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It’s been an exciting year thus far for patients with neuroendocrine tumors (NETs), with the FDA approving a new treatment regimen and more advancements on the horizon, according to James C. Yao, MD, a professor in the Department of Gastrointestinal (GI) Medical Oncology at The University of Texas MD Anderson Cancer Center.

In February, the FDA approved everolimus (Afinitor) as a treatment for patients with progressive, well-differentiated, non-functional NETs of GI or lung origin with unresectable, locally advanced or metastatic disease. The mTOR inhibitor has been approved since 2011 for unresectable or advanced pancreatic NETs.

Meanwhile, the agency is evaluating Lutathera (177Lutetium DOTA-octreotate), a peptide receptor radionuclide therapy (PRRT), for patients with gastroenteropancreatic NETs under its priority review program. Similarly, telotristat etiprate, a small-molecule tryptophan hydroxylase inhibitor, also is being considered under the FDA’s priority review program for carcinoid syndrome in patients with metastatic NETs.

: Please summarize the highlights of neuroendocrine advancements.

 at the 2016 World Congress on Gastrointestinal Cancers in July to discuss developments in the field.

: The last year has been a big year for neuroendocrine tumors. There were 3 positive phase III studies. One included everolimus in lung and GI neuroendocrine tumors; that study has already been published in Lancet in March.

 It received the FDA approval late in February.

Another study that was reported was the NETTER- 1 study.

 This is a prospective randomized phase III study of Lutathera (177-Lu-Dotatate) versus octreotide. The study showed significant benefit in terms of progression-free survival. The publication and the health authority decisions are still pending at this time.

Finally, there was a phase III study of telotristat.

 This is an agent targeting a fairly rare condition, refractory carcinoid syndrome. The study did meet its primary endpoint. It was a fairly short 12-week study that compared, essentially, either placebo plus standard treatment of a somatostatin analog or 2 doses of telotristat at 250 mg and 500 mg.

What can you tell us about next-generation somatostatin analogs, particularly pasireotide?

The study showed a significant decrease in bowel movement count. It is less than, on average, 1 BM per day from their baseline. But nonetheless, it was a positive study. Publication and health authority decisions are still pending.

The current generation of somatostatin analogs mostly target somatostatin receptor-2 and, to some extent, somatostatin receptor-5, and these are octreotide and lanreotide. There are new agents on the horizon that target a broader profile of somatostatin receptors. These include pasireotide, which targets somatostatin receptors 1,2,3, and 5. Its utility in refractory carcinoid syndrome was also explored in a phase III study, but because of the study design and the limited number of patients, I don’t think this study really fully answered the question.

Nonetheless, in that study there were some hints that it may have some efficacy in terms of further delaying disease progression compared with regular somatostatin analogs. Whether those will be explored in future trials remains an unanswered question at this time.

Telotristat is a little bit different. It doesn’t target the somatostatin receptor, but specifically serotonin. So I really think this is going to be limited to control refractory diarrhea.

What is the potential for PRRTs in development for NETs?

There are 2 components for carcinoid syndrome, and that’s flushing and diarrhea, with serotonin being the one that’s responsible for the diarrhea component. So that’s where telotrisat will likely have its role—in patients with refractory diarrhea from neuroendocrine tumors.

Peptide receptor radiotherapy is using this somatostatin receptor in a different way. You can use the ligand in the peptide, in this case to deliver a radioisotope to the tumor.

Because some of the isotopes that are being used include indium, yttrium, and lutetium, the agent probably best studied is the lutetium label compound. That’s what the NETTER-1 study was about. Previously, there have been a lot of single-arm case series or potentially single-arm studies showing activity. NETTER-1 showed a randomized control study for midgut neuroendocrine tumors and the utility of PRRT in this group of tumors in delaying tumor growth.

There are some other compounds out there, including yttrium-labelled dotatoc, and also I think a compound that looks at a somatostatin receptor antagonist that may better deliver isotopes. So clinical trials with these other agents are also ongoing.

One limitation for peptide-receptor radiotherapy is that, even though it works, you cannot keep repeating the treatment because you will get trouble in terms of bone marrow toxicity, secondary malignancies, and renal issues.

How do you think treatment of neuroendocrine tumors will be different in 5 or 10 years?

There are also other ways of targeting somatostatin receptor that are being explored. For example, there is a study that will look at an agonist peptide targeting somatostatin receptor-2, but instead of being tied to a radioisotope, it is tied to a DM1, which is a peptide—drug conjugate. [The goal is] to use the peptide to deliver a toxin rather than deliver a radioisotope. There are many of these options out there.

