Patent Application
20200325229
This application claims the benefit of priority of SG provisional application No. 10201708262R, filed 6 Oct. 2017, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention relates generally to the field of molecular biology. In particular, the present invention relates to the use of biomarkers for the detection and treatment of cancer.
Natural-killer/T cell lymphoma (NKTL) is an uncommon and aggressive malignancy with a predilection for Asian, Mexican and South American populations. With the exception of Japan, it is the most common mature T-cell lymphoma in Asia. Neoplastic cells are invariably infected by the Epstein Barr virus (EBV) and are characterized by a cytotoxic phenotype.
Immune checkpoint inhibitors have changed the landscape for treatment of many cancers, including some hematologic malignancies. Investigations on several solid tumours, including non-small-cell lung carcinoma, melanoma and bladder cancer, have generally concluded that immunohistochemistry (IHC) PD-L1 positivity coincides with greater likelihood of response to PD-1/PD-L1 blockade. However, there was also a lower but definite response rate in patients with PD-L1-negative tumours. These observations highlight the many pitfalls of adopting PD-L1 immunohistochemistry, based on a single tumour specimen per patient, as an absolute selection criterion for PD-1 blockade therapy.
Extranodal natural killer/T-cell nasal-type lymphoma (ENKL) is an uncommon and aggressive malignancy with a predilection for Asian, Mexican and South American populations. To date, there is no targeted therapy available for the treatment of ENKL. As anthracycline-based regimens with ENKL are associated with dismal results, L-asparaginase-based regimens, like the SMILE (dexamethasone, methotrexate, ifosfamide. L-asparaginase, etoposide) regimen, have significantly improved clinical outcomes, especially for patients with disseminated disease. However, SMILE or SMILE-like regimens still fail in up to 40 to 50% of the cases and the toxicities associated with SMILE also preclude its use in older patients.
Furthermore, there is still no FDA approved targeted regime to manage natural-killer/T-cell lymphoma (NKTL) as the disease is uncommon, and made it challenging to identify biomarker of response to therapy. Thus, there is an unmet need for methods of identifying natural-killer/T-cell lymphoma and for methods of treating the same.
In one aspect, the present invention refers to a method of treating natural killer/T-cell lymphoma in a subject, the method comprising administering to a subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterised by the presence of at least one JAK3-activating mutation or at least one PD-L1 structural rearrangement.
In another aspect, the present invention refers to a method of determining response of a subject suffering from natural killer/T-cell lymphoma to pembrolizumab treatment, the method comprising obtaining a sample from the subject; detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement; wherein the presence of at least one JAK activating mutation or at least one PD-L1 structural rearrangement indicates that the subject will respond to pembrolizumab treatment.
In yet another aspect, the present invention refers to a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement, the kit comprising a detection agent, and at least one pair of primers; wherein the primers enrich for the genomic regions of the JAK3 and PD-L1 genes.
In a further aspect, the present invention refers to a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement for next-generation sequencing.
In another aspect, the present invention refers to a kit as disclosed herein for use according to the method as disclosed herein.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
In recent years, immune checkpoint (ICP) inhibitors have shown promising objective response rates (ORR) in the treatment of many malignancies. Of note, one result shows 80% objective response rates from the use of programmed death-1 (PD-1 or CD279) inhibitors in relapsed or refractory (RR) Hodgkin lymphoma (HL). Currently, clinical studies involving non-small-cell lung carcinoma, melanoma and bladder cancer have generally concluded that immunohistochemistry (IHC) positivity of programmed death-ligand 1 (PD-L1) coincides with greater likelihood of response to PD-1/PD-L1 blockade. Intriguingly, there was also a lower but definite response rate in patients with PD-L1-negative tumours. These observations suggest that more information could be harnessed from the tumours and augment the current de facto criteria of selecting patients for PD-1 blockade therapy.
The inventors have identified recurrent genetic alterations in relapsed or refractory natural killer/T-cell lymphoma (RR NKTL) patients who have achieved complete response (CR) with programmed cell death 1 (PD-1) blockade therapy.
With the advancements in sequencing technologies, recurring somatic mutations altering the JAK-STAT pathway, epigenetic modifiers, DDX3X gene and germline genetic predisposition in the HLA-DPB1 gene have been found in natural killer/T-cell lymphoma (NKTL) patients, but none of these studies have used whole genome sequencing (WGS) techniques. In order to explore the natural killer/T-cell lymphoma genomes in a high sequencing throughput and genome-wide fashion for targetable genomic alterations, whole genome sequencing data was employed to study the somatic alterations of 11 pre-treated natural killer/T-cell lymphoma tumours that have subsequent clinical response data to pembrolizumab treatment.
Through whole-genome sequencing of paired natural killer/T-cell lymphoma (NKTL) tumour-normal samples, it was shown that a somatic breakpoint-cluster is present within the programmed cell death ligand 1 (PD-L1/CD274) gene that is highly recurrent in 36% (4 of 11) of the tumours. These structural rearrangements (SRs) are validated to disrupt the 3′ untranslated region (UTR) of the PD-L1 gene, which result in the aberrant expression of PD-L1 chimeric transcripts.
