Patent Grant
10982279
This document relates to methods and materials for assessing and/or treating mammals (e.g., humans) having cancer. For example, this document provides methods and materials for identifying a mammal as having cancer (e.g., a localized cancer). For example, this document provides methods and materials for monitoring and/or treating a mammal having cancer.
Much of the morbidity and mortality of human cancers world-wide is a result of the late diagnosis of these diseases, where treatments are less effective (Torre et al., 2015 CA Cancer J Clin 65:87; and World Health Organization, 2017 Guide to Cancer Early Diagnosis). Unfortunately, clinically proven biomarkers that can be used to broadly diagnose and treat patients are not widely available (Mazzucchelli, 2000 Advances in clinical pathology 4:111; Ruibal Morell, 1992 The International journal of biological markers 7:160; Galli et al., 2013 Clinical chemistry and laboratory medicine 51:1369; Sikaris, 2011 Heart, lung & circulation 20:634; Lin et al., 2016 in Screening for Colorectal Cancer: A Systematic Review for the U.S. Preventive Services Task Force. (Rockville, Md.); Wanebo et al., 1978 N Engl J Med 299A48; and Zauber, 2015 Dig Dis Sci 60:681).
Recent analyses of cell-free DNA suggests that such approaches may provide new avenues for early diagnosis (Phallen et al., 2017 Sci Transl Med 9; Cohen et al., 2018 Science 359:926; Alix-Panabieres et al., 2016 Cancer discovery 6:479; Siravegna et al., 2017 Nature reviews. Clinical oncology 14:531; Haber et al., 2014 Cancer discovery 4:650; Husain et al., 2017 JAMA 318:1272; and Wan et al., 2017 Nat Rev Cancer 17:223).
This document provides methods and materials for determining a cell free DNA (cfDNA) fragmentation profile in a mammal (e.g., in a sample obtained from a mammal). In some cases, determining a cfDNA fragmentation profile in a mammal can be used for identifying a mammal as having cancer. For example, cfDNA fragments obtained from a mammal (e.g., from a sample obtained from a mammal) can be subjected to low coverage whole-genome sequencing, and the sequenced fragments can be mapped to the genome (e.g., in non-overlapping windows) and assessed to determine a cfDNA fragmentation profile. This document also provides methods and materials for assessing and/or treating mammals (e.g., humans) having, or suspected of having, cancer. In some cases, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile. In some cases, this document provides methods and materials for monitoring and/or treating a mammal having cancer. For example, one or more cancer treatments can be administered to a mammal identified as having cancer (e.g., based, at least in part, on a cfDNA fragmentation profile) to treat the mammal.
Described herein is a non-invasive method for the early detection and localization of cancer. cfDNA in the blood can provide a non-invasive diagnostic avenue for patients with cancer. As demonstrated herein, DNA Evaluation of Fragments for early Interception (DELFI) was developed and used to evaluate genome-wide fragmentation patterns of cfDNA of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancers as well as 245 healthy individuals. These analyses revealed that cfDNA profiles of healthy individuals reflected nucleosomal fragmentation patterns of white blood cells, while patients with cancer had altered fragmentation profiles. DELFI had sensitivities of detection ranging from 57% to >99% among the seven cancer types at 98% specificity and identified the tissue of origin of the cancers to a limited number of sites in 75% of cases. Assessing cfDNA (e.g., using DELFI) can provide a screening approach for early detection of cancer, which can increase the chance for successful treatment of a patient having cancer. Assessing cfDNA (e.g., using DELFI) can also provide an approach for monitoring cancer, which can increase the chance for successful treatment and improved outcome of a patient having cancer. In addition, a cfDNA fragmentation profile can be obtained from limited amounts of cfDNA and using inexpensive reagents and/or instruments.
In general, one aspect of this document features methods for determining a cfDNA fragmentation profile of a mammal. The methods can include, or consist essentially of, processing cfDNA fragments obtained from a sample obtained from the mammal into sequencing libraries, subjecting the sequencing libraries to whole genome sequencing (e.g., low-coverage whole genome sequencing) to obtain sequenced fragments, mapping the sequenced fragments to a genome to obtain windows of mapped sequences, and analyzing the windows of mapped sequences to determine cfDNA fragment lengths. The mapped sequences can include tens to thousands of windows. The windows of mapped sequences can be non-overlapping windows. The windows of mapped sequences can each include about 5 million base pairs. The cfDNA fragmentation profile can be determined within each window. The cfDNA fragmentation profile can include a median fragment size. The cfDNA fragmentation profile can include a fragment size distribution. The cfDNA fragmentation profile can include a ratio of small cfDNA fragments to large cfDNA fragments in the windows of mapped sequences. The cfDNA fragmentation profile can be over the whole genome. The cfDNA fragmentation profile can be over a subgenomic interval (e.g., an interval in a portion of a chromosome).
In another aspect, this document features methods for identifying a mammal as having cancer. The methods can include, or consist essentially of, determining a cfDNA fragmentation profile in a sample obtained from a mammal, comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile, and identifying the mammal as having cancer when the cfDNA fragmentation profile in the sample obtained from the mammal is different from the reference cfDNA fragmentation profile. The reference cfDNA fragmentation profile can be a cfDNA fragmentation profile of a healthy mammal. The reference cfDNA fragmentation profile can be generated by determining a cfDNA fragmentation profile in a sample obtained from the healthy mammal. The reference DNA fragmentation pattern can be a reference nucleosome cfDNA fragmentation profile. The cfDNA fragmentation profiles can include a median fragment size, and a median fragment size of the cfDNA fragmentation profile can be shorter than a median fragment size of the reference cfDNA fragmentation profile. The cfDNA fragmentation profiles can include a fragment size distribution, and a fragment size distribution of the cfDNA fragmentation profile can differ by at least 10 nucleotides as compared to a fragment size distribution of the reference cfDNA fragmentation profile. The cfDNA fragmentation profiles can include position dependent differences in fragmentation patterns, including a ratio of small cfDNA fragments to large cfDNA fragments, where a small cfDNA fragment can be 100 base pairs (bp) to 150 bp in length and a large cfDNA fragments can be 151 bp to 220 bp in length, and where a correlation of fragment ratios in the cfDNA fragmentation profile can be lower than a correlation of fragment ratios of the reference cfDNA fragmentation profile. The cfDNA fragmentation profiles can include sequence coverage of small cfDNA fragments, large cfDNA fragments, or of both small and large cfDNA fragments, across the genome. The cancer can be colorectal cancer, lung cancer, breast cancer, bile duct cancer, pancreatic cancer, gastric cancer, or ovarian cancer. The step of comparing can include comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile in windows across the whole genome. The step of comparing can include comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile over a subgenomic interval (e.g., an interval in a portion of a chromosome). The mammal can have been previously administered a cancer treatment to treat the cancer. The cancer treatment can be surgery, adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy, targeted therapy, or any combinations thereof. The method also can include administering to the mammal a cancer treatment (e.g., surgery, adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy, targeted therapy, or any combinations thereof). The mammal can be monitored for the presence of cancer after administration of the cancer treatment.