That’s a loaded question in some ways. Given the time horizon for drug development, what can be affected in terms of standard-of-care treatment 5 years from now are mostly the ongoing phase II studies that we’re talking about. For example, there’s also a randomized phase II trial of pazopanib versus placebo that has just finished accrual, so we’re waiting for those studies. Further investigation of VEGF inhibitors in neuroendocrine tumors outside the pancreas is already approved. Sunitinib is approved for pancreatic tumors. Whether VEGF inhibitors have a role outside the pancreas hopefully be answered in that sort of time frame.

Yao JC, Fazio N, Singh S, et al. Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or gastrointestinal tract (RADIANT-4): a randomised, placebocontrolled, phase 3 study. 

Strosberg J, Wolin E, Chasen B, et al. NETTER-1 phase III: efficacy and safety results in patients with midgut neuroendocrine tumors treated with 177Lu-dotatate. 

. 2016;27(suppl 2):ii118- ii128. Abstract O-009.

Kulke MH, Hörsch D, Caplin M, et al. Telotristat etiprate is effective in treating patients with carcinoid syndrome that is inadequately controlled by somatostatin analog therapy (the phase 3 TELESTAR clinical trial). Presented at: 2015 European Cancer Congress; September 25-29; Vienna, Austria. Abstract 37LBA.

Articles

James C. Yao, MD, discusses developments in the field of neuroendocrine tumors.

Excitement Continues to Build for Advances in Neuroendocrine Tumor Field

Ovarian cancer, still known as the “silent killer,” continues to present a significant clinical challenge despite recent therapeutic advances that include several biologically targeted drugs. The universally applied treatment paradigm is clearly unsuited to the histological, clinical, and molecular heterogeneity of this disease.

Genome sequencing studies have uncovered an array of distinct genomic drivers underlying various ovarian cancer subtypes. If researchers can capitalize on these discoveries, it may offer a path to more individualized and effective treatment options.

With more than 14,000 deaths expected in the United States in 2016, ovarian cancer remains the most lethal gynecologic malignancy and weighs in at No. 6 among the leading causes of cancer-related mortality. The ambiguity of early symptoms and lack of effective screening techniques result in more than 70% of patients being diagnosed in the advanced stages of disease.

The current standard of care has remained unchanged for many years and involves surgery and platinum- and taxane-based chemotherapy. Although the vast majority of patients will initially respond, disease recurrence is almost universal. Several targeted therapies have been developed for the treatment of recurrent disease. The realization that angiogenesis, the formation of new blood vessels from preexisting ones, is a hallmark of ovarian cancer led researchers to investigate the potential of bevacizumab (Avastin) and other antiangiogenic therapies.

The addition of bevacizumab to standard chemotherapy in platinum-resistant patients significantly improved progression-free survival in the phase III AURELIA trial and sealed its approval as second-line treatment for ovarian cancer in 2014.

Meanwhile, as in breast cancer, germline mutations in the breast cancer susceptibility genes 

 have also been discovered in ovarian cancer. These mutations impair the homologous recombination (HR) pathway used to fix DNA damage. Inhibitors of the poly(ADP)-ribose polymerase (PARP) enzyme, which plays a role in an alternative DNA repair pathway, have been developed to capitalize on the inability of ovarian cancer cells with defective HR pathways to repair damaged DNA.

 In December 2014, olaparib (Lynparza) became the first PARP inhibitor to be granted approval by the FDA for the treatment of patients with 

 mutation-positive ovarian cancer following the demonstration of response rates of more than 30% in a single-arm, phase II trial.

The addition of olaparib and bevacizumab to the ovarian cancer armamentarium marked the first new therapies approved for the treatment of ovarian cancer in almost a decade. Yet, only short-term survival gains have been achieved and long-term survival has remained stubbornly low, with 5-year rates of just 45%.

Broadly, ovarian tumors are classed according to the anatomic structures from which they are presumed to originate. Between 90% and 95% of cases are epithelial ovarian carcinomas, while nonepithelial tumors such as sex cord-stromal tumors and malignant ovarian germ cell tumors are considerably rarer.

Epithelial ovarian cancers can be further divided into several different histological subtypes. Most common are serous tumors, responsible for at least 65% of epithelial ovarian cancer cases, followed by endometrioid, mucinous, and clear cell. Historically, it was thought that epithelial ovarian carcinomas were all derived from the ovarian surface epithelium but more recent studies have defined a new model of carcinogenesis that divides epithelial ovarian cancers into 2 categories, designated as type I and type II.

