All cancers are believed to be due to mutations. Testing for myeloid differentiation primary response gene 88 (MYD88) has significant therapeutic and diagnostic value in a range of cancer types, including Waldenström's Macroglobulinemia (WM), diffuse large B-Cell lymphoma (DLBCL), monoclonal gammopathy of unknown significance (MGUS), and splenic marginal zone lymphoma (SMZL), collectively, “MYD88-associated cancers”. MYD88 is an adaptor molecule in a toll-like receptor and interleukin-1 receptor signaling pathway. Mutation in MYD88 results in over-activation of toll-interleukin-receptor pathways, subsequent phosphorylation of IRAK1/4, and release of nuclear factor-kappa-B (NF-κB) drive cell survival and proliferation. It has been demonstrated in DLBCL and WM that inhibition of MYD88 signaling results in decreased NF-κB activity and reduced cell survival. MYD88 mutations are also associated with greater disease burden in patients with DLBCL and poor overall survival following initial and secondary therapy options. MYD88 mutations are detected in 39% of activated B-cell-like (ABC) DLBCL. These mutations are, however, rarely discovered in germinal center B-cell-like (GCB) DLBCL and primary medastinal B-cell lymphoma (PMBL). Therefore, MYD88 mutation status may serve as a surrogate marker for the ABC-subtype. Recent work on ABC-DLBCL and WM has demonstrated increased response to therapy—in both disease types—by combination therapy with a toll-like receptor agonist (IMO-8400) and Rituximab in mouse models. As such, MYD88 mutation status is a useful marker in determining prognosis and in guiding current and future therapy options.
MYD88 mutations are found in almost all cases of WM and ˜50% of patients with Immunoglobulin M (IgM)-secreting MGUS, while these mutations are rarely detected in patients with SMZL (0-6%) and are absent in multiple myeloma (0%). Differential diagnosis of WM from SMZL and IgM-multiple myeloma is often difficult because of overlapping morphologic, immunophenotypic, cytogenetic, and clinical characteristics. MYD88 is, therefore, a useful marker for accurate diagnosis given its positive mutation status presenting primarily in WM. MYD88's diagnostic, prognostic, and therapeutic power necessitates the development of high-throughput, high-sensitivity assays.
Most mutations in MYD88 occur at codon L265, converting leucine to proline (L265P); however, mutations at M232, P258, L103, and Q143 have also been reported. Allele-specific (AS) polymerase chain reaction (PCR) based assays have been developed for MYD88 L265P and demonstrated the ability to detect minute fractions of L265P positive cells. However, inherent limitations in this methodology prohibit AS-PCR from detecting variants other than those previously described or specifically designed for this purpose. Thus, the need for an alternative methodology that can be used in routine lab work at larger volumes is desired.
Wild-type blocking PCR (WTB-PCR) using locked nucleic acid (LNA) has demonstrated high sensitivity and versatility in the detection of low percentage mutant populations. By adding an LNA oligo (10-12 NT), complementary to the region of the hotspot, amplification of the WT allele is inhibited, leading to experimentally driven positive selection for mutant alleles. This is accomplished by designing the LNA oligo so that it anneals to the template strand during the primer annealing step of PCR and melts from mutant template DNA—but not WT DNA—during extension. Because a single nucleotide mismatch in the LNA-DNA hybrid greatly decreases its melting temperature, only mutant template DNA is free to complete its extension. Therefore, WT DNA is amplified linearly but mutant DNA is amplified exponentially. Traditional Sanger sequencing can then be performed.
Sanger sequencing has traditionally been the gold standard in testing for both known and unknown somatic mutations. One of the limitations of Sanger sequencing is its limit of detection (˜10-20% mutant allele in a background of WT). This level of sensitivity is inappropriate for detecting low level somatic mutations that may be present in samples from premalignant tissues or patients with few circulating tumor cells, or when bone marrow (BM) is patchy. This also makes assessing residual disease after therapy or detecting emerging resistance mutations during therapy difficult by conventional sequencing alone. By replacing conventional PCR with LNA-mediated wild-type blocking PCR (WTB-PCR) in Sanger sequencing, sensitivities of up to 0.1% mutant allele in a background of WT can be achieved. In WTB-PCR enrichment for mutant alleles is achieved via the addition of a short (˜10-14 NT) LNA oligonucleotide that binds preferentially to WT DNA thereby preventing amplification of WT DNA. The mutant enriched WTB-PCR product can then be sequenced. By blocking WT DNA rather than selecting for specific mutations WTB-PCR allows for enrichment of both known and unknown mutations present in minute cell fractions.
Among the most prevalent methods for detecting mutations in small cell fractions are allele-specific PCR (AS-PCR) and real-time quantitative PCR (qPCR). Both are limited by false-positives and the ability to only detect one mutation for which the assay was designed. WTB-PCR, however, allows the user to visualize sequencing traces, which enables the detection of multiple mutation types and can aid in ruling out false positives due to artifacts or deamination events. Next-generation sequencing (NGS) may offer a suitable alternative to conventional sequencing, however, substantially greater costs, complexity, and longer assay time render it an unnecessary option for many disease types with few distinct molecular markers or for monitoring patients on therapy for emerging resistance mutations. Furthermore, high false positive rate when detecting variants with mutant allele frequencies of less than 5% can pose a problem for amplicon-based NGS.
The utility of WTB-PCR over AS-PCR lies in its ability to block WT allele amplification rather than amplifying one specific variant allele. In addition, these variant alleles can then be visualized and confirmed via sequencing—contrary to AS-PCR—thereby avoiding false positives. This enrichment of the mutant DNA is particularly useful in testing clinical samples that may contain relatively few neoplastic cells. DLBCL or WM cells may constitute a small percentage of the total cells in bone marrow or peripheral blood samples, leading to false negative results.