In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, identifying the mammal as having cancer, where the identifying includes determining a cfDNA fragmentation profile in a sample obtained from the mammal, comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile, and identifying the mammal as having cancer when the cfDNA fragmentation profile obtained from the mammal is different from the reference cfDNA fragmentation profile; and administering a cancer treatment to the mammal. The mammal can be a human. The cancer can be colorectal cancer, lung cancer, breast cancer, gastric cancers, pancreatic cancers, bile duct cancers, or ovarian cancer. The cancer treatment can be surgery, adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy, targeted therapy, or combinations thereof. The reference cfDNA fragmentation profile can be a cfDNA fragmentation profile of a healthy mammal. The reference cfDNA fragmentation profile can be generated by determining a cfDNA fragmentation profile in a sample obtained from a healthy mammal. The reference DNA fragmentation pattern can be a reference nucleosome cfDNA fragmentation profile. The cfDNA fragmentation profile can include a median fragment size, where a median fragment size of the cfDNA fragmentation profile is shorter than a median fragment size of the reference cfDNA fragmentation profile. The cfDNA fragmentation profile can include a fragment size distribution, where a fragment size distribution of the cfDNA fragmentation profile differs by at least 10 nucleotides as compared to a fragment size distribution of the reference cfDNA fragmentation profile. The cfDNA fragmentation profile can include a ratio of small cfDNA fragments to large cfDNA fragments in the windows of mapped sequences, where a small cfDNA fragment is 100 bp to 150 bp in length, where a large cfDNA fragments is 151 bp to 220 bp in length, and where a correlation of fragment ratios in the cfDNA fragmentation profile is lower than a correlation of fragment ratios of the reference cfDNA fragmentation profile. The cfDNA fragmentation profile can include the sequence coverage of small cfDNA fragments in windows across the genome. The cfDNA fragmentation profile can include the sequence coverage of large cfDNA fragments in windows across the genome. The cfDNA fragmentation profile can include the sequence coverage of small and large cfDNA fragments in windows across the genome. The step of comparing can include comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile over the whole genome. The step of comparing can include comparing the cfDNA fragmentation profile to a reference cfDNA fragmentation profile over a subgenomic interval. The mammal can have previously been administered a cancer treatment to treat the cancer. The cancer treatment can be surgery, adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy, targeted therapy, or combinations thereof. The method also can include monitoring the mammal for the presence of cancer after administration of the cancer treatment.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for determining a cfDNA fragmentation profile in a mammal (e.g., in a sample obtained from a mammal). As used herein, the terms “fragmentation profile,” “position dependent differences in fragmentation patterns,” and “differences in fragment size and coverage in a position dependent manner across the genome” are equivalent and can be used interchangeably. In some cases, determining a cfDNA fragmentation profile in a mammal can be used for identifying a mammal as having cancer. For example, cfDNA fragments obtained from a mammal (e.g., from a sample obtained from a mammal) can be subjected to low coverage whole-genome sequencing, and the sequenced fragments can be mapped to the genome (e.g., in non-overlapping windows) and assessed to determine a cfDNA fragmentation profile. As described herein, a cfDNA fragmentation profile of a mammal having cancer is more heterogeneous (e.g., in fragment lengths) than a cfDNA fragmentation profile of a healthy mammal (e.g., a mammal not having cancer). As such, this document also provides methods and materials for assessing, monitoring, and/or treating mammals (e.g., humans) having, or suspected of having, cancer. In some cases, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the presence and, optionally, the tissue of origin of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for monitoring a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the presence of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying a mammal as having cancer, and administering one or more cancer treatments to the mammal to treat the mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal, and one or more cancer treatments can be administered to the mammal.
A cfDNA fragmentation profile can include one or more cfDNA fragmentation patterns. A cfDNA fragmentation pattern can include any appropriate cfDNA fragmentation pattern. Examples of cfDNA fragmentation patterns include, without limitation, median fragment size, fragment size distribution, ratio of small cfDNA fragments to large cfDNA fragments, and the coverage of cfDNA fragments. In some cases, a cfDNA fragmentation pattern includes two or more (e.g., two, three, or four) of median fragment size, fragment size distribution, ratio of small cfDNA fragments to large cfDNA fragments, and the coverage of cfDNA fragments. In some cases, cfDNA fragmentation profile can be a genome-wide cfDNA profile (e.g., a genome-wide cfDNA profile in windows across the genome). In some cases, cfDNA fragmentation profile can be a targeted region profile. A targeted region can be any appropriate portion of the genome (e.g., a chromosomal region). Examples of chromosomal regions for which a cfDNA fragmentation profile can be determined as described herein include, without limitation, a portion of a chromosome (e.g., a portion of 2q, 4p, 5p, 6q, 7p, 8q, 9q, 10q, 11q, 12q, and/or 14q) and a chromosomal arm (e.g., a chromosomal arm of 8q, 13q, 11q, and/or 3p). In some cases, a cfDNA fragmentation profile can include two or more targeted region profiles.
In some cases, a cfDNA fragmentation profile can be used to identify changes (e.g., alterations) in cfDNA fragment lengths. An alteration can be a genome-wide alteration or an alteration in one or more targeted regions/loci. A target region can be any region containing one or more cancer-specific alterations. Examples of cancer-specific alterations, and their chromosomal locations, include, without limitation, those shown in Table 3 (Appendix C) and those shown in Table 6 (Appendix F). In some cases, a cfDNA fragmentation profile can be used to identify (e.g., simultaneously identify) from about 10 alterations to about 500 alterations (e.g., from about 25 to about 500, from about 50 to about 500, from about 100 to about 500, from about 200 to about 500, from about 300 to about 500, from about 10 to about 400, from about 10 to about 300, from about 10 to about 200, from about 10 to about 100, from about 10 to about 50, from about 20 to about 400, from about 30 to about 300, from about 40 to about 200, from about 50 to about 100, from about 20 to about 100, from about 25 to about 75, from about 50 to about 250, or from about 100 to about 200, alterations).
In some cases, a cfDNA fragmentation profile can be used to detect tumor-derived DNA. For example, a cfDNA fragmentation profile can be used to detect tumor-derived DNA by comparing a cfDNA fragmentation profile of a mammal having, or suspected of having, cancer to a reference cfDNA fragmentation profile (e.g., a cfDNA fragmentation profile of a healthy mammal and/or a nucleosomal DNA fragmentation profile of healthy cells from the mammal having, or suspected of having, cancer). In some cases, a reference cfDNA fragmentation profile is a previously generated profile from a healthy mammal. For example, methods provided herein can be used to determine a reference cfDNA fragmentation profile in a healthy mammal, and that reference cfDNA fragmentation profile can be stored (e.g., in a computer or other electronic storage medium) for future comparison to a test cfDNA fragmentation profile in mammal having, or suspected of having, cancer. In some cases, a reference cfDNA fragmentation profile (e.g., a stored cfDNA fragmentation profile) of a healthy mammal is determined over the whole genome. In some cases, a reference cfDNA fragmentation profile (e.g., a stored cfDNA fragmentation profile) of a healthy mammal is determined over a subgenomic interval.
In some cases, a cfDNA fragmentation profile can be used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and/or an ovarian cancer).
A cfDNA fragmentation profile can include a cfDNA fragment size pattern. cfDNA fragments can be any appropriate size. For example, cfDNA fragment can be from about 50 base pairs (bp) to about 400 bp in length. As described herein, a mammal having cancer can have a cfDNA fragment size pattern that contains a shorter median cfDNA fragment size than the median cfDNA fragment size in a healthy mammal. A healthy mammal (e.g., a mammal not having cancer) can have cfDNA fragment sizes having a median cfDNA fragment size from about 166.6 bp to about 167.2 bp (e.g., about 166.9 bp). In some cases, a mammal having cancer can have cfDNA fragment sizes that are, on average, about 1.28 bp to about 2.49 bp (e.g., about 1.88 bp) shorter than cfDNA fragment sizes in a healthy mammal. For example, a mammal having cancer can have cfDNA fragment sizes having a median cfDNA fragment size of about 164.11 bp to about 165.92 bp (e.g., about 165.02 bp).
