# Patent

## Title
Discovery of a somatic mutation in MYD88 gene in lymphoplasmacytic lymphoma

## Abstract

Diagnostic assays for facilitating the diagnosis of lymphoplasmacytic lymphoma (LPL) are provided. The method comprises assessing a biological sample of the subject for the presence of a mutation at position 38182641 in chromosome 3p22.2, wherein presence of the mutation is indicative that the subject has LPL. Also, provided are targeted therapies, methods for monitoring the progression or recurrence of LPL, and a sensitive and inexpensive real-time allele specific polymerase chain reaction assay for reliable and quantitative assessments of the mutation.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 14/128,241, filed Mar. 18, 2014 which is a national stage filing under 35 U.S.C. § 371 of International Application PCT/US2012/044956, filed Jun. 29, 2012, which was published under PCT Article 21(2) in English, and which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/571,657, filed Jul. 1, 2011, the disclosure of each referenced application is incorporated by reference herein in its entirety.


BACKGROUND OF THE INVENTION
Waldenstrom's macroglobulinemia (WM) is a distinct clinicopathological entity resulting from the accumulation, predominantly in the bone marrow, of clonally related lymphoplasmacytic cells which secrete a monoclonal IgM protein. This condition is considered to correspond to lymphoplasmacytic lymphoma (LPL) as defined by the World Health Organization classification system. Genetic factors play an important role in the pathogenesis of WM, with 25% of patients demonstrating a family history. IgM monoclonal gammopathy of unknown significance (MGUS) often precedes the development of WM. The primary oncogenetic event resulting in malignant transformation in WM remains to be delineated. Knowledge of such genomic alteration(s) may permit advances in diagnostic testing, and development of targeted therapies.
SUMMARY OF THE INVENTION
It has been discovered, surprisingly, that a somatic mutation in the myeloid differentiation primary response (MYD88) gene is associated with lymphoplasmacytic lymphoma. Accordingly, in some aspects, the invention involves, facilitating the diagnosis of lymphoplasmacytic lymphoma in a subject by selecting a subject on the basis that the subject presents one or more of the following clinical features: anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein, obtaining a biological sample of the subject, and determining from the biological sample whether the subject has a mutation at position 38182641 in chromosome 3p22.2, wherein the presence of the mutation is indicative that the subject has lymphoplasmacytic lymphoma. In some embodiments, the subject presents two or more of the clinical features. In some embodiments, the subject presents three or more of the clinical features. In some embodiments, said determining comprises performing an assay to interrogate position 38182641 in chromosome 3p22.2. In some embodiments, the assay comprises allele specific polymerase chain reaction performed using an allele specific primer, wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5.
According to one aspect of the invention, a method to distinguish lymphoplasmacytic lymphoma from other B cell neoplasms is provided. The method comprises obtaining a biological sample from a subject that presents symptoms of a B cell lymphoma, which symptom does not exclude a diagnosis of lymphoplasmacytic lymphoma. Symptoms of a B cell lymphoma which do not exclude a diagnosis of LPL include asymptomatic localized or generalized peripheral lymphadenopathy, plasmacytic difference, bone marrow involvement, autoimmune thrombocytopenia, end organ damage (renal insufficiency), anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. In some embodiments, said determining comprises performing an assay to interrogate position 38182641 in chromosome 3p22.2. In some embodiments, the assay comprises allele specific polymerase chain reaction performed using an allele specific primer, wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5.
In some embodiments, the method comprises obtaining a biological sample from a subject that presents symptoms of both LPL and at least one B cell neoplasm selected from the group consisting of nodal marginal zone lymphomas, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), splenic B cell marginal zone lymphoma, monoclonal gammopathy of undetermined significance and plasma cell myeloma, determining from the biological sample whether the subject has a mutation at position 38182641 in chromosome 3p22.2, and providing a report whether the subject has a mutation at position 38182641 in chromosome 3p22.2, wherein the presence of the mutation is indicative that the subject has lymphoplasmacytic lymphoma.
According to one aspect of the invention, a method to treat lymphoplasmacytic lymphoma in a subject is provided. The method comprises selecting a subject on the basis that the subject has a mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the subject also presents one or more symptoms or clinical features of LPL, such as, anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. The subject is administered a myeloid differentiation primary response 88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4 (IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitor in an amount effective to treat lymphoplasmacytic lymphoma. In some embodiments, the MYD88 inhibitor is a peptidomimetic compound ST2825. In some embodiments, the IRAK-1/4 inhibitor is N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole. In some embodiments, the BTK inhibitor is Ibrutinib (PCI-32765).
According to one aspect of the invention, a method for monitoring progression or recurrence of lymphoplasmacytic lymphoma in a subject is provided. The method comprises obtaining multiple biological samples of a subject over a period of time, determining from the multiple biological samples the level of a transcript comprising a mutation at position 38182641 in chromosome 3p22.2, wherein a change in the level of the transcript over the period of time is indicative of the progression or recurrence of LPL in the subject. In some embodiments, the subject is undergoing chemotherapy to treat LPL. In some embodiments, the level of the transcript is measured using quantitative real time polymerase chain reaction. In some embodiments, the quantitative real time polymerase chain reaction is performed using an allele specific primer, wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5.
According to one aspect of the invention, a method for detecting a mutation at position 38182641 in chromosome 3p22.2 in a subject is provided. The method comprises obtaining a biological sample from the subject in need of such detection, determining from the biological sample whether the subject has a mutation at position 38182641 in chromosome 3p22.2 by allele specific polymerase chain reaction performed using an allele specific primer wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2.
