Jeanne Tie

Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies

Circulating tumor DNA can be used in a variety of clinical and investigational settings across tumor types and stages for screening, diagnosis, and identifying mutations responsible for therapeutic response and drug resistance. Circulating Tumor DNA for Early Detection and Managing Resistance Cancer evolves over time, without any warning signs. Similarly, the development of resistance to therapy generally becomes apparent only when there are obvious signs of tumor growth, at which point the patient may have lost valuable time. Although a repeat biopsy may be able to identify drug-resistant mutations before the tumor has a chance to regrow, it is usually not feasible to do many repeat biopsies. Now, two studies are demonstrating the utility of monitoring the patients’ blood for tumor DNA to detect cancer at the earliest stages of growth or resistance. In one study, Bettegowda and coauthors showed that sampling a patient’s blood may be sufficient to yield information about the tumor’s genetic makeup, even for many early-stage cancers, without a need for an invasive procedure to collect tumor tissue, such as surgery or endoscopy. The authors demonstrated the presence of circulating DNA from many types of tumors that had not yet metastasized or released detectable cells into the circulation. They could detect more than 50% of patients across 14 tumor types at the earliest stages, when these cancers may still be curable, suggesting that a blood draw could be a viable screening approach to detecting most cancers. They also showed that in patients with colorectal cancer, the information derived from circulating tumor DNA could be used to determine the optimal course of treatment and identify resistance to epidermal growth factor receptor (EGFR) blockade. Meanwhile, Misale and colleagues illustrated a way to use this information to overcome treatment resistance. These authors also found that mutations associated with EGFR inhibitor resistance could be detected in the blood of patients with colorectal cancer. In addition, they demonstrated that adding MEK inhibitors, another class of anticancer drugs, can successfully overcome resistance when given in conjunction with the EGFR inhibitors. Thus, the studies from Bettegowda and Misale and their colleagues show the effectiveness of analyzing circulating DNA from a variety of tumors and highlight the potential investigational and clinical applications of this novel technology for early detection, monitoring resistance, and devising treatment plans to overcome resistance. The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer.

It is hypothesized that BRAF mutant cancers represent a discrete subset of metastatic colorectal cancer (CRC) defined by poorer survival. This study investigates whether BRAF mutant CRC is further defined by a distinct pattern of metastatic spread and explores the impact of BRAF mutation and microsatellite instability (MSI) on prognosis in metastatic CRC.

Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer

Detection of circulating tumor DNA in patients with resected stage II colon cancer provides evidence of residual disease. Footprints of persistent cancer Stage II colon cancer, which has spread through the wall of the colon but has not metastasized to the lymph nodes, can present a therapeutic dilemma. On one hand, these tumors can usually be completely removed by surgery, and the majority does not recur even without chemotherapy. On the other hand, it is difficult to determine which of these tumors will recur and to identify patients who would benefit from adjuvant chemotherapy after surgery. Tie et al. show that the presence of circulating tumor DNA in a patient’s blood after surgery is a sign of persistent tumor and a greatly increased risk of relapse, suggesting that this group of patients may require chemotherapy to prevent recurrence. Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencing–based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of 164 patients with negative ctDNA [hazard ratio (HR), 18; 95% confidence interval (CI), 7.9 to 40; P < 0.001]. In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.

Circulating Tumor DNA as an Early Marker of Therapeutic Response in Patients with Metastatic Colorectal Cancer

