Biomarker tests with discrepant results—why the differences?

Charna Albert

February 2024—When multimodality testing reveals discordant biomarker results, which method is correct? Annette S. Kim, MD, PhD, and JinJuan Yao, MD, PhD, in a CAP23 session last fall used their cases to share strategies for resolving discrepancies—or, in some cases, what look like discrepancies.

“All pathologists must be prepared to understand the underlying causes of the discordance to advise clinicians appropriately,” said Dr. Kim, Henry Clay Bryant clinical professor of pathology and director of the Division of Diagnostic Genetics and Genomics at the University of Michigan. “This may mean resolving differences between positive and negative results or interpreting noncategorical data, such as weak or equivocal.”

Dr. Kim is vice chair of the CAP Personalized Health Care Committee. She presented with fellow committee member Dr. Yao, who is assistant attending at Memorial Sloan Kettering Cancer Center. Two of the four cases they presented are reported here. For more on this topic, see the CAP’s CME/CE program (https://shorturl.at/yAOZ6).

Case No. 1 is that of a 46-year-old woman with a midline intraventricular mass with possible invasion of the corpus callosum. Fig. 1 is composed of the low-power view, showing areas of viable and necrotic tumor (top), as well as the high-power view, showing the high-grade histopathologic features of the tumor (below), with an estimated tumor purity of 90 percent.

The mass was diagnosed as a high-grade glioma.

Testing for several genetic alterations must be performed on diffuse gliomas to diagnose these tumor types for proper clinical care, according to guidelines published in 2022 (Brat DJ, et al. Arch Pathol Lab Med. 2022;146[5]:547–574). On diffuse gliomas that are wildtype for IDH1/2 and H3, BRAF mutation testing (V600) may be performed. Though the evidence to support this recommendation was assessed as low, the recommendation was influenced by the availability of targeted therapy, in addition to a limited number of studies.

Immunohistochemistry for the R132H variant of IDH1—the most frequent IDH1 mutation—was found to be negative, Dr. Kim said. The IHC for BRAF V600E was positive.

“BRAF p.V600E is found at varying prevalence in gliomas,” she said. Though it’s more common in low-grade disease, “even in high-grade gliomas, up to eight or nine percent can have a BRAF mutation, so it would not be unreasonable to have a BRAF-positive high-grade glioma.” She and her colleagues elected to confirm the BRAF result with orthogonal testing by digital droplet PCR (ddPCR).

In the assay they used, each droplet contained, in addition to a single template molecule of the patient’s DNA, fluorescence resonance energy transfer probes, in which a fluorophore is attached to a quencher through an oligonucleotide. The oligonucleotide is complementary either to the wildtype strand of DNA for the locus of interest or the mutant strand. As polymerase is added, exonuclease activity releases the fluorophore from the quencher. “Your droplets will turn color, and you can measure the color of the fluorescence through a flow cytometer,” she said, denoting whether the DNA is mutant or wildtype.

The patient was negative for BRAF p.V600E by ddPCR. “So the IHC was positive, but the molecular was negative.”

“Our initial assumption was that something might be wrong with the molecular result,” she said. “We thought the molecular was a false-negative.” In the ddPCR assay validation, “we did find this particular assay would detect BRAF p.V600K”—the second-most common variant at that locus—“but at a lower fluorescence level, because there was a different affinity for the probe than the typical p.V600E variant.” They debated whether the tumor could be harboring a novel V600 variant, detectable by IHC but not by the molecular test. “One of the reasons we hypothesized that the molecular might be a false-negative is because through the CAP, we’ve published data showing that dinucleotide variants of BRAF at the V600 codon can be missed by many molecular assays.”

In the CAP study, laboratory-developed testing methods for BRAF performed better than FDA-approved companion diagnostics, with LDTs demonstrating 96.6 percent acceptability and FDA-approved tests 93 percent (Moncur JT, et al. Arch Pathol Lab Med. 2019;143[10]:1203–1211). The main cause of the discrepancy was p.V600K analysis, with LDT acceptability rates at 88 percent and FDA-approved assays at 66.1 percent.