According to one aspect of the invention, a method for facilitating the diagnosis of lymphoplasmacytic lymphoma in a subject is provided. The method comprises selecting a subject on the basis that the subject presents one or more of the following clinical features: anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein, obtaining a biological sample of the subject, determining from the biological sample whether the subject has a mutation at position 38182641 in chromosome 3p22.2, and providing a report summarizing statistically significant results indicating that the subject has lymphoplasmacytic lymphoma if the subject has the mutation.
The biological sample includes, but is not limited to, a sample of bone marrow, lymph node, spleen or blood. In some embodiments, the mutation results in a single nucleotide change from T to C in the myeloid differentiation primary response 88 (MYD88) gene. In some embodiments, the mutation results in an amino acid change from leucine to proline at position 265 in the myeloid differentiation primary response 88 protein.
The subject (individual) is a human. In some embodiments, the subject is suspected of LPL and presents one or more of the clinical features of LPL.
Without intending to be bound by the theory of the invention, it is believed that the presence of the mutation in a subject at position 38182641 in chromosome 3p22.2 is predictive of the risk of development of LPL. The presence of the mutation at position 38182641 in chromosome 3p22.2 in a subject having an elevated (abnormal) level of monoclonal IgM serum paraprotein is predictive of the risk of development of LPL. In some embodiments, a method for predicting the risk of development of LPL is provided. The method comprises selecting a subject on the basis that the subject has an elevated (abnormal) level of monoclonal IgM serum paraprotein, and determining whether the subject has a mutation at position 38182641 in chromosome 3p22.2, wherein the presence of the mutation is indicative that the subject is at an increased risk of developing LPL.
It may even be that the few instances where the mutation was present in a subject who is diagnosed with a B cell neoplasm (other than LPL and subtype ABC of diffuse large B cell lymphoma) was a mistaken diagnosis.
These and other aspects of the inventions, as well as various advantages and utilities will be apparent with reference to the Detailed Description. Each aspect of the invention can encompass various embodiments as will be understood.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D show acquired uniparental disomy (aUPD) at chromosome 3p encompassing MYD88 as determined by WGS. Chromosome wide data demonstrating copy number neutral loss of heterozygosity with corresponding allele imbalance for patients WM1 and WM3 indicating presence of an aUPD throughout the tumor clone (FIG. 1A). For patients WM2 and WM4, an aUPD was present in a subpopulation of tumor cells as demonstrated by sustained allele imbalance. Confirmatory Sanger sequencing of the MYD88 mutation mirrors allele imbalances observed by WGS (FIG. 1B). Chromosome wide data (FIG. 1C) and confirmatory Sanger sequencing for MYD88 L265 (FIG. 1D) using CD19-depleted PB mononuclear cells for normal tissue comparisons in patients WM1 and WM2. Arrows (FIGS. 1B, 1D) denote variant allele position.
FIGS. 2A-2D show agarose gel-based AS-PCR assay for detection of MYD88 L265P. The reverse primers with an internal mismatch in the third position from the 3′-end and the common forward primer were indicated by the arrows (FIG. 2A). Sanger sequencing confirmed that DLBCL cell line OCI-LY3 carried homozygous MYD88 L265P, whereas OCI-LY19 was wild-type for MYD88 L265P. The position of L265P was indicated by arrow (FIG. 2B). Sensitivity of the AS-PCR assay was assessed by a serial dilution of the MYD88 L265P mutant DNA from OCI-LY3 with the wild-type DNA from OCI-LY19. The PCR products (159-bp) were separated on 2% agarose gel and indicated by arrows (FIG. 2C). The mutant MYD88 L265P allele can consistently be detected at a dilution of 0.1%. An example of WM patients who carried homozygous MYD88 L265P. This patient had 70% BM involvement (FIG. 2D).
FIGS. 3A-3D show the sensitivity and specificity of the real-time AS-PCR and scatter plot of comparison of MYD88 L265P positive and negative patients and healthy donors. The MYD88 L265P mutant DNA (OCI-LY3) was diluted with the wild-type DNA (OCI-LY19) at the concentration as indicated in the amplification plot. The mutant MYD88 L265P allele can be detected at a dilution of 0.08% (FIG. 3A). Correlation coefficient of the standard curve was 0.998 with a slope value of −3.48 (FIG. 3B). Melting curve analysis revealed that the MYD88 L265P mutant-specific amplicon melted at 84° C. A minor non-specific amplification was only found in the dilution of 0.4% or lower with a melting peak at 80° C. (FIG. 3C). Scatter plot showed two major clusters of WM patients that were separated by 3.2 cycles. The cluster with delta CT values ranging from 9.6 to 15.2 cycles was similar to healthy donors (delta CT values ranging from 10.7 to 16.9 cycles). This cluster was determined as MYD88 L265P negative, whereas another cluster with delta CT values ranging from −0.2 to 6.4 cycles was determined as MYD88 L265P positive (FIG. 3D).
FIG. 4 shows increased phosphorylation of BTK by western blotting with phospho-specific antibody in Waldenstrom's Macroglobulinemia (WM) cell lines, BCWM.1 and MWCL-1, compared to Multiple myeloma cell lines, ANBL6 and INA6. Antibody against total BTK was used as loading control. PCI-32765 significantly blocked the BTK phosphorylation in WM cells.
FIG. 5 shows that PCI-32765 blocked the downstream NF-kB, MAPK, Stat3 signaling by significantly reducing the phosphorylation of IKBα, ERK1/2 and Stat3 proteins in WM cell lines, BCWM.1 and MWCL-1, compared to multiple myeloma cell lines, ANBL6 and INA6. Antibodies against corresponding total proteins and GAPDH were used as loading controls.
FIG. 6 shows that knockdown of MYD88 by lentiviral transduction, and/or use of a MYD88 inhibitor leads to decreased BTK phosphorylation. Antibodies against total BTK and/or GAPDH were used as loading control.
FIGS. 7A-7C demonstrate that BTK inhibitor PCI-32765 induces apoptosis of MYD88 L265P expressing WM cells alone and in combination with MYD88 pathway inhibitor and IRAK 1/4 kinase inhibitor. Apoptosis analysis was performed using Annexin V and PI staining after PCI-32765 treatment for 24 hrs (FIG. 7A). In FIG. 7B, apoptosis analysis was performed using annexin V and PI staining after PCI-32765 and MYD88 homodimerization inhibitor treatment for 24 hrs. 1: DMSO; 2: PCI-32765 (1.0 μM); 3: MYD88 inhibitory peptides (100 μM); 4: PCI-32765+MYD88 inhibitory peptides. PCI-32765 shows synergistic tumor cell killing in combination with an IRAK 1/4 kinase inhibitor (FIG. 7C).