A cfDNA fragmentation profile can include a cfDNA fragment size distribution. As described herein, a mammal having cancer can have a cfDNA size distribution that is more variable than a cfDNA fragment size distribution in a healthy mammal. In some case, a size distribution can be within a targeted region. A healthy mammal (e.g., a mammal not having cancer) can have a targeted region cfDNA fragment size distribution of about 1 or less than about 1. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is longer (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 or more bp longer, or any number of base pairs between these numbers) than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is shorter (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 or more bp shorter, or any number of base pairs between these numbers) than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is about 47 bp smaller to about 30 bp longer than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution of, on average, a 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20 or more bp difference in lengths of cfDNA fragments. For example, a mammal having cancer can have a targeted region cfDNA fragment size distribution of, on average, about a 13 bp difference in lengths of cfDNA fragments. In some case, a size distribution can be a genome-wide size distribution. A healthy mammal (e.g., a mammal not having cancer) can have very similar distributions of short and long cfDNA fragments genome-wide. In some cases, a mammal having cancer can have, genome-wide, one or more alterations (e.g., increases and decreases) in cfDNA fragment sizes. The one or more alterations can be any appropriate chromosomal region of the genome. For example, an alteration can be in a portion of a chromosome. Examples of portions of chromosomes that can contain one or more alterations in cfDNA fragment sizes include, without limitation, portions of 2q, 4p, 5p, 6q, 7p, 8q, 9q, 10q, 11q, 12q, and 14q. For example, an alteration can be across a chromosome arm (e.g., an entire chromosome arm).
A cfDNA fragmentation profile can include a ratio of small cfDNA fragments to large cfDNA fragments and a correlation of fragment ratios to reference fragment ratios. As used herein, with respect to ratios of small cfDNA fragments to large cfDNA fragments, a small cfDNA fragment can be from about 100 bp in length to about 150 bp in length. As used herein, with respect to ratios of small cfDNA fragments to large cfDNA fragments, a large cfDNA fragment can be from about 151 bp in length to 220 bp in length. As described herein, a mammal having cancer can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) that is lower (e.g., 2-fold lower, 3-fold lower, 4-fold lower, 5-fold lower, 6-fold lower, 7-fold lower, 8-fold lower, 9-fold lower, 10-fold lower, or more) than in a healthy mammal. A healthy mammal (e.g., a mammal not having cancer) can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) of about 1 (e.g., about 0.96). In some cases, a mammal having cancer can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) that is, on average, about 0.19 to about 0.30 (e.g., about 0.25) lower than a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) in a healthy mammal.
A cfDNA fragmentation profile can include coverage of all fragments. Coverage of all fragments can include windows (e.g., non-overlapping windows) of coverage. In some cases, coverage of all fragments can include windows of small fragments (e.g., fragments from about 100 bp to about 150 bp in length). In some cases, coverage of all fragments can include windows of large fragments (e.g., fragments from about 151 bp to about 220 bp in length).
In some cases, a cfDNA fragmentation profile can be used to identify the tissue of origin of a cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, or an ovarian cancer). For example, a cfDNA fragmentation profile can be used to identify a localized cancer. When a cfDNA fragmentation profile includes a targeted region profile, one or more alterations described herein (e.g., in Table 3 (Appendix C) and/or in Table 6 (Appendix F)) can be used to identify the tissue of origin of a cancer. In some cases, one or more alterations in chromosomal regions can be used to identify the tissue of origin of a cancer.
A cfDNA fragmentation profile can be obtained using any appropriate method. In some cases, cfDNA from a mammal (e.g., a mammal having, or suspected of having, cancer) can be processed into sequencing libraries which can be subjected to whole genome sequencing (e.g., low-coverage whole genome sequencing), mapped to the genome, and analyzed to determine cfDNA fragment lengths. Mapped sequences can be analyzed in non-overlapping windows covering the genome. Windows can be any appropriate size. For example, windows can be from thousands to millions of bases in length. As one non-limiting example, a window can be about 5 megabases (Mb) long. Any appropriate number of windows can be mapped. For example, tens to thousands of windows can be mapped in the genome. For example, hundreds to thousands of windows can be mapped in the genome. A cfDNA fragmentation profile can be determined within each window. In some cases, a cfDNA fragmentation profile can be obtained as described in Example 1. In some cases, a cfDNA fragmentation profile can be obtained as shown in
In some cases, methods and materials described herein also can include machine learning. For example, machine learning can be used for identifying an altered fragmentation profile (e.g., using coverage of cfDNA fragments, fragment size of cfDNA fragments, coverage of chromosomes, and mtDNA).
In some cases, methods and materials described herein can be the sole method used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and/or an ovarian cancer). For example, determining a cfDNA fragmentation profile can be the sole method used to identify a mammal as having cancer.
In some cases, methods and materials described herein can be used together with one or more additional methods used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and/or an ovarian cancer). Examples of methods used to identify a mammal as having cancer include, without limitation, identifying one or more cancer-specific sequence alterations, identifying one or more chromosomal alterations (e.g., aneuploidies and rearrangements), and identifying other cfDNA alterations. For example, determining a cfDNA fragmentation profile can be used together with identifying one or more cancer-specific mutations in a mammal's genome to identify a mammal as having cancer. For example, determining a cfDNA fragmentation profile can be used together with identifying one or more aneuploidies in a mammal's genome to identify a mammal as having cancer.
In some aspects, this document also provides methods and materials for assessing, monitoring, and/or treating mammals (e.g., humans) having, or suspected of having, cancer. In some cases, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying the location (e.g., the anatomic site or tissue of origin) of a cancer in a mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the tissue of origin of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying a mammal as having cancer, and administering one or more cancer treatments to the mammal to treat the mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal, and administering one or more cancer treatments to the mammal. In some cases, this document provides methods and materials for treating a mammal having cancer. For example, one or more cancer treatments can be administered to a mammal identified as having cancer (e.g., based, at least in part, on the cfDNA fragmentation profile of the mammal) to treat the mammal. In some cases, during or after the course of a cancer treatment (e.g., any of the cancer treatments described herein), a mammal can undergo monitoring (or be selected for increased monitoring) and/or further diagnostic testing. In some cases, monitoring can include assessing mammals having, or suspected of having, cancer by, for example, assessing a sample (e.g., a blood sample) obtained from the mammal to determine the cfDNA fragmentation profile of the mammal as described herein, and changes in the cfDNA fragmentation profiles over time can be used to identify response to treatment and/or identify the mammal as having cancer (e.g., a residual cancer).
Any appropriate mammal can be assessed, monitored, and/or treated as described herein. A mammal can be a mammal having cancer. A mammal can be a mammal suspected of having cancer. Examples of mammals that can be assessed, monitored, and/or treated as described herein include, without limitation, humans, primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having, or suspected of having, cancer can be assessed to determine a cfDNA fragmentation profiled as described herein and, optionally, can be treated with one or more cancer treatments as described herein.
Any appropriate sample from a mammal can be assessed as described herein (e.g., assessed for a DNA fragmentation pattern). In some cases, a sample can include DNA (e.g., genomic DNA). In some cases, a sample can include cfDNA (e.g., circulating tumor DNA (ctDNA)). In some cases, a sample can be fluid sample (e.g., a liquid biopsy). Examples of samples that can contain DNA and/or polypeptides include, without limitation, blood (e.g., whole blood, serum, or plasma), amnion, tissue, urine, cerebrospinal fluid, saliva, sputum, broncho-alveolar lavage, bile, lymphatic fluid, cyst fluid, stool, ascites, pap smears, breast milk, and exhaled breath condensate. For example, a plasma sample can be assessed to determine a cfDNA fragmentation profiled as described herein.