“When challenged with a dinucleotide variant such as V600K, the FDA-approved assays did significantly worse,” Dr. Kim said.

The FDA-approved platform with the lowest pickup rate, she said, has a sensitivity of about 10 percent neoplastic cellularity to detect p.V600E, but “the probe designed to bind to V600E does not bind well at all to V600K. As a result, the threshold to be able to pick up this variant is 60 percent neoplastic cellularity, and in that case the variant would be misidentified as a V600E. This explains why we had qualms about some of these molecular assays that might involve hybridization of a mutation-specific oligo,” she said. It seemed possible that their molecular assay, too, could have missed some other novel or uncommon dinucleotide variant of V600.

Next-generation sequencing, which is a methodology that should identify all the different variants at this site agnostically, also was negative. “So the conclusion from this study was actually that the IHC was a false-positive,” she said. The IHC revealed lysosomal staining with a strong granular staining pattern (Fig. 2), “as opposed to a BRAF-positive control, which tends to have a diffuse, almost muddy-looking staining.”

One review of the literature reported high but not perfect sensitivity and specificity in IHC for BRAF (Ritterhouse LL, et al. Semin Diagn Pathol. 2015;32[5]:400–408). Compared with molecular testing in melanoma, thyroid, and colon cancer, IHC had a sensitivity range of 71 to 100 percent and specificity range of 62 to 100 percent. “So there was a significant false-positive rate,” she said. According to the review, a contribution to the wide range in specificity was from the results of one study that reported high numbers of false-positives, in part because of tumors scored positive when as little as 10 percent of the tumor demonstrated moderate to strong staining. “In which case it might be positive by IHC but negative by molecular,” Dr. Kim said.

High background staining, too, can cause issues, as with the lysosomal background staining in the false-positive IHC for BRAF from the case. “Another thing that should be taken into consideration is that BRAF IHC assays almost always are validated against melanomas,” she said. “They don’t take into account the background noise you might get in nontumor cells in some of these other tissue types.”

The final resolution: The mass was a diffuse glioma, IDH-wildtype and BRAF-negative. “And although the ddPCR for BRAF was initially thought to be a false-negative, in fact the IHC was a false-positive.”

Case No. 2 involved a partially obstructive rectal mass, discovered in a colonoscopy for blood in stool.

The patient, a 52-year-old woman, had a complicated past medical history. She was diagnosed at age 44 with POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, skin changes) syndrome after a two-year history of bilateral weakness, skin hyperpigmentation, thrombocytosis, and monoclonal gammopathy with elevated vascular endothelial growth factor levels.

“The patient did undergo an autologous stem cell transplant at age 45 and was in hematologic remission but still had a lot of neuropathy,” Dr. Kim said. She was diagnosed with Barrett’s esophagitis at age 49.

Initial radiology staging was T4N2M1. The liver MRI identified a segment 7 (0.7 cm) and 4a (3.2 × 3.8 cm) small liver metastases. The pelvic MRI found a T4N2 tumor involving the peritoneum and uterus.

The patient was diagnosed with invasive rectal adenocarcinoma by rectosigmoid biopsy and subsequent resection (post-neoadjuvant therapy). Residual tumor remained after therapy (stage ypT3NO) (Fig. 3).

As in all colorectal cancer cases, Dr. Kim said, a hereditary cancer syndrome could have been at play, of which Lynch syndrome is the most common.

Of the genes associated with hereditary colorectal carcinoma, MLH1, MSH2, MSH6, and PMS2 usually are examined by IHC. Mutations in the epithelial cell adhesion molecule, located near the MSH2 gene, also can result in loss of MSH2. “Those are the ones we typically evaluate up front in Lynch syndrome,” she said.