DETAILED DESCRIPTION OF THE INVENTION
The present invention, in one aspect, relates to the surprising discovery of a somatic mutation in the myeloid differentiation primary response (MYD88) gene in patients with lymphoplasmacytic lymphoma. In particular, the invention is based on the identification of a somatic mutation at position 38182641 in chromosome 3p22.2 which results in a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P). While previous work has identified the same mutation in subtype ABC of diffuse large B cell lymphoma, this previous work did not make any association between the mutation and lymphoplasmacytic lymphoma.
Since the molecular mechanism of LPL was unknown, the differential diagnosis of many diseases that are morphologically similar to LPL was hampered. The discovery set forth in the instant application helps to discriminate LPL from other overlapping entities, and allows for disease specific treatment targeting.
According to one aspect, the present invention provides diagnostic assays for facilitating or aiding in the diagnosis of lymphoplasmacytic lymphoma (LPL) in a subject. LPL is a neoplasm of small B lymphocytes, plasma cytoid lymphocytes, and plasma cells, usually involving bone marrow (BM) and sometime lymph nodes and spleen. Waldenstrom macroglobulinemia (WM) is found in a significant subset of patients with LPL and is defined as LPL with BM involvement and an IgM monoclonal gammopathy of any concentration.
According to one aspect of the invention, a subject is selected for assessment of a mutation at position 38182641 in chromosome 3p22.2 on the basis that the subject is suspected of having LPL, and a biological sample of the subject is assessed for the presence of a mutation. The presence of the mutation is indicative that the subject has LPL. As used herein, “selecting a subject” means identifying a subject that presents one or more clinical features of LPL for further diagnostic analysis. The one or more clinical features of LPL include anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. In some embodiments, a subject presenting two or more, three or more, four or more, five or more, six or more, or seven or more of these clinical features is selected. The subject is selected by a medical practitioner (e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.), a healthcare organization, or a clinical laboratory.
Non-limiting examples of the biological sample include bone marrow, lymph node, spleen or blood. Obtaining a biological sample of a subject means taking possession of a biological sample of the subject. Obtaining a biological sample from a subject means removing a biological sample from the subject. Therefore, the person obtaining a biological sample of a subject and determining the presence of the mutation in the sample does not necessarily obtain the biological sample from the subject. In some embodiments, the biological sample may be removed from the subject by a medical practitioner (e.g., a doctor, nurse, or a clinical laboratory practitioner), and then provided to the person determining the presence of the mutation. The biological sample may be provided to the person determining the mutation by the subject or by a medical practitioner (e.g., a doctor, nurse, or a clinical laboratory practitioner). In some embodiments, the person determining the mutation obtains a biological sample from the subject by removing the sample from the subject.
The term “mutation” means any change or difference in the nucleic acid or protein sequence of MYD88 as compared to the wild type sequence that results in the activation of MYD88 which leads to the activation of NF-κB. Mutations include, but are not limited to, nonsense mutations, missense mutations, frameshift mutations, rearrangement mutations, insertion mutations and deletion mutations. In some embodiments, the mutation is a somatic mutation at position 38182641 in chromosome 3p22.2 which results in a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P).
One skilled in the art will appreciate that many suitable methods, in addition to and including the ones discussed in the examples, can be used to detect mutations in the MYD88 gene. Detection methods that can be used include, but are not limited to, direct sequencing, DNAchip technologies, mass spectroscopy, polymerase chain reaction (PCR), allele specific polymerase chain reaction, real time polymerase chain reaction, reverse transcriptase PCR, electrophoretic mobility, nucleic acid hybridization, fluorescent in situ hybridization, and denaturing high performance liquid chromatography.
In some embodiments, mutations in the MYD88 gene may be detected by allele specific polymerase chain reaction (AS-PCR). For AS-PCR, allele specific primers are used which hybridize at or near their 3′ ends to a particular mutation in the MYD88 gene. If the mutation is not present, the 3′-terminal mismatched primer does not initiate replication, and an amplification product is not observed. In some embodiments, only the forward primer or the reverse primer hybridizes at or near its 3′ ends to a particular mutation in the MYD88 gene. In some embodiments, both the forward and the reverse primer hybridize at or near their 3′ ends to a particular mutation in the MYD88 gene. In some embodiments, the allele specific primer is SEQ ID NO: 5. In some embodiments, the mutation is a somatic mutation at position 38182641 in chromosome 3p22.2 which results in a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P).
In some embodiments, mutations in the MYD88 gene may be detected by the direct sequencing of nucleic acid molecules. Techniques for the direct sequencing of DNA are well known in the art. In one embodiment of the invention, mutations may be detected by Sanger sequencing, which may include the use of a thermostable polymerase enzyme, a sequencing primer, dNTPs and limiting amounts of chain terminating fluorescently or radioactively labeled ddNTPs. Polyacrylamide gel electrophoresis or another technique such as capillary electrophoresis may be used to separate the products of the sequencing reactions followed by the detection of the fluorescent or radioactive labels. In one example of this embodiment of the invention, mutations in MYD88 could be determined using automated sequencing on an Applied Biosystems 3700 DNA Analyzer or 3730×1 DNA AnalyzerTM. Mutations may be identified by comparing the sequence of a subject to that of a wildtype individual or to reference sequences found in the public databases.
Other embodiments of the invention contemplate the use of DNA chip technologies for the detection of mutations within the MYD88 gene. Among other applications, DNA chip technologies allow for the identification of mutations within the sequences of the intention through the analysis of the hybridization patterns of a nucleic acid sample onto a high-density spatially addressable microarray of predetermined sequences.
Another technique for the detection of mutations is denaturing HPLC analysis. Accordingly, one embodiment of the invention includes the use of a Transgenomic WaveTM machine for the dHPLC analysis of nucleic acids for the identification of heterozygous mutations or polymorphisms within the sequences of the invention.
The invention also contemplates the use of mass spectroscopy for the genotyping of mutations. Mutant and wildtype nucleic acid molecules may differ in mass due to the different composition of wildtype and mutant sequences, allowing for the identification of mutations on the basis of the molecular mass of different nucleotide sequences. The use of mass spectroscopy, and in particular Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) mass spectroscopy for the genotyping of mutations is well known by those skilled in the relevant art. For example, U.S. Pat. No. 6,043,031 describes a fast and highly accurate mass spectrometer based process for detecting a particular nucleic acid sequence. The MassARRAYTM platform from SEQUENOMTM is an example of a commercially available system capable of genotyping single nucleotide polymorphisms and detecting the mutations in genes.
According to one aspect, the present invention provides a method to distinguish lymphoplasmacytic lymphoma from other B cell neoplasms selected from the group consisting of nodal marginal zone lymphomas, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), splenic B cell marginal zone lymphoma, monoclonal gammopathy of undetermined significance and plasma cell myeloma. The method comprises selecting or identifying a subject that presents one or more symptoms or clinical features of LPL which overlap with one or more symptoms of at least one of the B cell neoplasms described above. Thus, the subject is an individual who is suspected of having either LPL or one of the other B cell neoplasm. The subject is selected for further diagnostic analysis by a medical practitioner (e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.), a healthcare organization, or a clinical laboratory.
The one or more symptoms or clinical features of LPL include anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. In addition, the subject may also present one or more of the following clinical features or symptoms of other B cell neoplasms: asymptomatic localized or generalized peripheral lymphadenopathy, plasmacytic difference, bone marrow involvement, autoimmune thrombocytopenia, peripheral blood villous lymphocytes, end organ damage (hypercalcemia, renal insufficiency, bone lesions), recurrent infections, elevated creatine, hyperuricemia, and hypoalbunemia. The subject suspected of having either LPL or one of the other B cell neoplasm is assessed for the presence of a mutation at position 38182641 in chromosome 3p22.2, wherein the presence of the mutation is indicative that the subject has LPL.