Type I tumors consist of low-grade serous, mucinous, endometrioid, and clear cell tumors, for which precursor lesions within the ovary have been clearly described. Type II tumors include predominantly high-grade serous cancers (HGSCs) as well as carcinosarcomas and high-grade endometrioid and poorly differentiated ovarian cancers.

Precursor ovarian lesions have not been well described for this group of tumors, and it is now thought that they can arise from the epithelium of both the ovarian surface and the fallopian tube. In addition to their histological differences, ovarian cancers also display distinct clinical behaviors.

Type II tumors are typically much more aggressive and more often found at an advanced stage, but they respond well to chemotherapy, with an average response rate of 70%. Type I tumors are more indolent, and are frequently identified earlier on in the course of disease, but are significantly more chemoresistant with a response rate of less than 30%.

Genomic Chaos in High-Grade Serous Subtype

Despite these histological and clinical differences, ovarian cancers are still tarred with the same brush when it comes to treatment. However, genome sequencing studies are beginning to reveal unique molecular underpinnings that might offer a path to more individualized treatments.

Since HGSC represent the vast majority of ovarian cancer cases, these have largely been the focus of molecular studies. In 2011, The Cancer Genome Atlas (TCGA) Research Project published a comprehensive analysis of 489 HGSC samples, which included whole-exome sequencing on more than 300 tumor samples and matched normal pairs.

The study involved hundreds of researchers from more than 80 different institutions and established that the most common molecular alteration in HGSC was a mutation in the guardian of the genome, the 

 gene, which was present in 96% of all samples. The pivotal role of 

 in maintaining genome stability, through its effects on cell cycle progression, apoptosis, DNA repair, and other important cellular functions, means that a mutation in this gene can generate genomic chaos.

. The TCGA identified just 8 other recurrently mutated genes and the frequency of mutations for individual genes were low. Germline mutations in

 were observed in 9% of patients, with somatic mutations in a further 3%, while 

Notably, large structural changes to the genome, in which genes are accidentally deleted or duplicated as a result, appear to be a major driver of ovarian cancer development, making it a so-called C-class tumor (a tumor driven by copy number alterations).

The most commonly amplified genes, each found in more than 20% of cases, were 

 encodes the cyclin E1 gene, with a major role in cell-cycle regulation. 

 are transcription factors that regulate the activity of numerous cellular signaling pathways involved in a variety of cancer hallmark processes. The most frequent losses were in the 

It has been suggested that cancers that share the molecular features of BRCA-mutant tumors may respond similarly to PARP inhibitors and other drugs designed to take advantage of defective HR pathways—a property dubbed 

Another significant finding of the TCGA Research Project’s study of ovarian cancer was that up to half of all cases of HGSC display HR defects. Although these are predominantly caused by germline and somatic mutations as well as epigenetic alterations in the 

 genes, other types of BRCAness also existed.

These included mutations in other components of the HR pathway such as in Fanconi anemia genes, principally 

In addition to HR pathway alterations, genes in other DNA repair pathways such as the nucleotide excision repair and mismatch repair pathways were mutated or deleted in a further 8% of HGSC samples. Interestingly, a recent study that used genome sequencing to analyze the possible mechanisms of resistance and recurrence in ovarian cancer found that restoration of the HR pathway was a common means of achieving chemoresistance— for example, through the reversion to wild-type BRCA1/2 genes or the removal of 

The other half of HGSC samples that do not display HR deficiency remain poorly characterized, although up to one-fifth of these tumors have a focal amplification of CCNE1. According to the study on resistance mechanisms, CCNE1 amplification is the most frequent cause of primary resistance to therapy, which could in part explain why patients with BRCA1/2 mutations have a more favorable response to chemotherapy.

The TCGA also performed gene expression profiling on HGSC samples to examine how sets of genes are expressed in ovarian cancer and what implications this could have for therapy or what it might mean for patient survival.

Researchers identified 4 transcriptional subtypes within ovarian cancers. An “immunoreactive” group was characterized by expression of the T-cell chemokine ligands CXCL11 and CXCL10 and the receptor CXCR3. So-called “differentiated” tumors were associated with an increased expression of MUC16 and MUC1 and expression of the secretory fallopian tube marker SLP1.

A “proliferative” subgroup displayed high expression of genes involved in proliferation, including MCM2 and PCNA, while those classed as “mesenchymal” had markers that suggested a high level of stromal components, such as fibroblasts, indicated by the expression of the 

The different gene expression groups did not translate into variations in patient survival. However, the researchers did identify a 193-gene expression signature that was predictive of overall survival.