BACKGROUND Early indicators of treatment response in metastatic colorectal cancer (mCRC) could conceivably be used to optimize treatment. We explored early changes in circulating tumor DNA (ctDNA) levels as a marker of therapeutic efficacy. PATIENTS AND METHODS This prospective study involved 53 mCRC patients receiving standard first-line chemotherapy. Both ctDNA and CEA were assessed in plasma collected before treatment, 3 days after treatment and before cycle 2. Computed tomography (CT) scans were carried out at baseline and 8-10 weeks and were centrally assessed using RECIST v1.1 criteria. Tumors were sequenced using a panel of 15 genes frequently mutated in mCRC to identify candidate mutations for ctDNA analysis. For each patient, one tumor mutation was selected to assess the presence and the level of ctDNA in plasma samples using a digital genomic assay termed Safe-SeqS. RESULTS Candidate mutations for ctDNA analysis were identified in 52 (98.1%) of the tumors. These patient-specific candidate tissue mutations were detectable in the cell-free DNA from the plasma of 48 of these 52 patients (concordance 92.3%). Significant reductions in ctDNA (median 5.7-fold; P < 0.001) levels were observed before cycle 2, which correlated with CT responses at 8-10 weeks (odds ratio = 5.25 with a 10-fold ctDNA reduction; P = 0.016). Major reductions (≥10-fold) versus lesser reductions in ctDNA precycle 2 were associated with a trend for increased progression-free survival (median 14.7 versus 8.1 months; HR = 1.87; P = 0.266). CONCLUSIONS ctDNA is detectable in a high proportion of treatment naïve mCRC patients. Early changes in ctDNA during first-line chemotherapy predict the later radiologic response.

KRAS Mutation Is Associated with Lung Metastasis in Patients with Curatively Resected Colorectal Cancer

Purpose: Oncogene mutations contribute to colorectal cancer development. We searched for differences in oncogene mutation profiles between colorectal cancer metastases from different sites and evaluated these as markers for site of relapse. Experimental Design: One hundred colorectal cancer metastases were screened for mutations in 19 oncogenes, and further 61 metastases and 87 matched primary cancers were analyzed for genes with identified mutations. Mutation prevalence was compared between (a) metastases from liver (n = 65), lung (n = 50), and brain (n = 46), (b) metastases and matched primary cancers, and (c) metastases and an independent cohort of primary cancers (n = 604). Mutations differing between metastasis sites were evaluated as markers for site of relapse in 859 patients from the VICTOR trial. Results: In colorectal cancer metastases, mutations were detected in 4 of 19 oncogenes: BRAF (3.1%), KRAS (48.4%), NRAS (6.2%), and PIK3CA (16.1%). KRAS mutation prevalence was significantly higher in lung (62.0%) and brain (56.5%) than in liver metastases (32.3%; P = 0.003). Mutation status was highly concordant between primary cancer and metastasis from the same individual. Compared with independent primary cancers, KRAS mutations were more common in lung and brain metastases (P < 0.005), but similar in liver metastases. Correspondingly, KRAS mutation was associated with lung relapse (HR = 2.1; 95% CI, 1.2 to 3.5, P = 0.007) but not liver relapse in patients from the VICTOR trial. Conclusions: KRAS mutation seems to be associated with metastasis in specific sites, lung and brain, in colorectal cancer patients. Our data highlight the potential of somatic mutations for informing surveillance strategies. Clin Cancer Res; 17(5); 1122–30. ©2011 AACR.

Detection and localization of surgically resectable cancers with a multi-analyte blood test

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancers rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science, this issue p. 926; see also p. 866 A blood test that combines protein and DNA markers may allow earlier detection of eight common cancer types. Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.

Optimizing targeted therapeutic development: Analysis of a colorectal cancer patient population with the BRAFV600E mutation

BRAFV600E mutations are found in 10% of colorectal cancers (CRCs). The low frequency of this mutation therefore makes it a challenging target for drug development, unless subsets of patients with higher rates of BRAFV600E can be defined. Knowledge of the concordance between primary–metastasis pairs and the impact of BRAFV600E on outcome would also assist in optimal drug development. We selected primary CRCs from 525 patients (stages I–IV) evenly matched for age (<70 and ≥70), gender and tumor location (right, left and rectum), and 81 primary–metastasis pairs. BRAFV600E, KRAS mutation and microsatellite instability (MSI) were determined and correlated with clinical features and patient outcomes. In multivariate analyses, increasing patient age (p = 0.04), female gender (p = 0.0005) and right‐sided tumor location (p < 0.0001) were independently associated with BRAFV600E. The prevalence of BRAFV600E was considerably higher in older (age > 70) females with KRAS wild‐type right‐sided colon cancers (50%) compared to the unselected cohort (10%). BRAFV600E was associated with inferior overall survival in metastatic CRC (HR = 2.02; 95% CI 1.26–3.26), particularly evident in patients treated with chemotherapy, and is independent of MSI status. BRAF status was concordant in all primary tumors and matched metastases (79 wild‐type pairs and two mutant pairs). Clinicopathological and molecular features can identify CRC patients with a higher prevalence of BRAFV600E. Patients with BRAFV600E wild‐type primary tumor do not appear to acquire the mutation in their metastases, and BRAFV600E is associated with poorer outcomes in metastatic patients. Our findings are timely and will help inform the rational development of BRAFV600E inhibitors in CRC.