A report summarizing the results of the analysis, i.e. the presence or absence of the mutation and any other information pertaining to the analysis could optionally be generated as part of the analysis (which may be interchangeably referred to herein as “providing” a report, “producing” a report, or “generating” a report). Examples of reports may include, but are not limited to, reports in paper (such as computer-generated printouts of test results) or equivalent formats and reports stored on computer readable medium (such as a CD, computer hard drive, or computer network server, etc.). Reports, particularly those stored on computer readable medium, can be part of a database (such as a database of patient records, which may be a “secure database” that has security features that limit access to the report, such as to allow only the patient and the patient's medical practitioners to view the report, for example). In addition to, or as an alternative to, generating a tangible report, reports can also be displayed on a computer screen (or the display of another electronic device or instrument).
A report can further be transmitted, communicated or reported (these terms may be used herein interchangeably), such as to the individual who was tested, a medical practitioner (e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.), a healthcare organization, a clinical laboratory, and/or any other party intended to view or possess the report. The act of ‘transmitting’ or ‘communicating’ a report can be by any means known in the art, based on the form of the report, and includes both oral and non-oral transmission. Furthermore, “transmitting” or “communicating” a report can include delivering a report (“pushing”) and/or retrieving (“pulling”) a report. For example, non-oral reports can be transmitted/communicated by such means as being physically transferred between parties (such as for reports in paper format), such as by being physically delivered from one party to another, or by being transmitted electronically or in signal form (e.g., via e-mail or over the internet, by facsimile, and/or by any wired or wireless communication methods known in the art), such as by being retrieved from a database stored on a computer network server, etc.
According to one aspect of the invention, a method to treat LPL is provided. The method comprises selecting a subject on the basis that the subject has a mutation at position 38182641 in chromosome 3p22.2, and administering to the subject a myeloid differentiation primary response 88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4 (IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitor in an amount effective to treat lymphoplasmacytic lymphoma. A non-limiting example of an MYD88 inhibitor includes the peptidomimetic compound ST2825 (WO 2006/06709). A non-limiting example of an IRAK-1/4 inhibitor is N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole. In some embodiments, BTK inhibitors useful in the instant invention block MYD88 L265P and BTK signaling. A non-limiting example of a BTK inhibitor includes Ibrutinib (PCI-32765).
In some embodiments, the method comprises selecting a subject on the basis that the subject has a mutation at position 38182641 in chromosome 3p22.2 and presents one or more symptoms of LPL. The selected subject is treated using an effective amount of bortezomib (Velcade®), bendamestine, alemtuzumab, and/or rituximab, but not bisphosphonates or fludarabine.
The MYD88 inhibitor, IRAK-1/4 inhibitor, and/or BTK inhibitor are administered in an effective amount. An effective amount is a dose sufficient to provide a medically desirable result and can be determined by one of skill in the art using routine methods. In some embodiments, an effective amount is an amount which results in any improvement in the condition being treated. In some embodiments, an effective amount may depend on the type and extent of LPL being treated and/or use of one or more additional therapeutic agents. However, one of skill in the art can determine appropriate doses and ranges of therapeutic agents to use, for example based on in vitro and/or in vivo testing and/or other knowledge of compound dosages.
When administered to a subject, effective amounts of the therapeutic agent will depend, of course, on the particular disease being treated; the severity of the disease; individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum dose is used, that is, the highest safe dose according to sound medical judgment.
In the treatment of LPL, an effective amount is that amount which slows the progression of the disease, halts the progression of the disease, or reverses the progression of the disease. An effective amount includes, but is not limited to, that amount necessary to slow, reduce, inhibit, ameliorate or reverse one or more symptoms associated with LPL. In some embodiments, such terms refer to a reduction in the levels of IgM serum paraprotein, anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, and adenopathy.
An effective amount of a compound typically will vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations, for one or several days (depending of course of the mode of administration and the factors discussed above).
Actual dosage levels of the therapeutic agent can be varied to obtain an amount that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular compound, the route of administration, the tissue being treated, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.
Pharmaceutical preparations and compounds comprising MYD88 inhibitor, IRAK-1/4 inhibitor, and/or BTK inhibitor are administered to a subject by any suitable route. For example, compositions can be administered orally, including sublingually, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically and transdermally (as by powders, ointments, or drops), bucally, or nasally. The pharmaceutical preparations of the present invention may include or be diluted into a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible fillers, diluants or other such substances, which are suitable for administration to a human or other mammal such as a dog, cat, or horse. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability. Carriers suitable for oral, subcutaneous, intravenous, intramuscular, etc. formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
According to one aspect of the invention, a method for monitoring progression or recurrence of lymphoplasmacytic lymphoma in a subject is provided. The method comprises obtaining multiple biological samples of a subject over a period of time, determining from the multiple biological samples the level of a transcript comprising a mutation at position 38182641 in chromosome 3p22.2, wherein an increase in the level of the transcript over the period of time is indicative of the progression or recurrence of LPL in the subject.
As used herein, a transcript comprising a mutation means MYD88 nucleic acid or protein that has a mutation at position 38182641 in chromosome 3p22.2. The level of the transcript comprising a mutation at position 38182641 in chromosome 3p22.2 can be determined by any means known to one skilled in the art, including, but not limited to Western blot, Northern blot, and quantitative real time polymerase chain reaction. In some embodiments, quantitation of the transcript levels is accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art. In some embodiments, the quantitative real time polymerase chain reaction is performed using an allele specific primer, wherein the allele specific primer hybridizes at or near its 3′ end to a mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5.
The level of the transcript comprising the mutation is indicative of the state of the disease. In normal (healthy) subjects the mutation is absent. In diseased subjects, the transcript comprising the mutation is expressed at a level consistent with the status (progression) of the disease. Although it is believed that most of the transcription of the MYD88 gene occurs in the bone marrow, levels of the transcript and protein will be present in the circulation because of the normal turnover and presence of dead cells in the blood.
As used herein, a change in the level of the transcript comprising a mutation means that the amount or concentration of the transcript sufficiently changes over time. A change in the level of the transcript over the period of time may be any statistically significant change which is detectable. Such a change may include, but is not limited to, about a 1%, about a 10%, about a 20%, about a 40%, about a 80%, about a 2-fold, about a 4-fold, about an 8-fold, about a 20-fold, or about a 100-fold change over the time. An increase in the level of the transcript indicative of unfavorable progression of the disease, while a decrease in the level of the transcript is indicative of a favorable progression of the disease.
As used herein, a “period of time” is intended to include a period of days, weeks, months or even years. Multiple biological samples of the subject are obtained over a period of time, i.e. a biological sample is obtained periodically over time at various intervals. A biological sample can be obtained at any interval. For example, a biological sample can be taken every day for weeks, months or years. Alternatively, a biological sample can be obtained once a week, or six times a week for a period of weeks, months or years. In one embodiment, a biological sample is obtained once a week over a period of three months. In one embodiment, a biological sample is obtained once a month for a period of months, or years.
