Characterization of clonal Ig DNA in plasma of AIDS-related NHL patients
Authors: Nina Wagner-Johnston, Dilber Nurmemet, Lan Lin, Victor Lemas, Linda Post, Richard Ambinder
Journal: Clinical Cancer Research (2007)

B17  Background:  In B cell lymphoma there is a special opportunity to develop plasma tumor markers. The presence of rearranged immunoglobulin (Ig) genes means that every tumor has a distinctive Ig DNA sequence. We describe a novel technique for detecting clonal Ig rearrangements in the plasma of AIDS lymphoma patients (pts).  Methods:  We evaluated serial (n=29) plasma specimens from 8 pts with AIDS-related lymphoma (ARL) and biopsy specimens from 2 of the 8 pts. Reactions containing 5 µl of the extracted DNA in a 50 µl volume underwent first round PCR. The multiplex primers (InVivoScribe Technologies; San Diego, CA) target several Fr regions and are labeled with fluorochrome. PCR products were analyzed on an ABI 310 capillary electrophoresis machine with TAMRA size standards. The expected size range of amplified products for Fr3 is 100-170 bp. A specimen control size ladder which targets multiple genes and generates a series of amplicons ensured adequate input DNA. Clonality was defined as one or two peaks of a constant molecular weight (within one base pair) in all 3 replicates of a sample. In two cases, second round PCR products from both the plasma and tumor were separately purified and cloned into a sequencing vector and colonies screened by PCR using vector primers. Those with the expected size inserts were sequenced in the DNA Analysis Core Research Facility at JHU.  Results:  All 8 pts had diffuse large B-cell histologies, 3 pts had stage II, 1 had stage III, and 4 had stage IV disease. Half the pts had B symptoms. In 4 pts with reported Ki67 index, the proliferative rate was high (>60%) in all tumor specimens. 4 pts had elevated LDH levels (range 366-1909 U/L) at diagnosis. The median CD4 at time of diagnosis was 137/cu mm (range 9-234). Of the 8 pts, three (38%) had clonal spikes at baseline and the presence or absence of the spike in subsequent specimens corresponded with disease activity. Serial specimens (n=6) during treatment from one of these pts showed clonal rearrangement prior to treatment, disappearance with chemotherapy including rituximab, then reappearance presaging relapse and death. The other two pts had serial specimens (n=12) which remained positive during the course of their treatments corresponding with their refractory disease. In 2 of these positive pts, the amplification product from plasma was then sequenced and the sequence confirmed that the amplified product in the pt’s plasma was identical to the sequence of the clonotypic IgH VDJ recombination in tumor. For purposes of comparison, we also surveyed healthy donors (n=20) and pts with Kaposi’s Sarcoma (n=10) and did not detect any clonal Ig DNA rearrangements.  Conclusions:  More than half of pts with AIDS-related lymphomas (ARL) present with high-risk disease and their outcomes are poor even after aggressive chemotherapy. The characterization of new tumor markers offers the possibility that new treatments may be more rapidly assessed in the future and that pts’ therapies may be individually tailored.