In one example, among 11 individuals with relapsed or refractory natural killer/T-cell lymphoma (NKTL) were treated with pembrolizumab, PD-L1 3′UTR structural rearrangements were found in all four responders but absent in the four non-responders. Without being bound by theory, it was thought that PD-L1 3′UTR structural rearrangement was associated with response to PD-1 blockade and reduced M2-macrophage signature, thereby allowing the use of PD-1 blockade therapy for PD-L-rearranged natural killer/T-cell lymphomas and, in turn, improving treatment outcome for these patients.
Disclosed herein is a method of treating natural killer/T-cell lymphoma in a subject, the method comprising administering to a subject a therapeutically effective amount of pembrolizumab, wherein the molecular genomic profile of the subject is characterised by the presence of at least one PD-L1 structural rearrangement. In one example, there is disclosed a method of treating natural killer/T-cell lymphoma in a subject, the method comprising administering to a subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterised by the presence of at least one JAK3-activating mutation or at least one PD-L1 structural rearrangement.
Thus, in one example, the structural rearrangement disrupts the 3′ untranslated region (3′ UTR) of the PD-L1 gene. In another example, the PD-L1 structural rearrangement is a mutation in the PD-L1 gene. In yet another example, the mutation in the PD-L1 gene disrupts the 3′ UTR of the PD-L1 gene.
In one example, the JAK3-activating mutation is, but is not limited to, any one or more of the following mutations: M511I, A572V, A573V, R657Q, V722I, V674A, L857P, R403H, Q501H, E958K. In another example, the JAK3-activating mutation is a single-nucleotide substitution (p.A572V or p.A573V) in the JAK3 gene (JAK3 RefSeq Gene ID: NM_000215). In other words, in one example, the JAK3 activating mutation is A572V. The terms “JAK3-activating” mutation” and “JAK3 mutation” are considered to be interchangeable.
As used herein, the term “mutation” refers to permanent alteration of the nucleotide sequence of the genome of an organism or a genetic element. The mutation can be, but is not limited to, insertions, deletions, substitutions, translocations, inversions, micro-inversions, duplications, tandem repeats, breakpoint(s) (mutations), and combinations thereof.
As used herein, the term “structural rearrangement” refers to one or more mutations that result in a change in the overall structure of the nucleic acid sequence of interest. A “structural rearrangement” spans across a genomic region and the boundaries of this mutation are known as breakpoints. For example, in the event that a breakpoint resides in a gene, the mutations as disclosed herein result in a change in the structure of said gene. Such structural rearrangements can also refer to changes in the chromosomal structure that encompasses the gene or nucleic acid sequence of interest. Thus, in one example, the mutation is a micro-inversion, inversion, translocation, tandem repeat, or a breakpoint (mutation), or combinations thereof.
As used herein, the term “inversion” refers to an inversion of a nucleic acid sequence within a specific sequence, whereby the sequence is excised and inserted in the reverse orientation compared to the orientation it was in before. In other words, the nucleic acid sequence of interest is reversed end to end as the result of a mutation. The term “micro-inversion” refers to nucleic acid sequences from 50 to 1000 bp (base pairs) in length. In one example, the mutation is a micro-inversion of between 150 to 250 bp in length. In one example, the mutation is a micro-inversion of between 200 to 210 bp in length. In another example, the mutation is a micro-inversion of about 206 bp in length.
As used herein, the term “relapse” or “recidivism” refers to a recurrence of a past condition, such as, for example, a medical condition. There are medical conditions known for having extended relapse periods (for example, malaria). In the present context, the term “relapse” refers to the scenario where a medical condition previously existed (for example, the presence of a particular disease), which had been treated or was no longer present in a subject, which has now reoccurred or re-surfaced in the subject.
As used herein, the term “refractory” refers to a disease or condition which does not respond to any attempted forms of treatment. For example, a cancer is said to be refractory when it does not respond to (or is resistant to) cancer treatment. Refractory cancer is also known as resistant cancer.
Thus, in one example, the natural killer/T-cell lymphoma described herein is a relapsed and/or refractory natural killer/T-cell lymphoma. In one example, the natural killer/T-cell lymphoma is a relapsed natural killer/T-cell lymphoma. In another example, the natural killer/T-cell lymphoma is a refractory natural killer/T-cell lymphoma.
Eleven natural killer/T-cell lymphoma patients from Singapore, China and Hong Kong who were relapsed or refractory (RR) to L-asparaginase containing chemotherapy regimens, after a median of two (range between 1 to 5 lines of treatment) lines of treatments, were included into this study (Table 1). These eleven pembrolizumab-treated patients had a median age of 42 years old at diagnosis (range between 27 to 66 years of age) and a median follow-up time of eleven months (range between 2 to 25 months) since treated with pembrolizumab. Sixty-four percent (64%; 7 of 11 cases) of the patients achieved complete responses (CR) while 36% (4 of 11 cases) of the patients had progressive disease (PD). Two patients (NKTL26 & NKTL31) remained in remission from pembrolizumab for more than two years, which is considered to be a rare occurrence in relapsed or refractory natural killer/T-cell lymphoma (RR NKTL). The most recent pembrolizumab-treated case (NKTL28) achieved ongoing remission for at least 6 months. The median duration of response to pembrolizumab (for responding patients) was 14 months.