In some embodiments, the subject is undergoing chemotherapy to treat LPL. An increase in the level of the transcript over the period of time in a subject undergoing chemotherapy to treat LPL would indicate that there is progression of the disease and that the subject is not responding to the therapy. A decrease in the level of the transcript over the period of time in a subject undergoing chemotherapy to treat LPL would indicate that the subject is responding to the therapy.
According to one aspect of the invention, a method for detecting a mutation at position 38182641 in chromosome 3p22.2 in a subject is provided. The method comprises obtaining a biological sample from the subject in need of such detection, and determining from the biological sample whether the subject has a mutation at position 38182641 in chromosome 3p22.2 by allele specific polymerase chain reaction (AS-PCR). The AS-PCR is performed using an allele specific primer wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5. In some embodiments, the mutation is a somatic mutation at position 38182641 in chromosome 3p22.2 which results in a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P).
A subject in need of detection may be a subject suspected of having LPL. The subject may present one or more clinical features of LPL including, but not limited to anemia, hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. In some embodiments, a subject in need of detection may be a subject suspected of having subtype ABC of diffuse large B cell lymphoma. The subject may present one or more clinical features of subtype ABC of diffuse large B cell lymphoma including, but not limited to enlarged lymph node in the neck, groin or abdomen, fever, weight loss, and drenching night sweats. A subject in need of detection may be a subject suspected of having gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The subject may present one or more clinical features of MALT including, but not limited to chronic inflammation caused by Helicobacter pylori infection, stomach pain, dyspepsia, nausea, constipation and anemia.
The present invention is further illustrated by the following Example, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
Example 1
Materials and Methods
Thirty patients meeting consensus criteria for the diagnosis of WM were studied by whole genome sequencing (WGS). Their participation was approved by the Institutional Review Board at Dana-Farber Cancer Institute. The characteristics of these patients are summarized in Table 1. Nine of these patients had a familial history of a B-cell malignancy. Bone marrow (BM) and peripheral blood (PB) mononuclear cells from these individuals were obtained after density-gradient centrifugation. Magnetic bead sorting was used for tumor cell isolation (Miltenyi Biotec, Auburn, Calif.). The purity of isolated B-cells (CD19+) by this technique was over 90%, and the median clonal B-cell population by light chain restriction was 96% (Table 1). The median time from diagnosis of WM to BM collection for the 30 patients was 14.1 (range 0-138.2 months). CD19-depleted PB mononuclear cells were collected as matched normal tissue from 19 of the 30 patients, and paired tumor/normal tissue WGS performed for 10 of these patients. The baseline characteristics for these 10 patients did not differ significantly from unpaired patients.
Following cell isolation, high molecular weight DNA was isolated using the Allprep DNA/RNA mini kit (Qiagen, Valencia, Calif.). For 10 patients, WGS of tumor and matched normal samples was performed, and for 20 additional patients tumor samples alone were completely sequenced. Library construction and WGS of paired-end clones was performed by Complete Genomics Inc. (CGI; Mountain View, Calif.) as previously described. Read sequences were aligned to the reference genome (NCBI Build 37) and reads in potentially variant regions (SNVs, insertions, and deletions) were identified and subjected to local de novo assembly. The assembler then scored both variant and homozygous reference consensus calls in each sample using a Bayesian framework which considered read depth, individual base call quality, and mapping probabilities. A likelihood ratio test for each variant (variant score) expressed in decibels (dB) was generated and reported. High confidence somatic variants were identified using CGAT version 1.3 with a somatic score of 0.1 giving an estimated rate of one false somatic single nucleotide variant per 17.7 Mb of DNA. Copy number was estimated by % GC normalized read depth, and acquired uniparental disomy (aUPD) was identified as copy-neutral loss of heterozygosity. In addition allele imbalance was determined by the percentage of reads mapping to the minor allele at heterozygous SNPs which was averaged over 500 Kb.
To validate WGS results, a set of PCR primers were designed to amplify a 726-bp fragment that covers the MYD88 L265P mutation (forward: 5′-ggg ata tgc tga act aag ttg cca c-3′(SEQ ID NO: 1) and reverse: 5′-gac gtg tct gtg aag ttg gca tct c-3′ (SEQ ID NO: 2)). Twenty nanograms of genomic DNA were used for PCR amplification. After initial denaturation at 95° C. for 2 minutes, 40 cycles of PCR amplification were performed, each consisting of a denaturing step of 94° C. for 30 seconds, annealing at 65° C. for 30 seconds, and extension at 68° C. for 1 minute, followed by a final step at 68° C. for 10 minutes. The amplified fragments were isolated by QIAquick gel extraction kit (Qiagen, CA) and sequenced using the forward primer 5′-gct gtt gtt aac cct ggg gtt gaa g-3′ (SEQ ID NO: 3), and the reverse primer 5′-gac gtg tct gtg aag ttg gca tct c-3′ (SEQ ID NO: 4).
Sanger sequencing was used to validate WGS results in tumor samples from all 30 WM patients, and also in normal paired tissue from 19 patients, including the 10 individuals with paired WGS data. In as well, CD19+ and CD138+ isolated BM cells from an unrelated cohort of 12 other WM patients; CD19+ isolated BM cells from 8 IgM MGUS patients; CD138+ isolated BM cells from 8 multiple myeloma (MM) patients; CD19+ isolated PB mononuclear cells from 12 healthy donors; as well as the BCWM.1, MWCL-1, and WM-WSU WM cell lines; the IgM secreting Ramos cell line, and the MM1.S, RPMI 8226, and U266 MM cell lines were also Sanger sequenced for MYD88. TA cloning and sequencing of at least 100 clones was also performed using the above primers for CD19+ isolated BM cells for 4 patients with IgM MGUS (Genewiz, S. Plainfield, N.J.).
Results
Tumor and normal genomes were both sequenced to an average of 66× (range 60-91×) coverage of mapped individual reads. The average gross mapped yield for these genomes was 186.89 (range 171.56-262.03 Gb). In our initial analysis of the 10 paired genomes, we identified a recurring sequence variant with a variant score of 189 (range 74-345) at position 38182641 in chromosome 3p22.2 which results in a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P). This variant was the most common of a median of 3,419 (range 2,540-4,011) somatic variants identified by WGS in the 10 paired patients using a somatic score of 0.1. By WGS, the MYD88 L265 variant was found as a somatic mutation in tumor cells from all 10 paired patients, as it was not found in their matched normal tissues, and was also seen in tumor cells from 16 of the 20 unpaired WM patients. The presence versus absence of the MYD88 L265 mutation in these patients was not impacted by time from WM diagnosis (14.1 versus 14.8 months, respectively; p=0.40). For 22 of 26 patients, the MYD88 L265P variant was heterozygous, whereas in 4 patients an acquired UPD event (median 49.5 MB, range 48.5-50.0 MB) at 3p22.2 resulted in homozygous presence of the variant in at least a subset of tumor cells (FIGS. 1A, 1B). These UPD events were absent in normal tissue for the 2 of 4 patients who had paired samples (FIGS. 1C, 1D). No distinguishing clinical and laboratory features for patients with homozygous versus heterozygous MYD88 L265 mutation were observed, though patients with homozygous MYD88 L265 had a considerably longer interval from WM diagnosis (56.4 versus 11.1 months; p=0.30), and invariably a larger patient series will be required to determine the significance of this finding.
Sanger sequencing confirmed the presence of the MYD88 L265P variant in all 26 tumor samples revealed by WGS, as well as in one additional patient's tumor sample which had read level evidence of the MYD88 L265P variant but was not called with adequate confidence. In this patient, a subpopulation (approximately 10%) of tumor cells exhibited the MYD88 L265P mutation. By Sanger sequencing, the L265P MYD88 variant was therefore present in tumors of 27 of 30 (90%) WM patients, which included patients with sporadic as well as familial disease in whom the variant was observed at the same frequency. Sanger sequencing also confirmed the absence of the MYD88 L265P mutation in normal tissue from the 10 paired patients who underwent WGS, as well as in 9 other unpaired patients who had the MYD88 L265 mutation by WGS, and for whom normal tissue was available.
