Biomarkers for Non-Hodgkin Lymphomas and Uses Thereof

Abstract

The disclosure provides a method of identifying a subject as having B-cell non-Hodgkin lymphoma (NHL) such as testing a sample from a subject for a mutation in one or more biomarkers. Also described are methods for classifying or monitoring a subject having, or suspected of having, B-cell non-Hodgkin lymphoma comprising testing the sample for a mutation in one or more biomarkers.

Description

RELATED APPLICATIONS[0001]The present application is a Continuation-In-Part of U.S. patent application Ser. No. 13 / 805,504 filed on Dec. 19, 2012, which is a U.S. national phase application filed under 35 U.S.C. §371 claiming benefit to International patent Application No. PCT / CA2011 / 000724, filed Jun. 23, 2011, which is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 357,813 filed Jun. 23, 2010, and U.S. Provisional Application No. 61 / 420,065 filed Dec. 6, 2010, each of which application is hereby incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING[0002]A computer readable form of the Sequence Listing “3158-P39718US01_SequenceListing.txt” (16,384), submitted via EFS-WEB and created on Mar. 12, 2013 is herein incorporated by reference.FIELD OF THE DISCLOSURE[0003]The disclosure relates to methods of testing for cancer and more specifically to methods of testing samples for somatic mutations indicative of B-cel...

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[0032]FIG. 4 shows the N-terminal truncation of FOXO1 protein with mutation affecting initial codon. (A) The RNA-seq data of cell lines and patient samples revealed mutations in 3 samples affecting the initial codon of FOXO1. To determine the effect of such mutations on FOXO1 protein, we assayed FOXO1 by Western blot in DLBCL cell lines using an antibody raised against full-length FOXO1 (2H8.2). In the cell line containing a mutation at the initiator methionine (OCI-Ly1), we observed a FOXO1 band of reduced molecular weight, compared to FOXO1 wild-type cell lines (size indicated in Kilodaltons on the left). The reduced size is consistent with the use of a second methionine codon in the FOXO1 gene, producing a protein shortened at the amino terminus by 70 amino acids. The same blot was also probed with an antibody that recognizes an N-terminal epitope (L27) and lack of a band in OCI-Ly1 cells is consistent with the notion that the lower band in this cell line corresponds to FOXO1 protein lacking its N-terminus. Absence of the protein in the DB cell line was noted, which showed significantly reduced mRNA levels as measured by RNA-seq (upper bar chart; RPKM=Reads Per Kilobase of gene model per Million mapped reads).

[0033]FIG. 5 shows the effect of GNA13 mutations at the protein level. (A) A western blot revealed the expected lack of GNA13 protein in DOHH2, the cell line with a truncating point mutation detected in the RNA-seq data. The lack of protein in Karpas422, SU-DHL-6 and WSU-DLCL2 was surprising, as protein-truncating mutations were not detected in these cells. (B) Further analysis of the aligned sequence from these three cell lines and additional analysis utilizing a de-novo transcript assembly approach (Trans-ABySS; Methods), revealed multiple aberrations that may explain the lack of protein. Firstly, in Karpas422 reads were observed to map the first intron, suggesting that the intron is retained in a significant proportion of GNA13 transcripts (compare Karpas422 on the left to WSU-DLCL2 on the right). Inspection of sequence contigs from this case revealed the likely cause of intron reads to be a deletion of 87 nt that removes the canonical splicing donor from this exon (Panel C, top). Splicing still appears to occur to a lesser extent using a non-GT donor. Assembled reads from SU-DHL-6 revealed a 2 nt deletion and a large 1028 nt deletion. The former would affect the reading frame and the latter removes the terminal stop codon. Finally, in WSU-DLCL2, the splicing donor after the third exon was apparently mutated, converting the GT donor to a GC sequence (not shown). As in the Karpas422 case, there was clear evidence for retention of this intron in GNA13 transcripts in WSU-DLCL2. Intron retention has previously been linked to nonsense-mediated transcript degradation [76] and if that is the case here, could explain the lack of GNA13 protein in these cells.

[0034]FIG. 6 shows the predicted impact of recurrently mutated genes on BCR signalling and downstream messengers. (A) Autocrine and paracrine stimulation of IL-21R induces the dimerizati...

Problems solved by technology

Current knowledge of the specific genetic events leading to DLBCL and FL is limited to the presence of a few recurrent genetic abnormalities [2].

Despite the disparity...

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