1DOR: Durability of response as of January 2018; + indicates ongoing survival
Thus, in one example, the subject had previously not responded to SMILE (dexamethasone, methotrexate, ifosfaminde, L-asparaginase and etoposide) therapy. In another example, the subject had previously responded to SMILE. That is to say that the subject had previously responses to SMILE therapy, however, that the disease has re-occurred or relapsed. In another example, the subject had been previously treated with any one or more of the compounds dexamethasone, methotrexate, ifosfaminde, L-asparaginase or etoposide, or combinations thereof.
PD-L1 Positivity could not Stratify Response to Pembrolizumab in Natural Killer/T-Cell Lymphoma (NKTL) Patients
To verify if PD-L1 positivity in natural killer/T-cell lymphoma (NKTL) tumours could predict response to pembrolizumab, the positivity of PD-L1 in all 11 pre-treated NKTL tumours was determined using immunohistochemistry (IHC). The same pathologist assessed PD-L1 positivity in all the tumours in this study to ensure consistency Table 2). The PD-L1 positivity in the tumour cells varied greatly in both patients who achieved complete responses and progressive disease. PD-L1 positivity in the pre-treated tumours of the patients with complete responses ranged from 6% to 100% while the PD-L1 staining intensity among patients with progressive disease ranged from 35% to 90%. Hence PD-L1 staining intensity could not differentiate between patients who achieved complete response and those who had progressive disease. Interestingly, NKTL29 had only 6% of tumour cells stained positive for PD-L1 but achieved complete response from pembrolizumab. Apart from this PD-Lllow complete response case, all four progressive disease cases were strongly stained for PD-L1, with an average of 69% (range: 50% to 90%) tumour cells stained positively for PD-L1. This is concordant with clinical trials reporting that anti-tumor activity from PD-1 blockade therapy was also observed in melanoma and non-small cell lung carcinoma patients with low baseline PD-L1 positivity. In contrast, the method disclosed herein shows that some patients with low PD-L1 positivity may have good responses to PD-1 blockade. In summary and without being bound by theory, this goes against what is known in the art, as based on the immunohistochemistry staining as shown in the art, subjects that achieved complete response to PD-1 blockade should significantly associate with higher PD-L1 positivity in their tumours than of those who did not.
To identify genomic biomarkers of response to PD-1 blockade therapy in natural killer/T-cell lymphoma (NKTL), whole genome sequencing was performed on tumour-normal paired samples obtained from eleven patients who were subsequently treated with pembrolizumab. The natural killer/T-cell lymphoma (NKTL) tumours and, whole blood or buccal swabs, were sequenced to an average depth of 66.6× and 37.5×, respectively (Table 3). Somatic variant calling yielded an average of 1.15 single nucleotide variants (SNVs) and microlndels per Mb for each paired sample. An average of 39 (range: 1 to 80) somatic non-silent protein-coding variants per sample was identified and is comparable to previous reports on whole-exome sequencing of fresh-frozen natural killer/T-cell lymphoma (NKTL) samples (range: 41 to 42). In total, 10 genes were found to be recurrently mutated (
Therefore, in one example, there is disclosed a method of treating natural killer/T-cell lymphoma in a subject, the method comprising administering to a subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterised by the presence of at least one JAK3-activating somatic mutation. In another example, the at least one JAK3 activating mutation is an activating somatic mutation. In a further example, there is one JAK3 activating mutation present. In yet another example, the JAK3-activating mutation is p.A573V.
Thus, in one example, the mutation referred to herein is a micro-inversion, inversion, translocation, tandem repeat, or a breakpoint (mutation). In another example, the mutation is a translocation, a tandem repeat (or tandem duplication), or a micro-inversion.
In NKTL28 and NKTL31, exon 7 of PD-L1 was translocated to 2q24.2 and intron 6 of PD-L1 was translocated to 6p12.2, respectively (
Besides sequence analysis by the inventors' genomic pipeline, visual inspection was also performed for known recurrent mutated genes of natural killer/T-cell lymphoma (NKTL) to avoid artefacts. Mutations in genes associated with antigen presentation and interferon gamma pathways, which are known to associate resistance to immune checkpoint blockade in melanoma, are not found in the analysed cohort.