A sample from a mammal to be assessed as described herein (e.g., assessed for a DNA fragmentation pattern) can include any appropriate amount of cfDNA. In some cases, a sample can include a limited amount of DNA. For example, a cfDNA fragmentation profile can be obtained from a sample that includes less DNA than is typically required for other cfDNA analysis methods, such as those described in, for example, Phallen et al., 2017 Sci Transl Med 9; Cohen et al., 2018 Science 359:926; Newman et al., 2014 Nat Med 20:548; and Newman et al., 2016 Nat Biotechnol 34:547).
In some cases, a sample can be processed (e.g., to isolate and/or purify DNA and/or polypeptides from the sample). For example, DNA isolation and/or purification can include cell lysis (e.g., using detergents and/or surfactants), protein removal (e.g., using a protease), and/or RNA removal (e.g., using an RNase). As another example, polypeptide isolation and/or purification can include cell lysis (e.g., using detergents and/or surfactants), DNA removal (e.g., using a DNase), and/or RNA removal (e.g., using an RNase).
A mammal having, or suspected of having, any appropriate type of cancer can be assessed (e.g., to determine a cfDNA fragmentation profile) and/or treated (e.g., by administering one or more cancer treatments to the mammal) using the methods and materials described herein. A cancer can be any stage cancer. In some cases, a cancer can be an early stage cancer. In some cases, a cancer can be an asymptomatic cancer. In some cases, a cancer can be a residual disease and/or a recurrence (e.g., after surgical resection and/or after cancer therapy). A cancer can be any type of cancer. Examples of types of cancers that can be assessed, monitored, and/or treated as described herein include, without limitation, colorectal cancers, lung cancers, breast cancers, gastric cancers, pancreatic cancers, bile duct cancers, and ovarian cancers.
When treating a mammal having, or suspected of having, cancer as described herein, the mammal can be administered one or more cancer treatments. A cancer treatment can be any appropriate cancer treatment. One or more cancer treatments described herein can be administered to a mammal at any appropriate frequency (e.g., once or multiple times over a period of time ranging from days to weeks). Examples of cancer treatments include, without limitation adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy (e.g., chimeric antigen receptors and/or T cells having wild-type or modified T cell receptors), targeted therapy such as administration of kinase inhibitors (e.g., kinase inhibitors that target a particular genetic lesion, such as a translocation or mutation), (e.g. a kinase inhibitor, an antibody, a bispecific antibody), signal transduction inhibitors, bispecific antibodies or antibody fragments (e.g., BiTEs), monoclonal antibodies, immune checkpoint inhibitors, surgery (e.g., surgical resection), or any combination of the above. In some cases, a cancer treatment can reduce the severity of the cancer, reduce a symptom of the cancer, and/or to reduce the number of cancer cells present within the mammal.
In some cases, a cancer treatment can include an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors include nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (tecentriq), avelumab (bavencio), durvalumab (imfinzi), ipilimumab (yervoy). See, e.g., Pardoll (2012) Nat. Rev Cancer 12: 252-264; Sun et al. (2017) Eur Rev Med Pharmacol Sci 21(6): 1198-1205; Hamanishi et al. (2015) J. Clin. Oncol. 33(34): 4015-22; Brahmer et al. (2012) N Engl J Med 366(26): 2455-65; Ricciuti et al. (2017) J. Thorac Oncol. 12(5): e51-e55; Ellis et al. (2017) Clin Lung Cancer pii: S1525-7304(17)30043-8; Zou and Awad (2017) Ann Oncol 28(4): 685-687; Sorscher (2017) N Engl J Med 376(10: 996-7; Hui et al. (2017) Ann Oncol 28(4): 874-881; Vansteenkiste et al. (2017) Expert Opin Biol Ther 17(6): 781-789; Hellmann et al. (2017) Lancet Oncol. 18(1): 31-41; Chen (2017) J. Chin Med Assoc 80(1): 7-14.
In some cases, a cancer treatment can be an adoptive T cell therapy (e.g., chimeric antigen receptors and/or T cells having wild-type or modified T cell receptors). See, e.g., Rosenberg and Restifo (2015) Science 348(6230): 62-68; Chang and Chen (2017) Trends Mol Med 23(5): 430-450; Yee and Lizee (2016) Cancer J. 23(2): 144-148; Chen et al. (2016) Oncoimmunology 6(2): e1273302; US 2016/0194404; US 2014/0050788; US 2014/0271635; U.S. Pat. No. 9,233,125; incorporated by reference in their entirety herein.
In some cases, a cancer treatment can be a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include: amsacrine, azacitidine, axathioprine, bevacizumab (or an antigen-binding fragment thereof), bleomycin, busulfan, carboplatin, capecitabine, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, erlotinib hydrochlorides, etoposide, fiudarabine, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrxate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, procarbazine, all-trans retinoic acid, streptozocin, tafluposide, temozolomide, teniposide, tioguanine, topotecan, uramustine, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. Additional examples of anti-cancer therapies are known in the art; see, e.g. the guidelines for therapy from the American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), or National Comprehensive Cancer Network (NCCN).
When monitoring a mammal having, or suspected of having, cancer as described herein (e.g., based, at least in part, on the cfDNA fragmentation profile of the mammal), the monitoring can be before, during, and/or after the course of a cancer treatment. Methods of monitoring provided herein can be used to determine the efficacy of one or more cancer treatments and/or to select a mammal for increased monitoring. In some cases, the monitoring can include identifying a cfDNA fragmentation profile as described herein. For example, a cfDNA fragmentation profile can be obtained before administering one or more cancer treatments to a mammal having, or suspected or having, cancer, one or more cancer treatments can be administered to the mammal, and one or more cfDNA fragmentation profiles can be obtained during the course of the cancer treatment. In some cases, a cfDNA fragmentation profile can change during the course of cancer treatment (e.g., any of the cancer treatments described herein). For example, a cfDNA fragmentation profile indicative that the mammal has cancer can change to a cfDNA fragmentation profile indicative that the mammal does not have cancer. Such a cfDNA fragmentation profile change can indicate that the cancer treatment is working. Conversely, a cfDNA fragmentation profile can remain static (e.g., the same or approximately the same) during the course of cancer treatment (e.g., any of the cancer treatments described herein). Such a static cfDNA fragmentation profile can indicate that the cancer treatment is not working. In some cases, the monitoring can include conventional techniques capable of monitoring one or more cancer treatments (e.g., the efficacy of one or more cancer treatments). In some cases, a mammal selected for increased monitoring can be administered a diagnostic test (e.g., any of the diagnostic tests disclosed herein) at an increased frequency compared to a mammal that has not been selected for increased monitoring. For example, a mammal selected for increased monitoring can be administered a diagnostic test at a frequency of twice daily, daily, bi-weekly, weekly, bi-monthly, monthly, quarterly, semi-annually, annually, or any at frequency therein. In some cases, a mammal selected for increased monitoring can be administered a one or more additional diagnostic tests compared to a mammal that has not been selected for increased monitoring. For example, a mammal selected for increased monitoring can be administered two diagnostic tests, whereas a mammal that has not been selected for increased monitoring is administered only a single diagnostic test (or no diagnostic tests). In some cases, a mammal that has been selected for increased monitoring can also be selected for further diagnostic testing. Once the presence of a tumor or a cancer (e.g., a cancer cell) has been identified (e.g., by any of the variety of methods disclosed herein), it may be beneficial for the mammal to undergo both increased monitoring (e.g., to assess the progression of the tumor or cancer in the mammal and/or to assess the development of one or more cancer biomarkers such as mutations), and further diagnostic testing (e.g., to determine the size and/or exact location (e.g., tissue of origin) of the tumor or the cancer). In some cases, one or more cancer treatments can be administered to the mammal that is selected for increased monitoring after a cancer biomarker is detected and/or after the cfDNA fragmentation profile of the mammal has not improved or deteriorated. Any of the cancer treatments disclosed herein or known in the art can be administered. For example, a mammal that has been selected for increased monitoring can be further monitored, and a cancer treatment can be administered if the presence of the cancer cell is maintained throughout the increased monitoring period. Additionally or alternatively, a mammal that has been selected for increased monitoring can be administered a cancer treatment, and further monitored as the cancer treatment progresses. In some cases, after a mammal that has been selected for increased monitoring has been administered a cancer treatment, the increased monitoring will reveal one or more cancer biomarkers (e.g., mutations). In some cases, such one or more cancer biomarkers will provide cause to administer a different cancer treatment (e.g., a resistance mutation may arise in a cancer cell during the cancer treatment, which cancer cell harboring the resistance mutation is resistant to the original cancer treatment).