Although other types of ovarian cancer have not been as well characterized genomically as HGSC, what has emerged from genome sequencing studies is that the subtypes of ovarian cancer are as distinct in their genome as in their histology and clinical behavior. Since type I ovarian cancers are significantly less responsive to chemotherapy than type II tumors, therapies designed to target the underlying genomic drivers may be particularly important.

A key driver of clear cell carcinoma is the 

 gene, which encodes a member of SWI/ SNF family of proteins that acts as a chromatin remodeling complex. 

 is mutated or deleted in up to half of clear cell ovarian cancers. Clear cell cancers are also characterized by mutations in the 

 gene in 20% to 33% of cases, driving activation of the phosphatidylinositol- 3-kinase (PI3K)/AKT pathway. Other features of this subtype are overexpression of the proinflammatory cytokine interleukin-6 (IL-6) and of hypoxia inducible factor 1 alpha (HIF1α).

The activity of HIF1α supports the use of antiangiogenic therapy in ovarian cancer since one of the targets of HIF1α is vascular endothelial growth factor (VEGF). Gene expression profiling of clear cell carcinomas has further reinforced this idea, demonstrating activation of pathways involved in the response to hypoxia and oxidative stress.

Mucinous adenocarcinomas and low-grade serous carcinomas are characterized by aberrant activation of the mitogen-activated protein kinase (MAPK) pathway via activating mutations in the 

 genes. These mutations are found in up to 33% of low-grade serous tumors and between 50% and 75% of mucinous tumors.

The human epidermal growth factor receptor 2 (HER2) protein is also significantly overexpressed in around 20% of mucinous ovarian cancers. Similar to clear cell carcinomas, the ARID1A gene is also recurrently mutated in around 30% of endometrioid tumors. Endometrioid ovarian cancers are also distinguished by mutations in CTNNB1, particularly in low-grade tumors, a gene which encodes the beta-catenin protein that serves as the central regulator of the Wnt/beta-catenin pathway that plays a significant role in numerous cellular processes.

 mutations are present in around a fifth of endometrioid tumors, implicating the PI3K/AKT pathway as a significant regulator of this type of ovarian cancer. 

 mutations are also present in up to 24% of endometrioid tumors.

A greater understanding of the genomic basis of ovarian cancer and the differences between the various subtypes of this disease is steadily beginning to inform the development of targeted therapies. Given the importance of DNA repair defects in HGSC, drugs targeting these pathways have remained a central focus of research efforts. Beyond the approval of olaparib and the continued development of other PARP inhibitors, the CHEK1 and CHEK2 proteins also have emerged as promising targets.

Forming part of the DNA damage signaling response (DDR) pathway, CHEK1 and CHEK2 are activated by the ATR and ATM kinases in response to breaks in a cell’s DNA and subsequently regulate a number of cellular processes, including checkpoint activation, cell cycle arrest, and initiation of DNA repair.

Various CHEK1 inhibitors have been developed, including GDC-0425, MK-8776, and AZD7762. Unfortunately, few clinical responses have been observed in patients treated with single-agent CHEK1 inhibitor therapy. Only Eli Lilly’s LY2606368 continues to be evaluated in clinical trials (NCT02203513).

 is so highly mutated in HGSC also makes this an extremely promising therapeutic target. However, targeting p53 has proved to be a significant clinical challenge. Based on conventional drug development processes, the p53 protein, which is a tumor suppressor and transcription factor, is not an ideal target. Nevertheless, some progress has been made in developing drugs that reactivate the mutant protein. APR-246, an analog of PRIMA-1 (p53 reactivation and induction of massive apoptosis) is being evaluated in a phase I/II clinical trial in patients with recurrent high-grade serous ovarian cancer (NCT02098343).

The PI3K/AKT and MAPK pathways have been shown to be dysregulated through mutations in the genes encoding their component proteins, including PIK3CA, BRAF, and PTEN. Small-molecule inhibitors of some of these proteins are already available and are being evaluated in ovarian cancer, including BRAF and MEK inhibitors and PI3K and mammalian target of rapamycin (mTOR) inhibitors.

Jane de Lartigue, PhD, is a freelance medical writer and editor based in New Haven, Connecticut.

Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. 

Coward JIG, Middleton K, Murphy F. New perspectives on targeted therapy in ovarian cancer. 

Fadare O, Khabele D. Molecular profiling of epithelial ovarian cancer. My Cancer Genome. https://goo.gl/4kjiJM. Updated January 26, 2016. Accessed August 5, 2016.

