Nilofer Saba Azad

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.

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade

Predicting responses to immunotherapy Colon cancers with loss-of-function mutations in the mismatch repair (MMR) pathway have favorable responses to PD-1 blockade immunotherapy. In a phase 2 clinical trial, Le et al. showed that treatment success is not just limited to colon cancer (see the Perspective by Goswami and Sharma). They found that a wide range of different cancer types with MMR deficiency also responded to PD-1 blockade. The trial included some patients with pancreatic cancer, which is one of the deadliest forms of cancer. The clinical trial is still ongoing, and around 20% of patients have so far achieved a complete response. MMR deficiency appears to be a biomarker for predicting successful treatment outcomes for several solid tumors and indicates a new therapeutic option for patients harboring MMR-deficient cancers. Science, this issue p. 409; see also p. 358 A pan-cancer biomarker is identified that can predict successful response to cancer immunotherapy in human patients. The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor–1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair–deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair–deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers’ tissue of origin.

The future of epigenetic therapy in solid tumours—lessons from the past

The promise of targeting epigenetic abnormalities for cancer therapy has not been realized for solid tumours, although increasing evidence is demonstrating its worth in haematological malignancies. In fact, true clinical efficacy in haematopoietic-related neoplasms has only become evident at low doses of epigenetic-targeting drugs (namely, inhibitors of histone deacetylase and DNA methyltransferases). Describing data from preclinical studies and early clinical trial results, we hypothesize that in using low-dose epigenetic-modulating agents, tumour cells can be reprogrammed, which overrides any immediate cytotoxic and off-target effect observed at high dose. We suggest that such optimization of drug dosing and scheduling of currently available agents could give these agents a prominent place in cancer management—when used alone or in combination with other therapies. If so, optimal use of these known agents might also pave the way for the introduction of other agents that target the epigenome.

Single-center phase II trial of transarterial chemoembolization with drug-eluting beads for patients with unresectable hepatocellular carcinoma: initial experience in the United States.

Purpose:This prospective phase II pilot study evaluated safety and efficacy of transarterial chemoembolization (TACE) with drug-eluting beads (DEBs) loaded with doxorubicin in patients with unresectable hepatocellular carcinoma (HCC). Methods:Twenty patients with unresectable HCC (75% Childs A, 95% Eastern Cooperative Oncology Group performance status 0 to 1, 60% Barcelona Clinic Liver Cancer C, tumor size 6.9 cm) underwent 34 DEB-TACE sessions. Primary endpoints were tumor response, assessed by contrast-enhanced magnetic resonance imaging at 1 month after treatment, using size (response evaluation criteria in solid tumors [RECIST]), contrast-enhancement (European Association for the Study of the Liver) and apparent diffusion coefficient values, and safety assessed by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE). Secondary endpoints included feasibility, progression-free survival, and overall survival. Results:DEB-TACE was successfully performed in 34 sessions and demonstrated a favorable safety profile. On initial (1 month) postprocedural magnetic resonance imaging, treated lesions had a mean decrease in size of 4% (P = 0.1129). Using RECIST, partial response was achieved in 2 patients (10%), and 18 patients (90%) had stable disease. Treated tumors demonstrated a mean decrease in contrast enhancement of 64% (P < 0.0001). By European Association for the Study of the Liver criteria, 12 patients (60%) had objective tumor response, and 8 (40%) had stable disease. No patients had progression of a treated lesion while undergoing treatment. At 6 months, the disease control rate was 95% using RECIST. Overall survival rates at 1 and 2 years were 65% and 55%, respectively; median overall survival was 26 months. Discussion:DEB-TACE is safe and effective in achieving local tumor control in patients with unresectable HCC.

Incidence and prognostic impact of KRAS and BRAF mutation in patients undergoing liver surgery for colorectal metastases.

Molecular biomarkers offer the potential for refining prognostic determinants in patients undergoing cancer surgery. Among patients with colorectal cancer, KRAS and BRAF are important biomarkers, but their role in patients undergoing surgical therapy for liver metastases is unknown. In this study, the incidence and prognostic significance of KRAS and BRAF mutations were determined in patients undergoing surgical therapy of colorectal liver metastases (CRLM).

A Phase I study of veliparib (ABT-888) in combination with low-dose fractionated whole abdominal radiation therapy in patients with advanced solid malignancies and peritoneal carcinomatosis