When a mammal is identified as having cancer as described herein (e.g., based, at least in part, on the cfDNA fragmentation profile of the mammal), the identifying can be before and/or during the course of a cancer treatment. Methods of identifying a mammal as having cancer provided herein can be used as a first diagnosis to identify the mammal (e.g., as having cancer before any course of treatment) and/or to select the mammal for further diagnostic testing. In some cases, once a mammal has been determined to have cancer, the mammal may be administered further tests and/or selected for further diagnostic testing. In some cases, methods provided herein can be used to select a mammal for further diagnostic testing at a time period prior to the time period when conventional techniques are capable of diagnosing the mammal with an early-stage cancer. For example, methods provided herein for selecting a mammal for further diagnostic testing can be used when a mammal has not been diagnosed with cancer by conventional methods and/or when a mammal is not known to harbor a cancer. In some cases, a mammal selected for further diagnostic testing can be administered a diagnostic test (e.g., any of the diagnostic tests disclosed herein) at an increased frequency compared to a mammal that has not been selected for further diagnostic testing. For example, a mammal selected for further diagnostic testing can be administered a diagnostic test at a frequency of twice daily, daily, bi-weekly, weekly, bi-monthly, monthly, quarterly, semi-annually, annually, or any at frequency therein. In some cases, a mammal selected for further diagnostic testing can be administered a one or more additional diagnostic tests compared to a mammal that has not been selected for further diagnostic testing. For example, a mammal selected for further diagnostic testing can be administered two diagnostic tests, whereas a mammal that has not been selected for further diagnostic testing is administered only a single diagnostic test (or no diagnostic tests). In some cases, the diagnostic testing method can determine the presence of the same type of cancer (e.g., having the same tissue or origin) as the cancer that was originally detected (e.g., based, at least in part, on the cfDNA fragmentation profile of the mammal). Additionally or alternatively, the diagnostic testing method can determine the presence of a different type of cancer as the cancer that was original detected. In some cases, the diagnostic testing method is a scan. In some cases, the scan is a computed tomography (CT), a CT angiography (CTA), a esophagram (a Barium swallom), a Barium enema, a magnetic resonance imaging (MRI), a PET scan, an ultrasound (e.g., an endobronchial ultrasound, an endoscopic ultrasound), an X-ray, a DEXA scan. In some cases, the diagnostic testing method is a physical examination, such as an anoscopy, a bronchoscopy (e.g., an autofluorescence bronchoscopy, a white-light bronchoscopy, a navigational bronchoscopy), a colonoscopy, a digital breast tomosynthesis, an endoscopic retrograde cholangiopancreatography (ERCP), an ensophagogastroduodenoscopy, a mammography, a Pap smear, a pelvic exam, a positron emission tomography and computed tomography (PET-CT) scan. In some cases, a mammal that has been selected for further diagnostic testing can also be selected for increased monitoring. Once the presence of a tumor or a cancer (e.g., a cancer cell) has been identified (e.g., by any of the variety of methods disclosed herein), it may be beneficial for the mammal to undergo both increased monitoring (e.g., to assess the progression of the tumor or cancer in the mammal and/or to assess the development of one or more cancer biomarkers such as mutations), and further diagnostic testing (e.g., to determine the size and/or exact location of the tumor or the cancer). In some cases, a cancer treatment is administered to the mammal that is selected for further diagnostic testing after a cancer biomarker is detected and/or after the cfDNA fragmentation profile of the mammal has not improved or deteriorated. Any of the cancer treatments disclosed herein or known in the art can be administered. For example, a mammal that has been selected for further diagnostic testing can be administered a further diagnostic test, and a cancer treatment can be administered if the presence of the tumor or the cancer is confirmed. Additionally or alternatively, a mammal that has been selected for further diagnostic testing can be administered a cancer treatment, and can be further monitored as the cancer treatment progresses. In some cases, after a mammal that has been selected for further diagnostic testing has been administered a cancer treatment, the additional testing will reveal one or more cancer biomarkers (e.g., mutations). In some cases, such one or more cancer biomarkers (e.g., mutations) will provide cause to administer a different cancer treatment (e.g., a resistance mutation may arise in a cancer cell during the cancer treatment, which cancer cell harboring the resistance mutation is resistant to the original cancer treatment).
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Analyses of cell free DNA have largely focused on targeted sequencing of specific genes. Such studies permit detection of a small number of tumor-specific alterations in patients with cancer and not all patients, especially those with early stage disease, have detectable changes. Whole genome sequencing of cell-free DNA can identify chromosomal abnormalities and rearrangements in cancer patients but detection of such alterations has been challenging in part due to the difficulty in distinguishing a small number of abnormal from normal chromosomal changes (Leary et al., 2010 Sci Transl Med 2:20ra14; and Leary et al., 2012 Sci Transl Med 4:162ra154). Other efforts have suggested nucleosome patterns and chromatin structure may be different between cancer and normal tissues, and that cfDNA in patients with cancer may result in abnormal cfDNA fragment size as well as position (Snyder et al., 2016 Cell 164:57; Jahr et al., 2001 Cancer Res 61:1659; Ivanov et al., 2015 BMC Genomics 16(Suppl 13):S1). However, the amount of sequencing needed for nucleosome footprint analyses of cfDNA is impractical for routine analyses.
The sensitivity of any cell-free DNA approach depends on the number of potential alterations examined as well as the technical and biological limitations of detecting such changes. As a typical blood sample contains ˜2000 genome equivalents of cfDNA per milliliter of plasma (Phallen et al., 2017 Sci Transl Med 9), the theoretical limit of detection of a single alteration can be no better than one in a few thousand mutant to wild-type molecules. An approach that detects a larger number of alterations in the same number of genome equivalents would be more sensitive for detecting cancer in the circulation. Monte Carlo simulations show that increasing the number of potential abnormalities detected from only a few to tens or hundreds can potentially improve the limit of detection by orders of magnitude, similar to recent probability analyses of multiple methylation changes in cfDNA (
This study presents a novel method called DELFI for detection of cancer and further identification of tissue of origin using whole genome sequencing (
Materials and Methods
Patient and Sample Characteristics
Plasma samples from healthy individuals and plasma and tissue samples from patients with breast, lung, ovarian, colorectal, bile duct, or gastric cancer were obtained from ILSBio/Bioreclamation, Aarhus University, Herlev Hospital of the University of Copenhagen, Hvidovre Hospital, the University Medical Center of the University of Utrecht, the Academic Medical Center of the University of Amsterdam, the Netherlands Cancer Institute, and the University of California, San Diego. All samples were obtained under Institutional Review Board approved protocols with informed consent for research use at participating institutions. Plasma samples from healthy individuals were obtained at the time of routine screening, including for colonoscopies or Pap smears. Individuals were considered healthy if they had no previous history of cancer and negative screening results.
