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Blood, Vol. 113, Issue 19, 4677-4680, May 7, 2009
Primary central nervous system lymphoma: tumor-related clones exist in the blood and bone marrow with evidence for separate development
Blood McCann et al.
113: 4677
Supplemental materials for: McCann et al
Files in this Data Supplement:
- Table S1. V-gene usage and somatic hypermutation in patients with PCNSL (PDF, 36.1 KB)
- Figure S1. Expression of AID mRNA assessed by RT-qPCR assay (JPG, 44.5 KB)
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The expression of AID mRNA normalised to β actin was assessed in 12 PCNSLs by quantitative real-time PCR assay. AID mRNA transcripts were observed in 11 of 12 tumors; AID mRNA expression levels in tumor samples were expressed relative to the human Burkitt’s lymphoma cell line Ramos (=1.0). Analyses were performed according to the 2−ΔΔCT method. Samples were analyzed in triplicate. AID expression at the protein level, as assessed by IHC, was confirmed for 4 tumors, indicated by an *.
- Figure S2. Assessment of intraclonal variation in sequences identified from the biopsy, blood, and BM of PCNSL patients (JPG, 35.9 KB)
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The level of intraclonal variation was assessed in cloned sequences derived from the CNS-localized tumor and the systemic compartments (all n=<25). In general, sequences identified from the blood and BM tended towards increased intraclonal diversity. Intraclonal variation was expressed as a percentage of the total nucleotides sequenced (FR1 to CDR3); continuing SHM was confirmed if levels exceeded that of the previously determined Taq error rate of 0.05%, indicated by a broken line.
- Figure S3. Intraclonal variation in sequences identified from the blood of patient 11 (JPG, 149 KB)
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Cloning of individual PCR products revealed that in addition to 9 point mutations common to the primary tumor consensus sequence, 3 extra replacement mutations (aa positions 17, 27a, and 27b) featured in a major (21/25) subset of sequences isolated from the blood. However, intraclonal variants existed that lacked 1 (1/25) or all 3 mutations (4/25). None of these point mutations featured in sequences isolated from the primary tumor (n=25) or the BM (n=25). The tissue type and the observed frequency of the sequence is indicated on the left. The unique CDR3 sequence to which the specific primer was designed to anneal is underlined. Dots represent identity to germline. Mutations at the aa level are shown as upper case letters for replacement mutations or as lower case letters for silent mutations.
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