomes by providing more accurate diagnoses, particularly of relatively indolent or aggressive tumors that may require more or less aggressive treatment.
“Although we focused on just four common cancers here, we expect that DNA methylation analysis could be easily expanded to aid diagnoses of a much larger number of cancers,” said Zhang. “A great benefit is that this approach requires only a small amount of tissue to obtain adequate DNA for analysis, potentially allowing the use of less invasive biopsies or biopsies of metastatic lesions where the tumor is of unknown primary cancer type.”
He said more studies have been planned to fully explore the clinical applications and potential of DNA methylation and its role in future personalized cancer care.
Materials provided by University of California – San Diego. Note: Content may be edited for style and length.
When performed in tandem, two molecular biology laboratory tests distinguish, with near certainty, pancreatic lesions that mimic early signs of cancer but are completely benign. The lesions almost never progress to cancer, so patients may be spared unnecessary pancreatic cancer screenings or operations. The two-test combination is the only one to date that can accurately and specifically identify these benign pancreatic lesions. Its utility was described in one of the largest studies of patients with this form of pancreatic lesion by researchers from Indiana University, Indianapolis. The results of the study now appear in an “article in press” on the Journal of the American College of Surgeons website in advance of print publication.
Between 2 to 3 percent of all patients have some type of pancreatic lesions or cysts revealed on routine abdominal diagnostic radiology scans. Nearly all of these patients will later develop pancreatic cancer. The most common and deadliest form of pancreatic cancer — pancreatic adenocarcinoma — has a five-year survival rate of 12 to 14 percent for early-stage disease and 1 to 3 percent for advanced disease, according to the American Cancer Society.
A small percentage of patients have serous cystic neoplasms (SCN) that do not harbor malignant potential or progress to cancer. Nevertheless, these patients undergo imaging or other surveillance every six months to spot changes indicative of cancer, or they undergo an operation to remove part of the gland as a precaution because SCN are difficult to find using standard diagnostic methods. More than 60 percent of SCN are not predicted preoperatively and 50 to 70 percent are missed or incorrectly diagnosed on radiology scans.
However, the researchers determined that two proteins can play a significant role in ruling out precancer and cancer. Vascular endothelial growth factor A (VEGF-A) is a protein associated with promotion of new blood vessel formation. VEGF-A is upregulated in many tumors, and its expression can be correlated with a tumor’s stage. Its utility in the diagnosis of pancreatic cysts was discovered by researchers at Indiana University. Carcinoembryonic antigen (CEA) is a protein associated with cell adhesion. It is present in low levels in healthy individuals, but it is increased with certain types of cancers.
Tests for each of these proteins in pancreatic cyst fluid have accurately distinguished SCN from other types of pancreatic lesions. In the present study, VEGF-A, alone, singled out SCN with a sensitivity of 100 percent and specificity of 83.7 percent, and CEA had a 95.5 percent sensitivity and 81.5 percent specificity.
Together, however, the tests approached the gold standard of pathologic testing: The combination had a sensitivity of 95.5 percent and specificity of 100 percent for SCN. Authors of the study concluded that results of the VEGF-A/CEA test could have prevented 26 patients from having unnecessary surgery.
“Every day, surgeons follow patients who have pancreatic cysts that have no risk of cancer but are still worrisome. They perform surgery or conduct diagnostic tests just to make sure they’re not wrong. With VEGF-A and CEA, we believe we may have invented a test that can help that group of patients who don’t have a risk of cancer get off the testing cycle and avoid surgery which, in and of itself, has a risk of mortality or complications,” said C. Max Schmidt, MD, PhD, FACS, study author and professor of surgery and biochemical/molecular biology, Indiana University School of Medicine.
The study included 149 patients who underwent an operation to remove a pancreatic cystic lesion. Twenty-six of these patients had SCN. The diagnosis of each surgical specimen was confirmed by pathologic examination, and samples of pancreatic fluid from all patients were tested for VEGF-A and CEA according to testing protocols for enzyme-linked biochemical analysis of fluids.
The accuracy of the VEGF-A/CEA test needs to be confirmed in large prospective studies. The test itself needs to be performed under quality control conditions. Until a central laboratory is created to meet standards for cystic fluid analysis, Dr. Schmidt recommends that pancreatic surgical programs send specimens to the Indiana University Health Pancreatic Cyst and Cancer Early Detection Center in Indianapolis.
“Many investigators are looking for biological markers to help them understand which pancreatic cysts go on to form cancers. Our laboratory is doing that, but it’s also looking for markers to help determine which ones never have the chance of becoming cancers in the first place. These are benign, and they’re fooling us into thinking they could become cancers. If we can do that with confidence, we’ll find patients who can avoid potentially morbid surgery on the pancreas to remove something that never needed to be removed,” Dr. Schmidt said.
Materials provided by American College of Surgeons. Note: Content may be edited for style and length.