Plasma samples from individuals with breast, colorectal, gastric, lung, ovarian, pancreatic, and bile duct cancer were obtained at the time of diagnosis, prior to tumor resection or therapy. Nineteen lung cancer patients analyzed for change in cfDNA fragmentation profiles across multiple time points were undergoing treatment with anti-EGFR or anti-ERBB2 therapy (see, e.g., Phallen et cd, 2019 Cancer Research 15, 1204-1213). Clinical data for all patients included in this study are listed in Table 1 (Appendix A). Gender was confirmed through genomic analyses of X and Y chromosome representation. Pathologic staging of gastric cancer patients was performed after neoadjuvant therapy. Samples where the tumor stage was unknown were indicated as stage X or unknown.
Nucleosomal DNA Purification
Viably frozen lymphocytes were elutriated from leukocytes obtained from a healthy male (C0618) and female (D0808-L) (Advanced Biotechnologies Inc., Eldersburg, Md.). Aliquots of 1×106 cells were used for nucleosomal DNA purification using EZ Nucleosomal DNA Prep Kit (Zymo Research, Irvine, Calif.). Cells were initially treated with 100 μl of Nuclei Prep Buffer and incubated on ice for 5 minutes. After centrifugation at 200 g for 5 minutes, supernatant was discarded and pelleted nuclei were treated twice with 100 μl of Atlantis Digestion Buffer or with 100 μl of micrococcal nuclease (MN) Digestion Buffer. Finally, cellular nucleic DNA was fragmented with 0.5 U of Atlantis dsDNase at 42° C. for 20 minutes or 1.5 U of MNase at 37° C. for 20 minutes. Reactions were stopped using 5× MN Stop Buffer and DNA was purified using Zymo-Spin™ IIC Columns. Concentration and quality of eluted cellular nucleic DNA were analyzed using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif.).
Sample Preparation and Sequencing of cfDNA
Whole blood was collected in EDTA tubes and processed immediately or within one day after storage at 4° C., or was collected in Streck tubes and processed within two days of collection for three cancer patients who were part of the monitoring analysis. Plasma and cellular components were separated by centrifugation at 800 g for 10 min at 4° C. Plasma was centrifuged a second time at 18,000 g at room temperature to remove any remaining cellular debris and stored at −80° C. until the time of DNA extraction. DNA was isolated from plasma using the Qiagen Circulating Nucleic Acids Kit (Qiagen GmbH) and eluted in LoBind tubes (Eppendorf AG). Concentration and quality of cfDNA were assessed using the Bioanalyzer 2100 (Agilent Technologies).
NGS cfDNA libraries were prepared for whole genome sequencing and targeted sequencing using 5 to 250 ng of cfDNA as described elsewhere (see, e.g., Phallen et al., 2017 Sci Transl Med 9:eaan2415). Briefly, genomic libraries were prepared using the NEBNext DNA Library Prep Kit for Illumina [New England Biolabs (NEB)] with four main modifications to the manufacturer's guidelines: (i) The library purification steps used the on-bead AMPure XP approach to minimize sample loss during elution and tube transfer steps (see, e.g., Fisher et al., 2011 Genome Biol 12:R1); (ii) NEBNext End Repair, A-tailing, and adapter ligation enzyme and buffer volumes were adjusted as appropriate to accommodate the on-bead AMPure XP purification strategy, (iii) a pool of eight unique Illumina dual index adapters with 8-base pair (bp) barcodes was used in the ligation reaction instead of the standard Illumina single or dual index adapters with 6- or 8-bp barcodes, respectively; and (iv) cfDNA libraries were amplified with Phusion Hot Start Polymerase.
Whole genome libraries were sequenced directly. For targeted libraries, capture was performed using Agilent SureSelect reagents and a custom set of hybridization probes targeting 58 genes (see, e.g., Phallen et al., 2017 Sci Transl Med 9:eaan2415) per the manufacturer's guidelines. The captured library was amplified with Phusion Hot Start Polymerase (NEB). Concentration and quality of captured cfDNA libraries were assessed on the Bioanalyzer 2100 using the DNA1000 Kit (Agilent Technologies). Targeted libraries were sequenced using 100-bp paired-end runs on the Illumina HiSeq 2000/2500 (Illumina).
Analyses of Targeted Sequencing Data from cfDNA
Analyses of targeted NGS data for cfDNA samples was performed as described elsewhere (see, e.g., Phallen et al., 2017 Sci Transl Med 9:eaan2415). Briefly, primary processing was completed using Illumina CASAVA (Consensus Assessment of Sequence and Variation) software (version 1.8), including demultiplexing and masking of dual-index adapter sequences. Sequence reads were aligned against the human reference genome (version hg18 or hg19) using NovoAlign with additional realignment of select regions using the Needleman-Wunsch method (see, e.g., Jones et al., 2015 Sci Transl Med 7:283ra53). The positions of the sequence alterations have not been affected by the different genome builds. Candidate mutations, consisting of point mutations, small insertions, and deletions, were identified using VariantDx (see, e.g., Jones et al., 2015 Sci Transl Med 7:283ra53) (Personal Genome Diagnostics, Baltimore, Md.) across the targeted regions of interest.
To analyze the fragment lengths of cfDNA molecules, each read pair from a cfDNA molecule was required to have a Phred quality score≥30. All duplicate cfDNA fragments, defined as having the same start, end, and index barcode were removed. For each mutation, only fragments for which one or both of the read pairs contained the mutated (or wild-type) base at the given position were included. This analysis was done using the R packages Rsamtools and GenomicAlignments.
For each genomic locus where a somatic mutation was identified, the lengths of fragments containing the mutant allele were compared to the lengths of fragments of the wild-type allele. If more than 100 mutant fragments were identified, Welch's two-sample t-test was used to compare the mean fragment lengths. For loci with fewer than 100 mutant fragments, a bootstrap procedure was implemented. Specifically, replacement N fragments containing the wild-type allele, where N denotes the number of fragments with the mutation, were sampled. For each bootstrap replicate of wild type fragments their median length was computed. The p-value was estimated as the fraction of bootstrap replicates with a median wild-type fragment length as or more extreme than the observed median mutant fragment length.
Analyses of Whole Genome Sequencing Data from cfDNA
Primary processing of whole genome NGS data for cfDNA samples was performed using Illumina CASAVA (Consensus Assessment of Sequence and Variation) software (version 1.8.2), including demultiplexing and masking of dual-index adapter sequences. Sequence reads were aligned against the human reference genome (version hg19) using ELAND.
Read pairs with a MAPQ score below 30 for either read and PCR duplicates were removed. hg19 autosomes were tiled into 26,236 adjacent, non-overlapping 100 kb bins. Regions of low mappability, indicated by the 10% of bins with the lowest coverage, were removed (see, e.g., Fortin et al, 2015 Genome Biol 16:180), as were reads falling in the Duke blacklisted regions (see, e.g., hgdownload.cse.ucsc.edu/goldenpath/hg19/encodeDCC/wgEncodeMapability/). Using this approach, 361 Mb (13%) of the hg19 reference genome was excluded, including centromeric and telomeric regions. Short fragments were defined as having a length between 100 and 150 bp and long fragments were defined has having a length between 151 and 220 bp.
To account for biases in coverage attributable to GC content of the genome, the locally weighted smoother loess with span ¾ was applied to the scatterplot of average fragment GC versus coverage calculated for each 100 kb bin. This loess regression was performed separately for short and long fragments to account for possible differences in GC effects on coverage in plasma by fragment length (see, e.g., Benjamini et al., 2012 Nucleic Acids Res 40:e72). The predictions for short and long coverage explained by GC from the loess model were subtracted, obtaining residuals for short and long that were uncorrelated with GC. The residuals were returned to the original scale by adding back the genome-wide median short and long estimates of coverage. This procedure was repeated for each sample to account for possible differences in GC effects on coverage between samples. To further reduce the feature space and noise, the total GC-adjusted coverage in 5 Mb bins was calculated.