All four PD-L1 structural rearrangements were predicted to lose whole or part of the PD-L1 3′UTR, or the PD-L1 3′UTR function, except the micro-inversion that spanned across 206 bp and sits entirely within the 3′UTR of PD-L1. To determine the functional significance of this micro-inversion in regulating PD-L1 expression, the wild type and mutant (with 206 bp inversion) PD-L1 3′UTR were cloned into a luciferase reporter assay system and transfected into lymphoma and leukemia cell lines, namely, NK-S1, K-562 and Jurkat (
Although the mechanisms of response to PD-1 blockade from PD-L1 3′UTR structural rearrangements and JAK3-activating mutations remain to be elucidated, it was investigated if the clonality of these alterations could support the complete response in patients who had PD-L1 and JAK3 alterations in their pre-treated tumours, from the single-agent regime of pembrolizumab. From the somatic single-nucleotide variants, it was possible to obtain solutions for the clonal architectures for 10 cases (SciClone did not have a clonality solution for NKTL1). Five cases, four complete response cases and one progressive disease cases, had a clonal architecture (Table 4 and
Immunotherapy, in particular PD-1 blockade therapy, has shown promise in the treatment of several cancers, including natural killer/T-cell lymphoma. It is shown that four out of seven NKTL patients (57%) who achieved complete response to PD-1 blockade had a clonal architecture for the PD-L1 3′UTR structural rearrangement in their tumours. PD-L1 3′UTR structural rearrangements was also recently identified in a single case of ovarian cancer where the patient achieved complete response with pembrolizumab, further supporting its role as a potential biomarker of response to PD-1 blockade therapy in natural killer/T-cell lymphoma.
Also disclosed herein is a method of determining response of a subject suffering from natural killer/T-cell lymphoma to pembrolizumab treatment, the method comprising obtaining a sample from the subject; detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement. In another example, the presence of at least one JAK activating mutation or at least one PD-L1 structural rearrangement indicates that the subject will respond to treatment. In another example, the treatment is a compound or treatment as disclosed herein. In yet another example, the treatment is pembrolizumab.
As used herein, the term “response” can also be used interchangeably with susceptibility to a treatment. The term “susceptibility” refers to the propensity of something, for example a disease, to be likely affected by something else, for example, a treatment for said disease. This effect can be either positive or negative, depending on the feature or the treatment which is being referenced. For example, if a subject is sensitive to a particular treatment, then the susceptibility of said subject to a particular treatment is a positive effect. The term “susceptibility” can be interchanged with, for example, reactivity or sensitivity.
Thus, in one example, the method disclosed herein is a method of determining susceptibility of a subject suffering from natural killer/T-cell lymphoma to pembrolizumab treatment.
All natural killer/T-cell lymphomas are diagnostically EBER+(indicating the presence of the Epstein-Barr virus) and the Epstein-Barr virus (EBV) protein, LMPI can be considered to constitutively up-regulate PD-L1. Without being bound by theory, it speculated that natural killer/T-cell lymphomas will respond to PD-1 inhibitors, as they are innately PD-L1+. Indeed, relapsed/refractory natural killer/T-cell lymphoma patients in a previous clinical study had an initial response to pembrolizumab.
Thus, in one example, there is disclosed a method of treating natural killer/T-cell lymphoma in a subject. In another example, the method comprises administering to a subject an inhibitor selected from the group consisting of PD-1 inhibitor, CD279 inhibitor, PD-L1 inhibitor, CD274 inhibitor and combinations thereof. In yet another example, the subject is to be administered an inhibitor selected from the group consisting of PD-1 inhibitor, CD279 inhibitor, PD-L1 inhibitor, CD274 inhibitor and combinations thereof.
Also disclosed herein is the use of a compound or inhibitor as disclosed herein in the manufacture of a medicament for treating natural killer/T-cell lymphoma.
As used herein, the term “inhibitor” refers to compounds that are capable of inhibiting or blocking the activity of a specific receptor, or a group of related receptors. Various compounds and drugs are not limited to a single effect and can therefore be considered to be inhibitors of the same receptor, even if they are structurally and/or chemically different. That is to say, the inhibition of a specific receptor is the characteristic of these compounds in examples where more than one inhibitor is used.
Thus, in one example, the inhibitor as disclosed herein is an inhibitor that results in a blockade of the PD-1/PD-L1 axis. In another example, the inhibitor is, but is not limited to, PD-1 inhibitor, CD279 inhibitor, PD-L1 inhibitor, CD274 inhibitor, and combinations thereof. In yet another example, the method as disclosed herein comprises administering to a subject an inhibitor that is, but is not limited to, PD-1 inhibitor, CD279 inhibitor, PD-L1 inhibitor, CD274 inhibitor and combinations thereof.
As used therein, the term “treatment” refers to both prophylactic inhibition of initial infection or disease, and therapeutic interventions to alter the natural course of an untreated infection or disease process, such as a tumour growth or an infection with a bacteria. Treating a disease also refers to a therapeutic intervention that inhibits, or suppresses, for example, the growth of a tumour, eliminates a tumour, ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.
In one example, the treatment or the compound to be administered to the subject is a compound which impedes the PD-1/PD-L1 axis. In other words, these compounds target immune checkpoints that have an effect on subject response to treatment. In one example, these target immune checkpoints are co-inhibitory immune checkpoint molecules. In another example, these co-inhibitory immune checkpoint molecules are, but are not limited to CTLA-4, CD80/CD86, PD1, PD-L1/PD-L2, CD80, PD-L1, BTLA, HVEM, TIM3, and GAL9. In a further example, the treatment or the compound to be administered to the subject is a PD1/PD-L1 blockade therapy. In yet another example, the PD1/PD-L1 blockade therapy is a PD-1 blockade therapy.