In an unrelated cohort of 12 WM patients, we further observed by Sanger sequencing a heterozygous MYD88 L265P variant in 11 (92%) patients. Importantly, the MYD88 L265P variant was present in both CD19+ and CD138+ selected cells in these patients consistent with the known distribution of the malignant WM clone which extends from mature B-cells to plasma cells. By Sanger sequencing, we also detected a heterozygous MYD88 L265P variant in BCWM.1 and MWCL-1 WM cells. In contrast, the MYD88 L265P variant was not found in the IgM secreting cell lines WM-WSU and Ramos, both of which carry t(8; 14), and in none of the myeloma cell lines. Furthermore, we did not detect the MYD88 L265P variant in CD19+ selected B-cells from 12 healthy individuals, nor in tumor samples from 8 of 8 myeloma patients. By direct Sanger sequencing, the MYD88 L265 mutation was absent in 7 of 8 (87.5%) IgM MGUS patients whose characteristics are described in Table 2. In the sole IgM MGUS patient in whom the MYD88 L265P mutation was detected, increased LPC were noted in his BM aspirate, and a core biopsy showed an isolated nodule but no distinct infiltration by LPL cells required for the consensus diagnosis of WM. His subsequent course has been marked by progressively rising IgM levels, and a falling hematocrit. TA cloning and sequencing of at least 100 clones was also undertaken for 4 IgM MGUS patients whose direct Sanger sequencing did not reveal the MYD88 L265 variant. The MYD88 L265 variant was absent in all clones for these 4 IgM MGUS patients. These findings may potentially signify that acquisition of the MYD88 L265 variant represents a transforming event from IgM MGUS to WM, or that the frequency of the MYD88 L265 variant is too low to be picked up in IgM MGUS patients by either WGS or Sanger sequencing. Another consideration is that IgM MGUS might itself be heterogeneous, with acquisition of the MYD88 L265P leading to WM, while in other cases with wild type MYD88, a different oncogenic trajectory may result.
The discovery of a mutation in MYD88 is of significance given its role as an adaptor molecule in Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling. All of the TLRs except for TLR3 use MYD88 to facilitate their signaling. Following TLR or IL-1R stimulation, MYD88 is recruited to the activated receptor complex as a homodimer which then complexes with IRAK4, leading to its autophosphorylation. The MYD88/IRAK4 complex then recruits and activates IRAK1 and IRAK2. Tumor necrosis factor receptor associated factor 6 (TRAF-6) is then activated by IRAK1 leading to early NF-κB activation, whereas IRAK2 facilitates late NF-κB activation. The L265P mutation in MYD88 occurs at a residue that is highly conserved in evolution, and contributes to a (3-sheet at the hydrophobic core of the domain. Staudt and colleagues recently reported the presence of the MYD88 L265P mutation in a subset of tumors taken from patients with the ABC subtype of diffuse large B-cell lymphoma (DLBCL), and in a few patients with gastric mucosa-associated lymphoid tissue (MALT) lymphomas. In contrast, the MYD88 L265P mutation was absent in tumor samples from patients with the GCB subtype of DLBCL, and Burkitt's lymphoma. The frequency of 90% observed for the MYD88 L265 variant in our studies from 2 independent cohorts of WM patients is far more extensive than that observed in DLBCL (29%) and MALT (6%) lymphomas.
Staudt and colleagues also demonstrated that ectopic expression of MYD88 L265P, but not wild type MYD88, prevented apoptosis of L265P expressing ABC DLBCL cell lines that underwent total MYD88 knock down. Co-immunoprecipitation experiments in these cell lines with the L265P mutation also showed enhanced binding to and phosphorylation of IRAK1, which was absent in cells with wild type MYD88. Additionally, knock down studies of MYD88 or IRAK1, or inhibition of IRAK1 function with an IRAK1/4 kinase inhibitor led to loss of NF-κB signaling. Similarly, we have observed that knockdown of MYD88 by lentiviral transduction, and/or treatment with a MYD88 homodimerization inhibitory peptide17 or IRAK1/4 inhibitor18 of BCWM.1 and MWCL-1 WM cells which possess the MYD88 L265P mutation leads to loss of NF-κB signaling and enhanced apoptosis. Since NF-κB signaling represents a critical determinant of WM cell growth and survival, targeting of tonic MYD88/IRAK signaling is of potential relevance to WM therapy.
In summary, using WGS, a novel, widely expressed somatic variant (MYD88 L265P) has been identified in malignant LPC of WM patients, which helps to discriminate WM from other overlapping entities, and whose putative role in NF-κB signaling is of relevance to WM lymphomagenesis. These studies offer novel insights into the pathogenesis of WM, and provide a framework for the development of diagnostic tools and targeted therapies for patients with WM.
Example 2
Materials and Methods
Patient and Samples
Ninety seven patients meeting diagnostic criteria for WM and 40 healthy donors (10 BM and 30 PBMC) were included in this study. Their participation was approved by the IRB at the Dana-Farber Cancer Institute. Bone marrow mononuclear cells (BMMC) were sorted using magnetic beads. Purity of isolated B-cells (CD19+) was over 90%. DNA was extracted using Allprep DNA/RNA mini kit (Qiagen, Valencia, Calif.). DLBCL cell lines OCI-LY3 and OCI-LY19 were kindly provided by Dr. Mark Minden from University Health Network in Canada.
Allele-Specific Polymerase Chain Reaction (AS-PCR)
Two reverse primers were designed to differentiate the mutant and wild-type allele of MYD88 L265P. To optimize the specificity, an internal mismatch in the third position from the 3′-end was introduced. The mutant-specific reverse primer was 5′-CCT TGT ACT TGA TGG GGA aCG-3′ (SEQ ID NO: 5) and the wild-type-specific reverse primer was 5′-GCC TTG TAC TTG ATG GGG AaC A-3′ (SEQ ID NO: 6). The common forward primer was 5′-AAT GTG TGC CAG GGG TAC TTA G-3′ (SEQ ID NO: 7). PCR reaction was performed in a final volume of 25 ul with 50 nM of each primer and 50 ng DNA using PCR SuperMix High Fidelity (Life technology, CA). Thermal cycling conditions were: 2 min at 94° C., followed by 40 cycles of 94° C. for 30 s, 57° C. for 30 s, and 68° C. for 30 s, with a final extension at 68° C. for 5 min. The amplified PCR products (159-bp) were separated on 2% agarose gel. To confirm the sequence, PCR products were purified by QIA quick gel extraction kit (Qiagen, CA) and sequenced using both forward and reverse PCR primers.
Real-Time AS-PCR
Quantitative detection of the MYD88 L265P mutation was developed using the primers described above and Power SYBR® Green PCR Master Mix according to manufacturer's instruction on the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif.). Briefly, PCR reaction was performed in a final volume of 25 μl with 25 nM of each primer and 50 ng DNA. Thermal cycling conditions were: 10 min at 950 C, followed by 40 cycles of 95° C. for 15 s and 60° C. for 60 s. Each sample was assayed in triplicate. The standard curve for MYD88 L265P was generated by a serial dilution of the mutant DNA with the wild-type DNA (50%, 10%, 2%, 0.4%, 0.08%, and wild-type). For the corresponding reference PCR, the forward primer is same as the one used for the AS-PCR (5′-AAT GTG TGC CAG GGG TAC TTA G-3′; (SEQ ID NO: 7)) and the reverse primer is located at 53-bp downstream of the AS-PCR primer (5′-TGG TGT AGT CGC AGA CAG TGA-3′; (SEQ ID NO: 8)). Levels of the mutant MYD88 L265P in patient samples were calculated based on the value of delta CT and the standard curve.
Sanger Sequencing
The forward PCR primer 5′-GGG ATA TGC TGA ACT AAG TTG CCA C-3′ (SEQ ID NO: 1) and reverse PCR primer 5′-GAC GTG TCT GTG AAG TTG GCA TCT C-3′ (SEQ ID NO: 4) were designed to amplify a 726-bp fragment covering the MYD88 L265P site. Amplified PCR products were isolated by QIA quick gel extraction kit (Qiagen, CA) and sequenced using the reverse PCR primer and a sequencing primer 5′-GCT GTT GTT AAC CCT GGG GTT GAA G-3′ (SEQ ID NO: 3).
Statistical Analysis
Correlation between the MYD88 L265P status and clinical parameters was evaluated by non-parametric ANOVA. Correlation between the changes of BM involvement and levels of mutant MYD88 L265P was assessed by linear regression. All analyses were performed with R (R Foundation for Statistical Computing, Vienna, Austria).
Results
Conventional AS-PCR Design
The somatic mutation L265P in the MYD88 gene was found in approximately 90% of WM patients. To efficiently and quickly determine the MYD88 L265P status, an inexpensive AS-PCR assay was developed that can be easily implemented in any laboratories with conventional PCR technology. As shown in FIG. 2A, the AS primers are located in exon 5 while the common forward primer is located in intron 4. To enhance the specificity in the AS-PCR reaction, an additional mismatch (T>A) was introduced at the third position from the 3′ end of the AS primers. Sensitivity of the AS-PCR assay was assessed by a serial dilution of the DNA isolated from DLBCL cell line OCI-LY3 (homozygous MYD88 L265P) with the DNA from DLBCL cell line OCI-LY19 (wild-type MYD88 L265P). The MYD88 L265P status of the cell lines was confirmed by Sanger sequencing (FIG. 2B). The sensitivity assessments demonstrated that the mutant allele of MYD88 L265P can consistently be detected at a dilution of 0.1% in the gel-based AS-PCR assay (FIG. 2C).
This assay was then applied to 97 WM patients who had not received pharmacological interventions at the time of BM biopsies. 87/97 (89.7%) were found positive for the MYD88 L265P mutation. To further verify the AS-PCR assay, the entire cohort was sequenced for the MYD88 L265P position. Among the 87 patients positive for MYD88 L265P by AS-PCR, 82 were positive while 5 were negative by Sanger sequencing. The 5 patients negative for MYD88 L265P by Sanger sequencing showed weak signals in the gel-based AS-PCR assay. By contrast, all patients negative for MYD88 L265P by AS-PCR remained negative by Sanger sequencing. In addition, DNA from 40 healthy donors (10 BM and 30 PBMC) was analyzed and no MYD88 L265P mutation was detected by either of the methods. The overall results suggest that this AS-PCR assay is simple, reliable, and sensitive. In addition, Sanger sequencing analysis suggested that five patients carried homozygous MYD88 L265P because the mutant allele peak was significantly higher than the wild-type allele peak. FIG. 2D shows an example of the homozygous carriers. This patient had 70% of BM involvement, so that the wild-type allele still can be detected by Sanger sequencing. Homozygous MYD88 L265P could be caused by an acquired uniparental disomy (UPD) event at 3p22.2.