Multicenter retrospective analysis of metastatic colorectal cancer (CRC) with high-level microsatellite instability (MSI-H)

BACKGROUND The microsatellite instability-high (MSI-H) phenotype, present in 15% of early colorectal cancer (CRC), confers good prognosis. MSI-H metastatic CRC is rare and its impact on outcomes is unknown. We describe survival outcomes and the impact of chemotherapy, metastatectomy, and BRAF V600E mutation status in the largest reported cohort of MSI-H metastatic colorectal cancer (CRC). PATIENTS AND METHODS A retrospective review of 55 MSI-H metastatic CRC patients from two institutions, Royal Melbourne Hospital (Australia) and The University of Texas MD Anderson Cancer Center (United States), was conducted. Statistical analyses utilized Kaplan-Meier method, Log-rank test, and Cox proportional hazards models. RESULTS Median age was 67 years (20-90), 58% had poor differentiation, and 45% had stage IV disease at presentation. Median overall survival (OS) from metastatic disease was 15.4 months. Thirteen patients underwent R0/R1 metastatectomies, with median OS from metastatectomy 33.8 months. Thirty-one patients received first-line systemic chemotherapy for metastatic disease with median OS from the start of chemotherapy 11.5 months. No statistically significant difference in progression-free survival or OS was seen between fluoropyrimidine, oxaliplatin, or irinotecan based chemotherapy. BRAF V600E mutation was present in 14 of 47 patients (30%). BRAF V600E patients demonstrated significantly worse median OS; 10.1 versus 17.3 months, P = 0.03. In multivariate analyses, BRAF V600E mutants had worse OS (HR 4.04; P = 0.005), while patients undergoing metastatectomy (HR 0.11; P = <0.001) and patients who initially presented as stage IV disease had improved OS (HR 0.27; P = 0.003). CONCLUSIONS Patients with MSI-H metastatic CRC do not appear to have improved outcomes. BRAF V600E mutation is a poor prognostic factor in MSI-H metastatic CRC.

PIK3CA and PTEN Gene and Exon Mutation-Specific Clinicopathologic and Molecular Associations in Colorectal Cancer

Purpose: PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal–regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial. Experimental Design: A total of 1,093 stage I–IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12–13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3–8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n = 5,594). Results: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P ≤ 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P ≤ 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status. Conclusion: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAFmut), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRASmut) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes. Clin Cancer Res; 19(12); 3285–96. ©2013 AACR.

Differentiation of tumor recurrence from radiation necrosis in high-grade gliomas using 201Tl-SPECT

MRI is routinely performed to detect recurrence in patients with primary brain tumors, but it may not differentiate recurrent tumor from radiation-induced necrosis reliably. Thallium-201 single-photon emission computed tomography ((201)Tl-SPECT) might be useful in distinguishing between these two clinical entities. In a retrospective study (201)Tl-SPECT studies with corresponding MRI studies in 19 patients with clinical or radiological suspicion of high-grade tumor recurrence were reviewed. The diagnostic accuracies of both modalities were based on the subsequent histology or clinical course where biopsy was not performed. Post-scan histology was available in nine patients (43%) who underwent re-resection. The SPECT result determined management in six patients (29%). Post-SPECT survival was significantly better in patients with negative (201)Tl-SPECT studies compared to patients with positive studies (median survival 15+vs. 6 months) (p=0.04, log-rank test). The sensitivity and specificity of (201)Tl-SPECT in diagnosing tumor recurrence were 83% and 100%, respectively. (201)Tl-SPECT can accurately differentiate tumor recurrence from radiation necrosis in patients with high-grade gliomas and abnormal MRI findings post irradiation. This is reflected in a significantly longer post-scan survival time in patients with a negative (201)Tl-SPECT result.