Thus, in one example, the treatment or the compound to be administered to the subject is a compound which impedes the PD-1/PD-L1 axis. In another example, the treatment or the compound to be administered to the subject is a compound which targets PD-1. These compounds can be, but are not limited to, nivolumab (opdivo), pembrolizumab (keytruda), atezolizumab (tecentriq), avelumab (bavencio), durvalumab (imfinzi), pidilizumab (Cure Tech), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline), PDR001 (Norvartis), cemiplimab (Regeneron and Sanofi), and combinations thereof. In one example, the compound to be administered is pembrolizumab (keytruda) in combination with any other compounds as disclosed herein. In another example, the compound is pembrolizumab (keytruda).
Subsequently, four of the eleven patients have progressed and died of disease. Alterations of the PD-L1 and JAK3 genes in these progressive cases had not been found. Without being bound by theory, it is thought that this initial “pseudo-remission” could be attributed by exogenous factors, such as Epstein-Barr virus (EBV) up-regulating PD-L1 that was transiently blocked by initial dosages of pembrolizumab. Hence, high PD-L1 positivity in tumours will not necessarily equate to good response to PD-1 blockade. In addition, the PD-L1 immunohistochemistry scores also varied greatly (6%, 2+ to 100%, 3+) within the cohort, and both subjects NKTL25 and NKTL27 had progressive disease despite having high PD-L1 staining grade for their pre-treated tumours, resulting in questions being raised to the effectiveness of PD-L1 positivity alone as a biomarker of response to PD-1 blockade in natural killer/T-cell lymphoma. No rearrangements were identified within the PD-L2 gene, and PD-L1 always served as the 5′ rearrangement partner with regard to structural rearrangements. This is in contrast to other hematologic malignancies where the over-expression of PD-L1 and/or PD-L2 is achieved by diverse mechanisms such as genomic amplification, JAK2 or PD-L2 translocations (Table 5), suggesting that different tumours have evolved alternate mechanisms for immune evasion.
To determine the prevalence of PD-L1 and JAK3 alterations, whole-genome sequencing (WGS) was performed on 32 more paired tumour-normal natural killer/T-cell lymphoma (NKTL) tumours and corresponding peripheral blood lymphocytes, the clinicopathological information of which is listed in Table 6 below. The absence of malignant cells in the corresponding peripheral blood in these samples was verified by mapping the sequencing data to the EBV genome as the pathogenic virus is known to reside in the neoplastic cells. Similar to the cohort of 11 pembrolizumab-treated patients, in this extended cohort of 32 NKTL samples that had no subsequent pembrolizumab treatment; PD-L1 was also found to be the most recurrently altered gene in the cohort (
The presence of aberrant transcripts in tumours harbouring PD-L1 3′UTR structural rearrangement (SR) was determined. For each of the PD-L1 SR, with available whole transcriptomic sequencing (WTS) data, it was possible to identify and validate the PD-L1 chimeric transcripts by Sanger sequencing (
Also disclosed herein is a kit for performing the method described herein. Thus, in one example, there is disclosed a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement, the kit comprising a detection agent, and at least one pair of primers. In yet another example, there is disclosed a kit or detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement comprising a detection agent, and at least one pair of primers; wherein the primers enrich for the genomic regions of the JAK3 and PD-L1 genes.
In one example, the at least one pair of primers is, but is not limited to, the primer pairs as listed in Tables 8 and 9 of the present specification. In another example, the primer pairs are, but are not limited to, SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO:11 and 12, SEQ ID NO:13 and 14, SEQ ID NO:15 and 16, SEQ ID NO:17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO:27 and 28, SEQ ID NO:29 and 30, SEQ ID NO:31 and 32, SEQ ID NO:33 and 34, SEQ ID NO: 35 and 36, SEQ ID NO: 37 and 38, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44, SEQ ID NO: 45 and 46, and SEQ ID NO: 47 and 48. In yet another example, there is disclosed a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement for next-generation sequencing. In yet another example, the kit as disclosed herein is for use according to the method as disclosed herein.
In summary, in the full cohort of 43 natural killer/T-cell lymphoma (NKTL) samples (11 samples were subsequently treated with pembrolizumab and 32 samples were not), it is shown that frequent (27.9%, 12 of 43) somatic PD-L1 3′UTR structural rearrangement in extranodal natural killer/T-cell lymphomas can explain how some extranodal natural killer/T-cell lymphomas can evade immune surveillance, thereby providing the foundation to use PD-1 inhibitors to better treat these patients.
The presence of recurrent JAK3-activating mutations in the described complete response cases also coincide with a report showing the long-term benefit of PD-1 blockade in a single lung cancer patient with JAK3-activating mutations.
It is shown that genomic features correlate with response to PD-1 blockade therapy in natural killer/T-cell lymphoma using whole genome sequencing data and showed that patients can be better selected for PD-1 blockade therapy via genomic screening.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.
As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means+/−5% of the stated value, more typically +/−4% of the stated value, more typically +/−3% of the stated value, more typically, +/−2% of the stated value, even more typically +/−1% of the stated value, and even more typically +/−0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The following examples illustrate methods by which aspects of the invention may be practiced or materials that may be prepared which is suitable for the practice of certain embodiments of the invention.