Quantitative Detection of MYD88 L265P Mutation
Next, a SYBR green-based real-time PCR was developed to allow quantification of MYD88 L265P. Given the high frequency of the MYD88 L265P mutation in WM, quantitative assessment of MYD88 L265P has potential to be developed as a robust biomarker for monitoring disease progression and response to treatment. Sensitivity and specificity of the real-time AS-PCR were determined by a serial dilution of the mutant DNA with the wild-type DNA. CT values were recorded for mutant-specific and reference PCR and the corresponding delta CT values were calculated. As shown in FIG. 3A, this real-time AS-PCR can detect the MYD88 L265P mutation at a dilution of 0.08% with more than 2 cycle differences from the wild-type DNA background. Correlation coefficient of the standard curve was 0.998 with a slope value of −3.5 (FIG. 3B). The melting curve analysis revealed that the MYD88 L265P mutant-specific amplicon melted at 840 C (FIG. 3C). A minor non-specific amplification was only found in the dilution of 0.4% or lower with a melting peak at 80° C.
To gain more information on the performance of the assays, all samples that have been analyzed by the gel-based AS-PCR and Sanger sequencing were reanalyzed by the real-time AS-PCR. As shown in the scatter plot (FIG. 3D), the healthy donors (n=40) had delta CT values ranging from 10.7 to 16.9 cycles (mean, 13.9 cycles; median, 14.1 cycles), whereas the WM patients had delta CT values ranging from −0.2 to 15.2 cycles (mean, 3.3 cycles; median, 1.9 cycles). There were two major clusters of WM patients separated by 3.2 cycles. The cluster with delta CT values ranging from 9.6 to 15.2 cycles (n=10) looked similar to healthy donors. Thus, this cluster was determined as MYD88 L265P negative. By contrast, another cluster had delta CT values ranging from −0.2 to 6.4 cycles (n=87). This cluster was determined as MYD88 L265P positive. In addition, the gel-based AS-PCR and the real-time AS-PCR showed an exactly same result of determining the status of MYD88 L265P in this cohort. The overall results suggest that a small subset of WM patients may not carry the MYD88 L265P mutation. Furthermore, the entire coding region of the MYD88 gene was sequenced for the 10 patients who were negative for MYD88 L265P as determined by AS-PCR. No MYD88 mutation was found.
Correlation Between MYD88 L265P Mutation Status and Clinical Characteristics
MYD88 L265P was initially reported in DLBCL as a gain-of-function mutation that promotes NF-kB and JAK-STAT3 signaling through activating the IRAK family of serine-threonine kinases. Interestingly, MYD88 L265P was frequently mutated in the ABC subtype of DLBCL (29%) but rare in the GCB subtype. We sought to evaluate the differences of clinical characteristics between the two clusters of WM patients determined by AS-PCR. Non-parametric ANOVA analysis revealed that the MYD88 L265P positive patients tended to have greater BM involvement (p=0.001), lower serum IgG (p=0.011), and higher serum IgM (p=0.007) compared to the MYD88 L265P negative patients (Table 3). Due to small number of MYD88 L265P negative patients, the observations need to be confirmed in an independent study with large sample size.
Determination of Therapeutic Effect by Quantitative Assessments of MYD88 L265P
To explore the potential of using the real-time AS-PCR method to determine therapeutic effect and monitor residual disease in WM, concordance between the readouts of BM involvement and levels of mutant MYD88 L265P for seven patients who received pharmacological intervention and provided BM biopsies before and after treatment was examined. The results are summarized in Table 4. Patient A was a homozygous carrier of MYD88 L265P and had 70% BM involvement and high level of mutant MYD88 L265P before treatment. This patient exhibited a complete clinical remission after the treatment. Accordingly, the BM involvement and the levels of mutant MYD88 L265P became undetectable. Patient B and C showed approximately 90% decrease in the BM involvement with the treatments. Similarly, a marked decrease in the levels of mutant MYD88 L265P was also observed from the two patients (96% and 74%, respectively). Patient D showed 47% decrease in the BM involvement versus 45% decrease in the levels of mutant MYD88 L265P. However, the levels of mutant MYD88 L265P were little changed in patient E and F who showed 17% and 37% decreases in the BM involvement, respectively. The last patient (G) did not appear to respond to the treatment and showed slightly increase in both BM involvement and levels of mutant MYD88 L265P. Linear regression analysis revealed a high correlation between the percentage changes of BM involvement and levels of mutant MYD88 L265P (R2=0.90, p=0.001). Although the study sample size was very small, the results supported the hypothesis of using quantitative assessment of MYD88 L265P to determine therapeutic effect and monitor residual disease in WM.
BM Examination is Essential in the Clinical Staging of Non-Hodgkin's Lymphomas (NHLs)
Merli et al reported a comparison between histology and flow cytometry (FC) on the assessments of BM involvement in non-Hodgkin's lymphomas (NHLs) including LPL (Assessment of bone marrow involvement in non-Hodgkin's lymphomas: comparison between histology and flow cytometry, Eur J Haematol, 85(5):405-15, 2010). A high concordance between the two methods was observed for most NHLs. However, significant discordance was reported in LPL. It was speculated that FC may underestimate the extent of infiltrate with respect to histology. Quantitative assessment of MYD88 L265P in BM biopsy represents a novel tool contributing to clinical staging at diagnosis in WM.
In conclusion, a sensitive and inexpensive real-time AS-PCR method has been developed that permits reliable and quantitative assessments of MYD88 L265P. This is the first quantitative assay for MYD88 L265P and will facilitate testing the potential of MYD88 L265P as a biomarker to improve diagnosis and clinical staging and monitor disease progression and response to treatment in WM.
Example 3
Bruton's tyrosine kinase (BTK) promotes B-cell receptor signaling along with B-cell expansion and survival through NF-κB and MAPK. MYD88 L265P is a widely expressed somatic mutation in tumor cells from WM patients. MYD88 L265P promotes enhanced tumor cell survival through IRAK 1/4 mediated NF-κB and MAPK signaling. We therefore sought to clarify the role of BTK signaling in MYD88 L265P expressing WM cells, and the impact of BTK and MYD88/IRAK inhibition on WM cell signaling and survival.
Materials and Methods
Western blot analysis was performed using total and phospho-specific antibodies in MYD88 L265P expressing WM cells, BCWM.1 and MCWL-1 following MYD88 knockdown by lentiviral transduction, and/or use of MYD88 or IRAK signal inhibitors. Cells were also treated with the BTK inhibitor PCI-32765, in the presence or absence of MYD88 homodimerization or IRAK1/4 inhibitors. Annexin V/PI staining was used to assess cell survival, and synergism assessed with CalcuSyn software.
Results
BTK was highly expressed and phosphorylated in MYD88-L265P expressing WM cells and PCI-32765 significantly blocked the BTK activation (FIG. 4). Increased phosphorylation of BTK was confirmed by western blotting with phospho-specific antibody in Waldenstrom's Macroglobulinemia (WM) cell lines, BCWM.1 and MWCL-1, compared to Multiple myeloma cell lines, ANBL6 and INA6. Antibody against total BTK was used as loading control. PCI-32765 significantly blocked the BTK phosphorylation in WM cells.
PCI32765 significantly reduced downstream NF-kB, MAPK and STAT3 signaling in WM cells (FIG. 5). In addition to significantly blocking the BTK activation, PCI-32765 also blocked the downstream NF-kB, MAPK, Stat3 signaling by significantly reduced the phosphorylation of IKBα, ERK1/2 and Stat3 proteins in WM cell lines, BCWM.1 and MWCL-1, compared to multiple myeloma cell lines, ANBL6 and INA6. Antibodies against corresponding total proteins and GAPDH were used as loading controls.
Knockdown of MYD88 by lentiviral transduction, and/or use of a MYD88 inhibitor leads to decreased BTK phosphorylation (FIG. 6). MYD88 knockdown was confirmed by western blot in BCWM.1 and MWCL-1 cells. The knockdown of MYD88 reduced BTK phosphorylation compared with controls. MYD88 homodimerization inhibitory peptides significantly reduced BTK phosphorylation compared with control peptides. Antibodies against total BTK and/or GAPDH were used as loading control.
Treatment with PCI-32765 induces apoptosis of MYD88 L265P expressing WM cells (FIG. 7A). PCI-32765 shows robust tumor cell killing in combination with a MYD88 pathway inhibitor in primary WM patients bone marrow tumor cells (FIG. 7B). PCI-32765 shows synergistic tumor cell killing in combination with an IRAK 1/4 kinase inhibitor (FIG. 7C).
BTK activation is facilitated by MYD88 pathway signaling in MYD88 L265P expressing WM cells, and participates in MYD88 downstream signaling. Inhibition of BTK by PCI-32765 leads to robust tumor killing of MYD88 L265P expressing WM cells, which is potentiated by MYD88 pathway inhibitors.
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TABLE 1







