SAN DIEGO – , according to Gregory A. Hosler, MD, PhD.
At the annual Cutaneous Malignancy Update, Dr. Hosler, director of dermatopathology for ProPath, highlighted the following molecular tests currently used for the diagnosis of challenging melanocytic lesions:
Comparative genomic hybridization (CGH). This technique allows for the detection of chromosomal copy number changes throughout the tumor genome. “With CGH, test (tumor) DNA and normal DNA are differentially labeled and compared to a reference library. Gains and losses of portions of the tumor genome are determined by comparing the relative signals from these two groups,” said Dr. Hosler, clinical professor of pathology and dermatology at the University of Texas Southwestern Medical Center, Dallas.
“In the past, your library was a metaphase of spread of chromosomes, which introduced technical challenges and made performance of the assay labor intensive. Because of this, CGH is not routinely performed by clinical laboratories and is used more as an exploratory/research technique.”
Array CGH (also known as SNP array). Newer versions of CGH use short DNA sequences that are tiled onto a chip. “The interesting thing about these chips is that you can purchase them or design them on your own,” Dr. Hosler said. “The chips may cover the entire genome or cover specific areas of the genome at higher resolution.” One upside of array CGH, he continued, is that it allows one to detect essentially all gains or losses of chromosomal material in a single reaction. “It is not subject to the artifacts associated with cutting thin sections like with fluorescence in situ hybridization (FISH); it can detect copy number neutral loss of heterozygosity, and it is more scalable,” Dr. Hosler said at the meeting, which was hosted by Scripps MD Anderson Cancer Center.
One downside of array CGH is that does not allow one to analyze specific cells, “so if you have a tumor that’s heterogeneous, the assay is agnostic to this and spits out a result based on all the material provided,” he said. “You can’t parse out different areas of the lesion. It also does not track balanced translocations.” In addition, he said, “there are also questions about reimbursement and these are lab-developed tests, so each lab’s assay is different. Finally, it requires specialized equipment and expertise for interpretation.”
FISH. First-generation melanoma FISH assays, which became available in 2009, used six probes and four colors and had a sensitivity of about 87% and specificity of about 95%, Dr. Hosler said, but there were problems with those assays, particularly related to Spitz nevi. Spitz nevi often duplicate their chromosomes, “so instead of being diploid they’re tetraploid,” he said.
“The second-generation melanoma FISH assays addressed this by adding centromeres to the assay, and targeted probes could be compared to the centromeres on the same chromosome to determine if these were true copy number gains, due to genetic instability, or gains or losses of entire arms or whole chromosomes. This modification and the addition of new targets really improved upon the sensitivity and specificity (94% and 98%, respectively),” he said, noting that this assay is widely used.
Upsides of melanoma FISH assays are that they are a “fairly routine methodology” in large clinical laboratories, he said, and that many labs are familiar with interpretation. “I would say the biggest advantage to FISH is its ability to analyze specific cells, which is useful with small or heterogeneous tumors,” Dr. Hosler said. “Also, there is a genetic reimbursement code for it, and it yields diagnostic and potentially prognostic information.” For example, certain copy number changes have shown to portend a worse prognosis if they’re present in a melanocytic tumor, including alterations in CDKN2A, CCND1, MYC, topoisomerase, and BAP1.
Downsides of melanoma FISH assays are that they are expensive, labor-intensive, and require experts to interpret the results. “The stacking and truncation of cell nuclei innate to paraffin-embedded FISH make interpretation difficult,” he said. “Also, all colors cannot be viewed simultaneously, and each lab’s assay potentially is different, requiring validation. These are not [Food and Drug Administration]-approved tests.”
Next generation sequencing (NGS). Also known as high-throughput sequencing, this technique allows for the generation of millions of sequencing reads that are aligned to a standard human genome, and likely represents the wave of the future. “With NGS you can increase breadth, so you can sequence the entire genome if you want, but you can also increase depth, meaning increasing the number of reads over a single target of the genome,” Dr. Hosler said. “That’s useful if you’re looking for a low frequency mutation.”
For example, NGS allows one to detect alterations of BRAF and KIT and other potentially actionable alterations. It can also be used to detect mutations in benign and malignant melanocytic lesions, including historically diagnostically challenging Spitz and desmoplastic subgroups. Several different NGS technologies exist, and there are different strategies behind each assay, including whole genome sequencing, whole exome sequencing, transcriptome sequencing, and targeted panels. “I’ve seen panels of 10 and I’ve seen panels of 1,500; there’s a wide range,” Dr. Hosler said. “The biggest challenge with NGS, currently, is that it’s difficult to interpret. Trying to figure out what’s important and what’s not important can be challenging. Often you need a team of people who are experts in bioinformatics to interpret these results.”
Slow turnaround time is another downside. “It can take a month to get results, and sometimes clinicians don’t want to wait that long, especially if they think a lesion is melanoma, so that’s an area of focus for NGS laboratories,” he said. “And there are questions on reimbursement. If you run NGS on every unusual melanocytic lesion, that’s not a good use of health care dollars. Who’s paying for it? I don’t have an answer for you. It’s all over the map right now. Each lab’s test and billing practice is different.”
Dr. Hosler reported having no relevant financial disclosures. ProPath is a nationwide pathology practice.