Eleven relapsed or refractory (RR) natural-killer/T-cell lymphoma patients were treated with pembrolizumab. Responses were assessed by radiological scans with the RECIST criteria. Whole genome sequencing (WGS) was used to molecularly profile all the pre-pembrolizumab tumours and matching normals of the eleven patients.
For relapsed or refractory (RR) natural-killer/T-cell lymphoma, the study cohort consists of 11 patients with relapsed or refractory (RR) natural-killer/T-cell lymphoma who had failed L-asparaginase-based chemotherapy regimens from Singapore, China and Hong Kong. NKTL1, NKTL25, NKTL26, NKTL43, NKTL44 and NKTL45 which were not previously sequenced were included from the previous study. Patients were diagnosed with natural-killer/T-cell lymphoma according to the 2008 World Health Organization classification with cytotoxic, CD3ε+ and EBER+ phenotypes. Initial tumours and blood/buccal swabs samples of 43 extranodal natural killer/T-cell lymphoma patients were collected, of which, 11 of them who have failed L-asparaginase-based chemotherapy regimens were subsequently treated with pembrolizumab. Response assessment was performed using a combination of PET/CT or CT/MRI or EBV PCR. Whole genome sequencing was used to molecularly profile all the pre-pembrolizumab tumours and matching normal pairs. The duration of response (DoR) was calculated from the date of starting pembrolizumab to the date of progression or death. The median DoR was estimated using the Kaplan-Meier method. Institutional Review Boards from SingHealth (2004/407/F), National University of Singapore (NUS-IRB-10-250) and Sun Yat-sen University Cancer Center (YB2015-015-01) approved the study. All subjects in this study provided written informed consent. The study also adhered to the Declaration of Helsinki.
For extranodal natural killer/T-cell lymphoma, all subjects in the study provided written informed consent. Extranodal natural killer/T-cell lymphoma was diagnosed according to the 2008 World Health Organization classification with cytotoxic, CD3ε+ and EBER+ phenotypes 3. Institutional Review Boards from SingHealth (2004/407/F), National University of Singapore (NUS-IRB-10-250) and Sun Yat-sen University Cancer Center (YB2015-015-01) approved the study. Initial tumours and blood samples of 40 extranodal natural killer/T-cell lymphoma patients were collected, of which, six of them were also treated with pembrolizumab after they have progressed onto the relapsed or refractory (RR) status. Four of these pembrolizumab-treated patients were from Singapore and the remaining two patients were from China. A combination of physical signs (for example, peripheral blood EBV loads and, PET or CT scans) was used to determine clinical response for pembrolizumab-treated patients. Among these six patients, fresh-frozen tumours were available for one patient and formalin-fixed paraffin-embedded (FFPE) tissues were available for five patients. WGS data was generated for all 40 tumours-blood samples. Sequencing and alignment statistics can be found in Table 7.
Genomic DNA from snap frozen and formalin-fixed paraffin-embedded (FFPE) tumour tissues, and whole blood was extracted as previously described. Buccal swab genomic DNA was purified using E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer's instructions. The quality and quantity were assessed as described elsewhere.
Resting and Activated NK-cells were used as baseline to compare the relative expressions of PD-L1 in the tumours samples. NK-cell isolation was performed using human apheresis cone blood obtained from the Health Sciences Authority of Singapore. Peripheral blood mononuclear cells were acquired by density centrifugation at 400×g for 30 minutes using Ficoll-Paque Plus (GE Healthcare). NK-cells were isolated using EasySep Human NK Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's protocol. The purity of NK-cells was greater than 90% as determined by CD3- and CD56+ expression by flow cytometry.
The isolated cells were suspended in X-VIVO 15 medium (Lonza) supplemented with 5% heat-inactivated human serum (Innova Biosciences) with or without 200 U/ml IL-2 (Proleukin). 1×106 cells were seeded on a 48-well plate and the activation of NK-cells was determined after 48 hours by flow cytometry as up-regulation of CD25-FITC (clone: M-A251; BD Biosciences) and CD69-BV421 (clone: FN50; BioLegend).
NK-cell isolation was performed using human apheresis cone blood obtained from the Health Sciences Authority of Singapore. Peripheral blood mononuclear cells were acquired by density centrifugation at 400×g for 30 minutes using Ficoll-Paque Plus (GE Healthcare). Removal of platelets was performed by slow centrifugation at 120×g for 10 minutes. NK-cells were isolated using EasySep Human NK Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's protocol with the starting cell concentration of 1×108 cells/ml.
The isolated NK-cells were stained with Live/Dead Aqua viability dye (ThermoFisher Scientific) followed by surface staining with monoclonal antibodies specific for CD3-V500 (clone: UCHT1; BD Biosciences) and CD56-PeCy7 (clone: B159; BD Biosciences) to determine the efficiency of the isolation. The purity of NK-cells was greater than 90% as determined by CD3-CD56+ expression by flow cytometry.