Purpose: The combination of low-dose radiotherapy with PARP inhibition has been shown to enhance antitumor efficacy through potentiating DNA damage. We combined low-dose fractionated whole abdominal radiation (LDFWAR) with escalating doses of veliparib (ABT-888), a small-molecule PARP inhibitor, in patients with peritoneal carcinomatosis from advanced solid tumor malignancies. Experimental Design: Patients were treated with veliparib (80–320 mg daily) for a total of 3 cycles. LDFWAR consisted of 21.6 Gy in 36 fractions, 0.6 Gy twice daily on days 1 and 5 for weeks 1–3 of each cycle. Circulating tumor cells (CTC) were collected and evaluated for γ-H2AX. Quality of life (QoL) was assessed using the EORTC-QLQ-C30 questionnaire. Results: Twenty-two patients were treated. Treatment-related grade 3 and 4 toxicities included lymphopenia (68%), anemia (9%), thrombocytopenia (14%), neutropenia (4%), leukopenia (9%), ascites (4%), vomiting (4%), and dyspnea (4%). No objective responses were observed. Disease stabilization (≥24 weeks) was observed in 7 patients (33%). Median progression-free survival (mPFS) was 4.47 months and median overall survival (mOS) was 13.04 months. In the subset of 8 ovarian and fallopian cancers, mPFS was 6.77 months and mOS was 17.54 months compared with mPFS 2.71 months and mOS 13.01 months in others. Patients with ovarian and fallopian cancers had better QoL over time than those with other cancers. An increased percentage of γ-H2AX–positive CTCs was observed in a subset of patients (3/6 with >2 CTCs at baseline). Conclusions: Combined veliparib and LDFWAR is a well-tolerated regimen that resulted in prolonged disease stability for some patients with advanced solid tumors and carcinomatosis, particularly in the ovarian and fallopian cancer subpopulation. Clin Cancer Res; 21(1); 68–76. ©2014 AACR.

Mutational profiling of colorectal cancers with microsatellite instability

Microsatellite instability (MSI) is caused by defective mismatch repair in 15–20% of colorectal cancers (CRCs). Higher mutation loads in tumors with mismatch repair deficiency can predict response to pembrolizumab, an anti-programmed death 1 (PD-1) immune checkpoint inhibitor. We analyzed the mutations in 113 CRCs without MSI (MSS) and 29 CRCs with MSI-High (MSI-H) using the 50-gene AmpliSeq cancer panel. Overall, MSI-H CRCs showed significantly higher mutations than MSS CRCs, including insertion/deletion mutations at repeat regions. MSI-H CRCs showed higher incidences of mutations in the BRAF, PIK3CA, and PTEN genes as well as mutations in the receptor tyrosine kinase families. While the increased mutations in BRAF and PTEN in MSI-H CRCs are well accepted, we also support findings of mutations in the mTOR pathway and receptor tyrosine kinase family genes. MSS CRCs showed higher incidences of mutations in the APC, KRAS and TP53 genes, confirming previous findings. NGS assays may be designed to detect driver mutations for targeted therapeutics and to identify tumors with high mutation loads for potential treatment with immune checkpoint blockade therapies. Further studies may be warranted to elucidate potential targeted therapeutics against mutations in the mTOR pathway and the receptor tyrosine kinase family in MSI-H CRCs as well as the benefit of anti-PD-1 immunotherapy in hypermutated MSS CRCs or other cancers.

Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers.

Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2–20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2–4, 48% when testing for KRAS and NRAS exons 2–4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative measurement of mutant allele frequencies to predict tumor heterogeneity and mutant allele-specific imbalance.

Epigenetic therapy for solid tumors: from bench science to clinical trials.

The cancer epigenome is characterized by global DNA methylation and chromatin changes, such as the hypermethylation of specific CpG island promoters. Epigenetic agents like DNA methyltransferase or histone deacetylase inhibitors induce phenotype changes by reactivation of epigenetically silenced tumor suppressor genes. Despite initial promise in hematologic malignancies, epigenetic agents have not shown significant efficacy as monotherapy against solid tumors. Recent trials showed that epigenetic agents exert favorable modifier effects when combined with chemotherapy, hormonal therapy, or other epigenetic agents. Due to the novel nature of their mechanism, it is important to reconsider the optimal patient selection, drug regimen, study design, and outcome measures when pursuing future trials in order to discover the full potential of this new therapeutic modality.

Intensity-Modulated Radiation Therapy for Rectal Carcinoma Can Reduce Treatment Breaks and Emergency Department Visits

Purpose. To compare the acute toxicities of IMRT to 3D-conformal radiation therapy (3DCRT) in the treatment of rectal cancer. Methods and Materials. Eighty-six patients with rectal cancer preoperatively treated with IMRT (n = 30) and 3DCRT (n = 56) were retrospectively reviewed. Rates of acute toxicity between IMRT and 3DCRT were compared for anorexia, dehydration, diarrhea, nausea, vomiting, weight loss, radiation dermatitis, fatigue, pain, urinary frequency, and blood counts. Fishers exact test and chi-square analysis were applied to detect statistical differences in incidences of toxicity between these two groups of patients. Results. There were fewer hospitalizations and emergency department visits in the group treated with IMRT compared with 3DCRT (P = 0.005) and no treatment breaks with IMRT compared to 20% with 3DCRT (P = 0.0002). Patients treated with IMRT had a significant reduction in grade ≥3 toxicities versus grade ≤2 toxicities (P = 0.016) when compared to 3DCRT. The incidence of grade ≥3 diarrhea was 9% among 3DCRT patients compared to 3% among IMRT patients (P = 0.31). Conclusions. IMRT for rectal cancer can reduce treatment breaks, emergency department visits, hospitalizations, and all grade ≥3 toxicities compared to 3DCRT. Further evaluation and followup is warranted to determine late toxicities and long-term results of IMRT.