The genetic analysis of tumors has become a mainstay of cancer research. With the arrival of next-generation sequencing technologies, researchers can now map the entire genome of a tumor more quickly and inexpensively, looking for molecular alterations that cause normal cells to become cancerous.
However, scientists must distinguish between genetic changes that directly induce cells to become cancerous or spur their malignant behavior — called driver mutations — and those that play no significant role. Several important driver mutations have been identified; many of them have been implicated in multiple cancer types. But for the majority of common cancers, finding new driver mutations has proved increasingly difficult.
Now a study led by Memorial Sloan Kettering scientists has unearthed a new driver mutation in a rare type of salivary gland cancer called polymorphous low-grade adenocarcinoma (PLGA). Experimental pathologist Jorge Reis-Filho and Britta Weigelt, an assistant member in his laboratory, showed that mutations in a single gene, PRKD1, may cause PLGA. The researchers identified these driver mutations after performing genetic analysis of only six tumors, and they demonstrated in a large series of salivary gland cancers that these mutations are specific to PLGA. This could soon result in the development of a genetic diagnostic test for patients with PLGA.
The findings, reported in a recent issue of Nature Genetics, came through a collaboration with researchers at the Ontario Institute for Cancer Research, the University Health Network in Toronto, and the Cleveland Clinic.
Drs. Reis-Filho and Weigelt explain how a mere handful of tumor samples can provide compelling evidence of a driver mutation and discuss why analyzing rare cancer types can speed the identification of new cancer genes.
What first led you to focus on PLGA and suspect that it was caused by a single genetic aberration — compared with most cancers, which seem to involve many?
Jorge Reis-Filho: There are several distinct types of malignant salivary gland tumors — including adenoid cystic carcinoma, mucoepidermoid carcinoma, and mammary-analogue secretory carcinoma — which, like PLGAs, sometimes occur in other sites, such the lungs or breasts. In addition, tumors from each of these types look very distinctive under the microscope, and there is little variation in the appearance from tumor to tumor among cases of these rare types of salivary gland cancers. This is in stark contrast to common types of cancers, which have considerable variation in the way they look under the microscope and display tumor heterogeneity.
Recent studies have shown that each of these other salivary gland tumor types are caused by a frequently occurring single genetic alteration, and given the clinical similarities, we suspected the same might be true for PLGA. To test this, we subjected six PLGA tumors to next-generation sequencing. In five of the six tumors, we found mutations that result in exactly the same protein change within a gene called PRKD1, which has not until now been linked to cancer.Back to top
How did you know that these mutations were causing PLGA?
Britta Weigelt: We had to do a number of experiments to confirm this. First, we looked at the other salivary gland cancers and saw that PRKD1 mutations were not present — they are specific to PLGA. We also sequenced 53 additional PLGA tumors and found the same mutations in more than 70 percent of them.
Our findings strongly suggested the mutations are driving the cancer, but we also needed to demonstrate it in the lab. We designed experiments that allowed us to test the signaling activity of the mutated PRKD1 protein and found that was strongly increased compared with the normal PRDK1 protein without the mutation. We also forced the expression of the PRKD1 mutation in nonmalignant human cell lines, and it caused the cells to take on cancerous attributes. That gave us proof of cause and effect.Back to top
How can this discovery help patients in the short term?
JRF: It can have immediate applications in helping doctors diagnose PLGA and define the treatment for patients with salivary gland cancers. Often, only small biopsies are available, and some salivary gland cancers, such as adenoid cystic carcinoma, may look like PLGA but are more aggressive and require different types of treatment. But now we can test for PRKD1 mutations, which may help us distinguish a PLGA from other more aggressive types of salivary gland cancers. We think a clinical test for the mutation, which is simple to develop, will be available by 2015.Back to top
What does this finding suggest about finding new driver mutations in general?
JRF: For the more common types of cancer, which include a heterogeneous group of tumors, we are now seeing hundreds or even thousands of tumors being sequenced for the identification of a small number of new driver mutations. Beyond the major drivers, the picture gets very murky with a diverse array of mutations potentially being implicated in the biology or clinical behavior of common cancers. Two breast tumors, for example, could have very different sets of mutations that caused the disease.
For rare cancers — which, by contrast, are more homogeneous than common cancers — the number of driver mutations can be very, very limited. This was the case with PLGA, and also with small cell carcinoma of the ovary, hypercalcemic type, a rare ovarian cancer that MSK gynecologic oncologist Douglas Levine recently found was caused by one mutation after analyzing only 12 tumor samples.
So investigating the genetic underpinning of rare cancer types in a handful of tumors can help identify new driver mutations more quickly. This is complementary to the conventional “brute force” approach for more common cancers using huge numbers of tumors. When we find a driver mutation in a rare cancer, we can then test whether it applies in the more common cancers — at least specific subtypes of those cancers. It’s a very neat way of going about it. And if a mutation is found, genetic diagnostic tests can be rapidly developed.Back to top