Clinical and laboratory characteristics for 30 WM patients whose


lymphoplasmacytic cells were used in whole genome sequencing studies.










Median
Range





Age
62
41-87


Familial WM
9 (30%)
NA


Untreated
26 (87%)
NA


Bone Marrow Involvement (%)
65%
5-95%


Clonal B-cells(%)1
96.3
55.5-100


IgM (mg/dL)
3,970
590-6,580


IgMκ/IgMλ monoclonal protein
24/6
NA


IgA (mg/dL)
41
11-516


IgG (mg/dL)
519
215-3,120


Serum B2M (mg/L)
3.55
1.50-12.1


Hematocrit (%)
31.4
24.0-41.5





1Determined by light chain restriction using flow cytometry.













TABLE 2







Clinical and laboratory characteristics for the 8 IgM MGUS patients who


underwent Sanger sequencing for the MYD88 L265P variant.










Median
Range





Age
62
52-71


Familial WM
2 (25%)
NA


Untreated
8 (100%)
NA


Bone Marrow Involvement (%)
0%
0-2%


Clonal B-cells(%)1
0%
0-1


IgM (mg/dL)
682
142-1,190


IgMκ/IgMλ monoclonal protein
5/3
NA


IgA (mg/dL)
142
53-244


IgG (mg/dL)
772
523-1,040


Serum B2M (mg/L)
1.70
1.30-2.10


Hematocrit (%)
41.4
36.7-44.8





1Determined by light chain restriction using flow cytometry.













TABLE 3







Correlation between MYD88 L265P mutation status 


and clinical characteristics










MYD88 L265P mutation status













Clinical parameter

Positive

Negative
p-value*















Age at diagnosis
n = 87
60.26
n = 10
66.50 
0.064




(9.97)

(8.33)



Gender, % female
n = 86
40.91%
n = 10
50.00%
0.738**


Bone marrow 
n = 85
52.92 
n = 10
18.00 
0.001


involvement, %

(30.49)

(18.74)



IgA, mg/dL
n = 85
622.79 
n = 10
970.10 
0.106




(552.45)

(711.69)



IgG, mg/dL
n = 85
216.35 
n = 10
332.10 
0.011




(322.36)

(284.60)



IgM, mg/dL
n = 85
3093.62
n = 10
1536.20 
0.007




(1943.89)

(2039.79)





Values are mean (SD).


*Non-parametric ANOVA


**Fisher's exact test













TABLE 4







Concordance between the assessments of BM 


involvement and MYD88 L265P mutation levels.



















Levels

%







of

change







mutant
%
of






BM
MYD88
change
MYD88



Age at

Treatment
Involvement
L265P
of BM
L265P


Patient
diagnosis
Gender
status
(%)
(%)*
involvement
levels

















A**
61
Male
Untreated
70
156.90
100
100





Bendamustine 
Negative
Negative







rituxan






B
44
Male
Untreated
90
60.73
89
96.33





R-CD
10
2.23




C
52
Male
Untreated
50
78.12
90
73.61





R-CD
5
19.08




D
59
Male
Untreated
95
99.15
47
45.39





Everolimus
50
54.15




E
63
Male
Untreated
90
96.07
17
1.14





Everolimus
75
94.97




F
70
Male
Untreated
95
95.93
37
8.61





Everolimus
60
87.67




G
63
Male
Untreated
20
67.93
−25
−12.87





Everolimus
25
76.67







R-CD: combination of rituximab, cyclophosphamide, and dexamethasone.


BM biopsy was collected before and after treatment. % of mutation was calculated based on standard curve.


**Homozygous carrier of the MYD88 L265P mutation.

## Claims

1. A method to treat lymphoplasmacytic lymphoma in a subject, the method comprising:
selecting the subject with lymphoplasmacytic lymphoma on the basis that the subject has a mutation at position 38182641 in chromosome 3p22.2 in a biological sample from the subject,
administering to the subject with lymphoplasmacytic lymphoma a myeloid differentiation primary response 88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4 (IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitor in an amount effective to treat lymphoplasmacytic lymphoma.

2. The method of claim 1, wherein the biological sample is a sample of bone marrow, lymph node, spleen or blood.

3. The method of claim 1, wherein the mutation results in a single nucleotide change from T to C in the myeloid differentiation primary response 88 (MYD88) gene.

4. The method of claim 1, wherein the mutation results in an amino acid change from leucine to proline at position 265 in the myeloid differentiation primary response 88 protein.

5. The method of claim 1, wherein the MYD88 inhibitor is a peptidomimetic compound ST2825.

6. The method of claim 1, wherein the IRAK-1/4 inhibitor is N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole.

7. The method of claim 1, wherein the BTK inhibitor is Ibrutinib (PCI-32765).

8. A method for monitoring progression or recurrence of lymphoplasmacytic lymphoma (LPL) in a subject and treating the subject, comprising
obtaining multiple biological samples of the subject with lymphoplasmacytic lymphoma over a period of time,
determining from the multiple biological samples the level of a transcript comprising a mutation at position 38182641 in chromosome 3p22.2,
and administration of an effective amount of a myeloid differentiation primary response 88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4 (IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitor to the subject when a change in the level of the transcript over the period of time is indicative of the progression or recurrence of LPL in the subject.

9. The method of claim 8, wherein the subject is undergoing chemotherapy.

10. The method of claim 8, wherein the level of the transcript is measured using quantitative real time polymerase chain reaction.

11. The method of claim 10, wherein the quantitative real time polymerase chain reaction is performed using an allele specific primer, wherein the allele specific primer hybridizes at or near its 3′ end to the mutation at position 38182641 in chromosome 3p22.2.

12. The method of claim 11, wherein the allele specific primer is SEQ ID NO: 5.

13. The method of claim 8, wherein the biological sample is a sample of bone marrow, lymph node, spleen or blood.

14. The method of claim 8, wherein the mutation results in a single nucleotide change from T to C in the myeloid differentiation primary response 88 (MYD88) gene.

15. The method of claim 8, wherein the mutation results in an amino acid change from leucine to proline at position 265 in the myeloid differentiation primary response 88 protein.