The isolated cells were resuspended in X-VIVO 15 medium (Lonza) supplemented with 5% heat-inactivated human serum (Innova Biosciences) with or without 200 U/ml IL-2 (Proleukin). 1×106 cells were seeded on a 48-well plate and the activation of NK-cells was determined after 48 hours by flow cytometry as up-regulation of CD25-FITC (clone: M-A251; BD Biosciences) and CD69-BV421 (clone: FN50; BioLegend).
All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on HiSeq 2000 or HiSeq X Ten System (Illumina) as 2×101 bp or 2×151 bp, respectively. Due to high fragmentation of genomic DNA from FFPE material, a size selection step was conducted prior to library preparation for the FFPE tumour samples. Amplifiable DNA fragments of −200 bp from the FFPE samples are used for sequencing library construction to avoid false-negatives confidently in the discovery for SR within the PD-L1 gene.
Alternatively, for extranodal natural killer/T-cell lymphoma, whole-genome sequencing (WGS) was performed for all 40 pairs of tumours-normal samples described in this study. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Due to high fragmentation of genomic DNA in FFPE material, a size selection step was conducted prior to library preparation for the FFPE tumours samples. Paired-end sequencing was performed on HiSeq 2000 or HiSeq X Ten System (Illumina) as 2×101 bp or 2×151 bp, respectively. The mean WGS data coverages for the tumours and normal are 68.9× and 42.2×, respectively.
RNA extraction, and quality and quantity assessment were done as previously described. Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina) and whole-transcriptome sequencing (WTS) was performed on HiSeq 2500, HiSeq 3000 or HiSeq X Ten System (Illumina) with 2×101 bp, 2×151 bp or 2×151 bp read length, respectively.
RNA reads were aligned using STAR to a combined reference of hs37d5 and EBV-1 in a 2-pass mode. The gene counts were normalized by DESeq2 and the significance in differential expression was calculated using two-tailed analysed rank-sum test. Statistical significance was considered as p<0.05.
cDNA Synthesis and Real-Time
Reverse transcription was performed for samples with available RNA using SuperScript III Reverse Transcriptase (Invitrogen).
For extranodal natural killer/T-cell lymphoma, to generate WGS data from the extranodal natural killer/T-cell lymphoma specimen for this study, genomic DNA from snap frozen and FFPE tumours tissues, and whole blood was extracted as previously described. Buccal swab genomic DNA was purified using E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer's instructions. The quality and quantity were assessed as described elsewhere. Whole-genome sequencing was performed for all the tumours and, whole blood or buccal swab samples described in this study. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). A size selection step was conducted prior to library preparation for the FFPE tumours samples. RNA extraction, and quality and quantity assessment were done as previously described 2. Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina).
Sequencing reads were aligned using BWA-MEM to the hs37d5 human reference genome. Strelka2 and MuSE were used to detect somatic short variants. Short variants were subsequently annotated by wAnnovar.
Prior to all downstream analysis, gDNA sequencing reads were aligned using BWA-MEM to the hs37d5 human reference genome and PCR duplicates were marked by Sambamba. To identify somatic structural rearrangements (SR), Manta was applied on the aligned gDNA reads of tumours-blood paired samples. All predicted SRs within the genic region of PD-L1 were verified with Sanger Sequencing. To determine if the predicted SRs from the gDNA sequencing data yielded transcript products, cDNA was obtained from the available corresponding RNA using SuperScript III Reverse Transcriptase (Invitrogen) for PCR-based validation and Sanger sequencing.
DNA reads were aligned using BWA-MEM to the hs37d5 human reference genome and PCR duplicates were removed by Sambamba. Read pairs were marked as discordant if they did not align to the reference genome with the expected orientation and/or within the expected insert size. Reads were flagged as clipped when either end of the read did not match the reference genome.
Detection of somatic structural rearrangements (SR) was done by Manta and each candidate SR was subjected to the following filtering criteria: 1) SR is supported by at least 3 discordant read-pairs and at least 3 soft-clipped reads, and the sum of all supporting reads is at least 10; 2) zero discordant and soft-clipped reads present in the matching normal sample; 3) at least 20× coverage in both tumours and matching normal sample; and 4) SR is at least 1000 bp in size.
The histogram of unique samples having SR within a genomic region, i.e. the SR landscape, was tabulated using a 1 Mbp averaging sliding window in steps of 100 kbp along the main chromosomes of hs37d5. The breakpoints of putative SRs were converted to the BEDPE format and, together with the SR landscape, visualized as links using CIRCOS.
WGS data was nalysed using FreeBayes 6 (-X -u -C5 -m30 -q20) and variants with score less than 30 were filtered out. Single nucleotide variants are predicted to be somatic only if it is called from the tumours and not the matching normal data.
Somatic single nucleotide variants and indels in WGS data were called using FreeBayes. Candidate variants with a score of less than 30 were filtered away. Variants were predicted to be somatic only if it was called from the tumours and not the matching normal data.
SciClone was used to analyse the clonality architecture of the tumours. CANVAS was used to analyse copy number and loss of heterozygousity information for each tumour, which were used as input for the clonality analysis by SciClone.
