Microarrays Aid in Diagnosing Pathogenesis in Renal Transplant Rejection

Jacquelyn K Beals, PhD

November 28, 2007

November 28, 2007 — An innovative study has shown that microarray analysis can supplement histopathology in diagnosing renal transplant rejection by detecting disturbances in the expression of gene sets involved in allograft rejection. The study, published in the December issue of the American Journal of Transplantation, focuses on the patterns of gene expression that accompany rejection.

"Previous studies used analyses based on single genes," said senior author Philip F. Halloran, MD, PhD, director of the Alberta Transplant Applied Genomics Centre, founding editor-in-chief of the American Journal of Transplantation, and professor in the Departments of Medicine and Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, in an email to Medscape Pathology. "But genes don't usually move singly. They move in groups — 'herds' — reflecting biologic events."

The investigators initially characterized these "herd movements" — pathogenesis-based transcript sets (PBTs) — in mouse transplants. Thus, genes were selected and grouped by their biological functions rather than by patterns within the patient population to be analyzed. PBTs were defined for 3 biological processes that occur in allograft rejection: cytotoxic T-cell infiltration, interferon-gamma effects, and parenchymal deterioration with a loss of kidney transcripts.

The study then related these PBTs to histopathology and to clinical diagnoses in a consecutive series of human renal transplant biopsies. The original renal transplant biopsies (N = 143) for cause were from 109 patients, most with 1 biopsy (n = 85); the remainder of the patients had 2 to 4 biopsies obtained at different times for independent clinical indications. Control tissue was obtained from unaffected cortical areas during nephrectomies for carcinoma.

Histopathological diagnoses of rejection or nonrejection typically derive from the Banff criteria, which identify intimal arteritis and tubulitis as the principal lesions characterizing acute rejection. Cyclosporine toxicity is also indicated by the presence of arteriolar hyalinosis.

This study defined PBT scores as the log 2 of the fold change in PBT expression compared with control subjects, which reflected the biological processes intrinsic to allograft rejection. There were high correlations among expressions of PBTs representing the same process, as well as correlations across processes:

  • Cytotoxic T lymphocyte–associated transcripts (CAT1 and CAT2); correlation: 0.98

  • Interferon-gamma-dependent rejection-induced transcripts (GRIT1 and GRIT2); correlation: 0.95

  • Kidney transcripts (KT1 and KT2); correlation: 0.93

  • CAT1 and CAT2 correlated positively with GRITs (correlation: 0.93 and 0.94, respectively) and inversely with KT1 (−0.70 and −0.74, respectively) and KT2 (−0.55 and −0.61, respectively).

The presence of histopathological lesions such as interstitial inflammation, vasculitis, and tubulitis was correlated with higher CAT and GRIT scores and with lower KT scores. These results were validated with 51 additional biopsies, and finally with the entire set of 143 + 51 biopsies. The report observed that "PBTs were most disturbed when patients had clinical as well as histopathologic criteria for rejection episodes. Biopsies with minimal PBT disturbances had a very low incidence of rejection."

However, some Banff criteria appeared unreliable when compared with PBT results. As Dr. Halloran noted, "The pathologists have had questions about the reliability of histology based on their ability to reproduce it. But there was no external standard against which to compare [the results]." The greatest disagreements between PBTs and histopathology occurred in cases with isolated regions of pathology, borderline changes, atrophy, fibrosis, or treatment effects.

As to the future use of PBTs as a diagnostic tool in human kidney transplant biopsies, Dr. Halloran said, "I see them being used by the clinician and pathologists in parallel to the microscope.... We need to develop clinical platforms to support this in routine laboratories." He identified the goal as "accurate diagnosis and improved understanding and classification of disease. The microscope and the microarray or PCR will work together."

Medscape Pathology also spoke with Robert B. Colvin, MD, Benjamin Castleman Distinguished Professor of Pathology, Massachusetts General Hospital, Boston, whose editorial on the study appears in the same issue of the journal. Comparing histopathology with PBT analysis, Dr. Colvin noted: "They both have advantages and disadvantages, which are complementary.

"There are many ways that [PBT use] might be useful.... [It] could indicate...the mechanism of damage in a way that a specific drug might be recommended. We'll have to go a little further in developing pathway-specific drugs for transplantation, but it would not be too farfetched to say that this pattern will be used to choose among different specific drugs," he said.

"A biopsy studied this way could probably tell you that the patient was responding to a drug, better than pathology could, because it takes a while for the pathology to change, and the gene expression changes very quickly," Dr. Colvin said, adding, "Histology has an advantage in showing the accumulated changes, not just what's happening at that moment.... The other thing the pathology will be able to show better is very focal lesions that are not widely present and might be lost in the noise of the microarrays."

The study was supported by a variety of sources including Genome Canada, Roche Molecular Systems, Hoffmann-La Roche Canada Ltd, and the Roche Organ Transplant Research Foundation. Dr. Colvin has been a scientific advisor for the research group at the University of Alberta, Edmonton. Dr. Halloran and Dr. Colvin have disclosed no relevant financial relationships.

Am J Transplant. 2007;7(12):2712–2722.

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