To compare the variability of fragment lengths from healthy subjects to fragments in patients with cancer, the standard deviation of the short to long fragmentation profiles for each individual was calculated. The standard deviations in the two groups were compared by a Wilcoxon rank sum test.
Analyses of Chromosome Arm Copy Number Changes
To develop arm-level statistics for copy number changes, an approach for aneuploidy detection in plasma as described elsewhere (see, e.g., Leary et al., 2012 Sci Transl Med 4:162ra154) was adopted. This approach divides the genome into non-overlapping 50 KB bins for which GC-corrected log 2 read depth was obtained after correction by loess with span ¾. This loess-based correction is comparable to the approach outlined above, but is evaluated on a log 2 scale to increase robustness to outliers in the smaller bins and does not stratify by fragment length. To obtain an arm-specific Z-score for copy number changes, the mean GC-adjusted read depth for each arm (GR) was centered and scaled by the average and standard deviation, respectively, of GR scores obtained from an independent set of 50 healthy samples.
Analyses of Mitochondrial-Aligned Reads from cfDNA
Whole genome sequence reads that initially mapped to the mitochondrial genome were extracted from barn files and realigned to the hg19 reference genome in end-to-end mode with Bowtie2 as described elsewhere (see, e.g., Langmead et al., 2012 Nat Methods 9:357-359). The resulting aligned reads were filtered such that both mates aligned to the mitochondrial genome with MAPQ>=30. The number of fragments mapping to the mitochondrial genome was counted and converted to a percentage of the total number of fragments in the original barn files.
Prediction Model for Cancer Classification
To distinguish healthy from cancer patients using fragmentation profiles, a stochastic gradient boosting model was used (gbm; see, e.g., Friedman et al., 2001 Ann Stat 29:1189-1232; and Friedman et al., 2002 Comput Stat Data An 38:367-378). GC-corrected total and short fragment coverage for all 504 bins were centered and scaled for each sample to have mean 0 and unit standard deviation. Additional features included Z-scores for each of the 39 autosomal arms and mitochondrial representation (log 10-transformed proportion of reads mapped to the mitochondria). To estimate the prediction error of this approach, 10-fold cross-validation was used as described elsewhere (see, e.g., Efron et al., 1997 J Am Stat Assoc 92, 548-560). Feature selection, performed only on the training data in each cross-validation run, removed bins that were highly correlated (correlation>0.9) or had near zero variance. Stochastic gradient boosted machine learning was implemented using the R package gbm package with parameters n.trees=150, interaction.depth=3, shrinkage=0.1, and n.minobsinside=10. To average over the prediction error from the randomization of patients to folds, the 10-fold cross validation procedure was repeated 10 times. Confidence intervals for sensitivity fixed at 98% and 95% specificity were obtained from 2000 bootstrap replicates.
Prediction Model for Tumor Tissue of Origin Classification
For samples correctly classified as cancer patients at 90% specificity (n=174), a separate stochastic gradient boosting model was trained to classify the tissue of origin. To account for the small number of lung samples used for prediction, 18 cfDNA baseline samples from late stage lung cancer patients were included from the monitoring analyses. Performance characteristics of the model were evaluated by 10-fold cross-validation repeated 10 times. This gbm model was trained using the same features as in the cancer classification model. As previously described, features that displayed correlation above 0.9 to each other or had near zero variance were removed within each training dataset during cross-validation. The tissue class probabilities were averaged across the 10 replicates for each patient and the class with the highest probability was taken as the predicted tissue.
Analyses of Nucleosomal DNA from Human Lymphocytes and cfDNA
From the nuclease treated lymphocytes, fragment sizes were analyzed in 5 Mb bins as described for whole genome cfDNA analyses. A genome-wide map of nucleosome positions was constructed from the nuclease treated lymphocyte cell-lines. This approach identified local biases in the coverage of circulating fragments, indicating a region protected from degradation. A “Window positioning score” (WPS) was used to score each base pair in the genome (see, e.g., Snyder et al., 2016 Cell 164:57). Using a sliding window of 60 bp centered around each base, the WPS was calculated as the number of fragments completely spanning the window minus the number of fragments with only one end in the window. Since fragments arising from nucleosomes have a median length of 167 bp, a high WPS indicated a possible nucleosomic position. WPS scores were centered at zero using a running median and smoothed using a Kolmogorov-Zurbenko filter (see, e.g., Zurbenko, The spectral analysis of time series. North-Holland series in statistics and probability; Elsevier, New York, N Y, 1986). For spans of positive WPS between 50 and 450 bp, a nucleosome peak was defined as the set of base pairs with a WPS above the median in that window. The calculation of nucleosome positions for cfDNA from 30 healthy individuals with sequence coverage of 9× was determined in the same manner as for lymphocyte DNA. To ensure that nucleosomes in healthy cfDNA were representative, a consensus track of nucleosomes was defined consisting only of nucleosomes identified in two or more individuals. Median distances between adjacent nucleosomes were calculated from the consensus track.
Monte Carlo Simulation of Detection Sensitivity
A Monte Carlo simulation was used to estimate the probability of detecting a molecule with a tumor-derived alteration. Briefly, 1 million molecules were generated from a multinomial distribution. For a simulation with m alterations, wild-type molecules were simulated with probability p and each of them tumor alterations were simulated with probability (1−p)/m. Next, g*m molecules were sampled randomly with replacement, where g denotes the number of genome equivalents in 1 ml of plasma. If a tumor alteration was sampled s or more times, the sample was classified as cancer-derived. The simulation was repeated 1000 times, estimating the probability that the in silico sample would be correctly classified as cancer by the mean of the cancer indicator. Setting g=2000 and s=5, the number of tumor alterations was varied by powers of 2 from 1 to 256 and the fraction of tumor-derived molecules from 0.0001% to 1%
Statistical Analyses
All statistical analyses were performed using R version 3.4.3. The R packages caret (version 6.0-79) and gbm (version 2.1-4) were used to implement the classification of healthy versus cancer and tissue of origin. Confidence intervals from the model output were obtained with the pROC (version 1.13) R package (see, e.g., Robin et al., 2011 BMC bioinformatics 12:77). Assuming the prevalence of undiagnosed cancer cases in this population is high (1 or 2 cases per 100 healthy), a genomic assay with a specificity of 0.95 and sensitivity of 0.8 would have useful operating characteristics (positive predictive value of 0.25 and negative predictive value near 1). Power calculations suggest that an analysis of more than 200 cancer patients and an approximately equal number of healthy controls, enable an estimation of the sensitivity with a margin of error of 0.06 at the desired specificity of 0.95 or greater.
Data and Code Availability
Sequence data utilized in this study have been deposited at the European Genome-phenome Archive under study accession nos. EGAS00001003611 and EGAS00001002577. Code for analyses is available at github.com/Cancer-Genomics/delfi_scripts.
Results
DELFI allows simultaneous analysis of a large number of abnormalities in cfDNA through genome-wide analysis of fragmentation patterns. The method is based on low coverage whole genome sequencing and analysis of isolated cfDNA. Mapped sequences are analyzed in non-overlapping windows covering the genome. Conceptually, windows may range in size from thousands to millions of bases, resulting in hundreds to thousands of windows in the genome. 5 Mb windows were used for evaluating cfDNA fragmentation patterns as these would provide over 20,000 reads per window even at a limited amount of 1-2× genome coverage. Within each window, the coverage and size distribution of cfDNA fragments was examined. This approach was used to evaluate the variation of genome-wide fragmentation profiles in healthy and cancer populations (Table 1; Appendix A). The genome-wide pattern from an individual can be compared to reference populations to determine if the pattern is likely healthy or cancer-derived. As genome-wide profiles reveal positional differences associated with specific tissues that may be missed in overall fragment size distributions, these patterns may also indicate the tissue source of cfDNA.
