A multicenter study directly comparing the diagnostic accuracy of gene expression profiling and immunohistochemistry for primary site identification in metastatic tumors

Abstract

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

FIGURE 1

Study design: metastatic tumor specimens meeting the criteria were obtained as archived formalin-fixed, paraffin-embedded specimens from 4 institutions: the University of Tennessee (UT); Virginia Commonwealth University (VCU); Indiana University-Purdue University, Indianapolis (IUPUI); and Folio Biosciences (FB). The CLIA-certified laboratory at UT sectioned each block and sent digitized H&E images to 4 different EPs and to the pathology team at PWDL. The EPs and PWDL were blinded to the primary site. The EPs ordered stains from a panel of 84 IHC and histochemical stains. All staining was performed at the UT CLIA-certified laboratory, and digitized images of the requested stained slides were provided to only the ordering EP using the web interface. The EPs, who were trained on the Whole Slide Imaging system and the web interface before the trial and during a pilot study, could issue a final diagnosis at any stage in the workup. Coded slides sent to PWDL were analyzed using the Pathwork Tissue of Origin Test. The accuracy of IHC and GEP were compared using statistical methods described.

FIGURE 2

Confusion matrix: GEP and IHC. Known primary site versus test prediction/final diagnosis. Test predictions and final diagnoses with positive predictive value (PPV) of 98% or higher are highlighted in green. Bladder (BL), breast (BR), colorectal (CO), gastric (GA), hepatocellular (LI), kidney (KI), melanoma (ME), non-Hodgkin’s lymphoma (LY), non–small cell lung (LU), ovarian (OV), pancreas (PA), prostate (PR), sarcoma (SC), testicular germ cell (GC), and thyroid (TH).

FIGURE 3

Forest plot of comparative accuracy, IHC compared with GEP, overall, and subgroup analysis. ORs <1 favor IHC, >1 favor GEP. Statistically significant subgroups are shown in blue; lines indicate the 95% confidence interval.