For relapsed or refractory (RR) natural-killer/T-cell lymphoma, details about PCRconditions and sequencing were previously described. Primers were designed using Primer3 software and the sequences are listed in Table 6 for the discovery cohort ofthe 11 pembrolizumab-treated NKTL patients. Sanger sequences were aligned to hs37 reference genome and confirmed with BLAT. Alternatively, the primer sequences are also listed in Table 7 for the prevalence cohort of the 32 NKTL patients who were not subsequently treated with pembrolizumab.
For relapsed or refractory (RR) natural-killer/T-cell lymphoma, PD-L1 IHC analysis was performed with anti-PD-L1 rabbit monoclonal antibody (SP263, Ventana). PD-L1 positivity was evaluated as a percentage of positively stained tumour cells at the cell membrane. Alternatively, for extranodal natural killer/T-cell lymphoma, PD-L expression was evaluated as staining at the cell membrane and scored based on the percentage of positive tumours cells and staining intensity. The following grading was used: 0, no staining, 1+, weak, 2+ mild and 3+ strong staining. The same pathologist reviewed all PD-L1 IHC stainings. Available H-scores for the samples used in this study is included as Table 10.
b100%, 3+
15%, 2+
55%, 3+
30%, 2+
50%, 2+
55%, 2+
50%, 2+
15%, 2+
40%, 2+
100%, 3+
30%, 2+
25%, 3+
80%, 3+
80%, 3+
30%, 2+
60%, 2+
90%, 2+
80%, 3+
80%, 3+
60%, 3+
50%, 3+
70%, 3+
60%, 3+
20%, 1+
aAssessed by immunohistochemistry.
bClinical Response Reported in Kwong et al. Blood 2017.
K-562 and Jurkat cell lines was purchased from ATCC and NK-S1 was generated in-house. LGC Standards authenticated the K-562 and Jurkat cell lines. Jurkat cells were maintained in RPMI 1640 (Gibco) supplemented with 10% FBS (HyClone), and K-562 and NK-S1 were grown in DMEM (Gibco) supplemented with 10% FBS (HyClone), 10% horse serum (Gibco) and 2 mM L-glutamine (Gibco). The cells were grown at 37° C. in the presence of 5% CO2 and routinely checked for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).
For extranodal natural killer/T-cell lymphoma, K-562 and Jurkat cell lines from ATCC and in-house NK-S1 cell line was used to investigate the regulatory effect of the smallest structural rearrangement found within the 3′UTR of PD-L1 with the study cohort.
Wild type PD-L1 3′UTR (ENST00000381573.8) from SNK6 cell line was cloned into the XhoI and NotI sites of the psiCHECK-2 vector (Promega). For the partially inverted 3′UTR recapitulating the rearrangement identified in sample NKTL1, three individual pieces with overhangs were amplified from a wild type sample (SNK6) and ligated together by PCR. Cloning was performed using Q5 High-Fidelity 2× Master Mix (New England BioLabs). All cloning primers used to clone the full-length wild type and mutant PD-L1 3′UTR are described in Table 11.
GAGTCCAGC
GCCGCAACT
TCTCCCTCC
AGGCTCCC
GACTCTCAG
TCATGCAG
TACTTTCAA
ATGCCTGA
TTGGAACGG
GACAGTAT
TACTTTCAA
ATGCCTGA
GAGTCCAGC
GCCGCAACT
For relapsed or refractory (RR) natural-killer/T-cell lymphoma, for K-562 and Jurkat, 5×104 cells and 6×104 cells were seeded on a 48-well plate in triplicates, respectively, and transfected with 250 ng plasmid DNA using the Lipofectamine 3000 Reagent (Invitrogen). For NK-S1 cells, 2×105 cells were electroporated in triplicates on a 24-well plate with 1 μg plasmid DNA using the Neon Transfection System (Invitrogen). The pulse parameters used were the following: voltage 1300, width 10 and no. 3. Alternatively, for extranodal natural killer/T-cell lymphoma, for K-562 cells, 2.5×105 cells were seeded on a 48-well plate in triplicates and transfected with 250 ng plasmid DNA using the Lipofectamine 3000 Reagent (Invitrogen). For NK-S1 and Jurkat cells, 2.5×105 cells were electroporated in triplicates on a 24-well plate with 1 μg plasmid DNA using the Neon Transfection System (Invitrogen).
The cells were lysed with Passive Lysis Buffer (Promega) after 48 hours. Luminescence was measured using the Dual-Luciferase Reporter Assay System (Promega) and the GloMax-Multi+ Detection System (Promega). Renilla luciferase activities were divided by Firefly luciferase activities and the results were normalized to the empty vector control (mock). Statistical significance was calculated by two-sided t-test. Statistical significance was considered as P<0.05. All experiments were repeated at least twice.
The WGS data of 43 natural killer/T-cell lymphoma (NKTL)-normal/blood pairs and whole transcriptomic sequencing (WTS) data of 28 NKTL have been deposited in European Genome Archive (EGA) under the study accession code: EGAS00001002420.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201708262R | Oct 2017 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2018/050509 | 10/10/2018 | WO | 00 |