The fragmentation size of cfDNA was focused on as it was found that cancer-derived cfDNA molecules may be more variable in size than cfDNA derived from non-cancer cells. cfDNA fragments from targeted regions that were captured and sequenced at high coverage (43,706 total coverage, 8,044 distinct coverage) from patients with breast, colorectal, lung or ovarian cancer (Table 1 (Appendix A), Table 2 (Appendix B), and Table 3 (Appendix C)) were initially examined. Analyses of loci containing 165 tumor-specific alterations from 81 patients (range of 1-7 alterations per patient) revealed an average absolute difference of 6.5 bp (95% CI, 5.4-7.6 bp) between lengths of median mutant and wild-type cfDNA fragments is (
As targeted sequencing only analyzes a limited number of loci, larger-scale genome-wide analyses to detect additional abnormalities in cfDNA fragmentation were investigated. cfDNA was isolated from ˜4 ml of plasma from 8 lung cancer patients with stage I-III disease, as well as from 30 healthy individuals (Table 1 (Appendix A), Table 4 (Appendix D), and Table 5 (Appendix E)). A high efficiency approach was used to convert cfDNA to next generation sequencing libraries and performed whole genome sequencing at ˜9× coverage (Table 4; Appendix D). Overall cfDNA fragment lengths of healthy individuals were larger, with a median fragment size of 167.3 bp, while patients with cancer had median fragment sizes of 163.8 (p<0.01, Welch's t-test) (Table 5; Appendix E). To examine differences in fragment size and coverage in a position dependent manner across the genome, sequenced fragments were mapped to their genomic origin and fragment lengths were evaluated in 504 windows that were 5 Mb in size, covering ˜2.6 Gb of the genome. For each window, the fraction of small cfDNA fragments (100 to 150 bp in length) to larger cfDNA fragments (151 to 220 bp) as well as overall coverage were determined and used to obtain genome-wide fragmentation profiles for each sample.
Healthy individuals had very similar fragmentation profiles throughout the genome (
In contrast to healthy cfDNA, patients with cancer had multiple distinct genomic differences with increases and decreases in fragment sizes at different regions (
To determine whether cfDNA fragment length patterns could be used to distinguish patients with cancer from healthy individuals, genome-wide correlation analyses were performed of the fraction of short to long cfDNA fragments for each sample compared to the median fragment length profile calculated from healthy individuals (
Subsampling analyses of whole genome sequence data was performed at 9× coverage from cfDNA of patients with cancer at ˜2×, ˜1×, ˜0.5×, ˜0.2×, and ˜0.1× genome coverage, and it was determined that altered fragmentation profiles were readily identified even at 0.5× genome coverage (
The fragmentation profiles were examined in the context of known copy number changes in a patient where parallel analyses of tumor tissue were obtained. These analyses demonstrated that altered fragmentation profiles were present in regions of the genome that were copy neutral and that these may be further affected in regions with copy number changes (
These analyses were extended to an independent cohort of cancer patients and healthy individuals. Whole genome sequencing of cfDNA at 1-2× coverage from a total of 208 patients with cancer, including breast (n=54), colorectal (n=27), lung (n=12), ovarian (n=28), pancreatic (n=34), gastric (n=27), or bile duct cancers (n=26), as well as 215 individuals without cancer was performed (Table 1 (Appendix A) and Table 4 (Appendix D)). All cancer patients were treatment naïve and the majority had resectable disease (n=183). After GC adjustment of short and long cfDNA fragment coverage (
To determine if position dependent fragmentation changes can be used to detect individuals with cancer, a gradient tree boosting machine learning model was implemented to examine whether cfDNA can be categorized as having characteristics of a cancer patient or healthy individual and estimated performance characteristics of this approach by ten-fold cross validation repeated ten times (
To assess the contribution of fragment size and coverage, chromosome arm copy number, or mitochondrial mapping to the predictive accuracy of the model, the repeated 10-fold cross-validation procedure was implemented to assess performance characteristics of these features in isolation. It was observed that fragment coverage features alone (AUC=0.94) were nearly identical to the classifier that combined all features (AUC=0.94) (
As fragmentation profiles reveal regional differences in fragmentation that may differ between tissues, a similar machine learning approach was used to examine whether cfDNA patterns could identify the tissue of origin of these tumors. It was found that this approach had a 61% accuracy (95% CI 53%-67%), including 76% for breast, 44% for bile duct, 71% for colorectal, 67% for gastric, 53% for lung, 48% for ovarian, and 50% for pancreatic cancers (
As cancer-specific sequence alterations can be used to identify patients with cancer, it was evaluated whether combining DELFI with this approach could increase the sensitivity of cancer detection (
Overall, genome-wide cfDNA fragmentation profiles are different between cancer patients and healthy individuals. The variability in fragment lengths and coverage in a position dependent manner throughout the genome may explain the apparently contradictory observations of previous analyses of cfDNA at specific loci or of overall fragment sizes. In patients with cancer, heterogeneous fragmentation patterns in cfDNA appear to be a result of mixtures of nucleosomal DNA from both blood and neoplastic cells. These studies provide a method for simultaneous analysis of tens to potentially hundreds of tumor-specific abnormalities from minute amounts of cfDNA, overcoming a limitation that has precluded the possibility of more sensitive analyses of cfDNA. DELFI analyses detected a higher fraction of cancer patients than previous cfDNA analysis methods that have focused on sequence or overall fragmentation sizes (see, e.g., Phallen et al., 2017 Sci Transl Med 9:eaan2415; Cohen et al., 2018 Science 359:926; Newman et al., 2014 Nat Med 20:548, Bettegowda et al., 2014 Sci Transl Med 6:224ra24; Newman et al., 2016 Nat Biotechnol 34:547). As demonstrated in this Example, combining DELFI with analyses of other cfDNA alterations may further increase the sensitivity of detection. As fragmentation profiles appear related to nucleosomal DNA patterns, DELFI may be used for determining the primary source of tumor-derived cfDNA. The identification of the source of circulating tumor DNA in over half of patients analyzed may be further improved by including clinical characteristics, other biomarkers, including methylation changes, and additional diagnostic approaches (Ruibal Morell, 1992 The International journal of biological markers 7:160; Galli et al., 2013 Clinical chemistry and laboratory medicine 51:1369; Sikaris, 2011 Heart, lung & circulation 20:634; Cohen et al., 2018 Science 359:926). Finally, this approach requires only a small amount of whole genome sequencing, without the need for deep sequencing typical of approaches that focus on specific alterations. The performance characteristics and limited amount of sequencing needed for DELFI suggests that our approach could be broadly applied for screening and management of patients with cancer.
These results demonstrate that genome-wide cfDNA fragmentation profiles are different between cancer patients and healthy individuals. As such, cfDNA fragmentation profiles can have important implications for future research and applications of non-invasive approaches for detection of human cancer.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation application of International Application No. PCT/US2019/032914, filed May 17, 2019, which claims the benefit of U.S. Patent Application Ser. No. 62/673,516, filed on May 18, 2018, and claims the benefit of U.S. Patent Application Ser. No. 62/795,900, filed on Jan. 23, 2019. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
This invention was made with U.S. government support under grant No. CA121113 from the National Institutes of Health. The U.S. government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20200131571 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62795900 | Jan 2019 | US | |
| 62673516 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/032914 | May 2019 | US |
| Child | 16730938 | US |