Krzystyniak J, Ceppi L, Dizon DS, Birrer MJ. Epithelial ovarian cancer: the molecular genetics of epithelial ovarian cancer. 

Mittempergher L. Genomic characterization of high-grade serous ovarian cancer: dissecting its molecular heterogeneity as a road towards effective therapeutic strategies. 

Patch AM, Christie EL, Etemadmoghadam D, et al. Whole-genome characterization of chemoresistant ovarian cancer [published correction appears in 

. 2015;521(7578):398]. Nature. 2015;521(7553):489-493.

Verschraegen C, Lounsbury K, Howe A, Greenblatt M. Therapeutic implications for ovarian cancer emerging from the tumor Cancer Genome Atlas. 

Wei W, Dizon D, Vathipadiekal V, Birrer MJ. Ovarian cancer: genomic analysis. 

Genome sequencing studies have uncovered an
array of distinct genomic drivers underlying various
ovarian cancer subtypes. If researchers can capitalize
on these discoveries, it may offer a path to more
individualized and effective treatment options.

Genome Sequencing Is Mapping a Path to Personalized Treatment for Ovarian Cancer

CRISPR is a revolutionary new gene-editing technology that has taken the medical research community by storm.

Although researchers have been exploring gene editing for more than 40 years, scientists say the CRISPR technology offers game-changing methods for anticancer research as well as a host of other applications. CRISPR, which is an acronym for clustered regularly interspaced short palindromic repeats, is also referred to as the CRISPR-Cas9 system. It consists of repeating sequences of genetic code, interrupted by spaces or spacer sequences. The technology allows for rapid and precise editing or alteration of a cell or organism’s genome, inactivating or repairing genes as needed by changing the DNA sequences.

The system is “borrowed” from a biological phenomenon in bacteria, whereby bacteria can defend themselves against invading viruses using an enzyme called Cas9 and a piece of RNA called a guide RNA, or gRNA. The gRNA finds the target sequence and binds to it.

The Cas9 enzyme, which has been compared to a pair of extremely sharp scissors, follows the gRNA and makes a cut across the two DNA strands. Once the cut is made, the cell tries to repair itself, but it is at this point that scientists can step in and alter the sequence of the gene that can, in turn, change the function of the encoded protein.

The technology has quickly become the “newest and most widely used gene-editing technique,” according to a National Institutes of Health (NIH) committee that recently examined a number of developments in the broader field.

 CRISPR “has rapidly led to breakthroughs in the editing of genomes of many organisms, including plants, nematodes, flies, fish, monkeys, and human cells,” the panel said.

According to researchers, CRISPR and functional genomics have changed the cancer research landscape. Researchers have been working on genome engineering since the 1970s but the techniques were inefficient or too difficult to use. “The reason [CRISPR] is exciting is that we have not had the ability to so precisely and so simply manipulate the genomes of cells, mammalian cells in particular, before this time,” Tyler Jacks, PhD, said in an interview with 

. “It’s remarkably powerful and beautiful in its simplicity. It’s just such a simple, straightforward system to use, and that is why it’s so popular— because an eighth-grader could do it.”

Jacks is a Daniel K. Ludwig Scholar and investigtor at the Ludwig Center at MIT, and his laboratory has pioneered the use of CRISPR to construct in vivo models of human cancers. “We can think of older genome engineering technologies as similar to having to rewire your computer each time you want to run a new piece of software, whereas the CRISPR technology is like software for the genome. We can program it easily, using these little bits of RNA,” Jennifer Doudna, PhD, explained in a TED Talk late last year.

Doudna is a professor of Chemistry and of Molecular and Cell Biology at the Department of Chemistry and Chemical Engineering of the University of California, Berkeley, and also is a pioneer in CRISPR technology.

CRISPR was named as one of the Top 10 breakthrough technologies in 2014 and 2016 by MIT Technology Review, and it won the Science Magazine’s Breakthrough Award in 2015, after being runner up in 2012 and 2013.

“It’s swept the field, which is pretty remarkable because the first breakthrough paper was only published in the summer of 2012,” Jacks said. “The last I counted, there had been something like 5000 papers since then, using the technology, which is pretty phenomenal.”

The ability to make such precise alterations may have been exciting on its own; however, CRISPR has an even greater advantage: more than one gene can be altered at the same time. Considering that most genetic disorders, including cancer, are caused by more than one damaged gene, the ability for multiple manipulations increases CRISPR’s potential utility.

Tumors require at least 3 to 6 mutations for them to become malignant, starting with a mutation that allows them to activate and sustain continuous proliferation.

 The potential of CRISPR to help researchers target several mutations at once speeds up the possibility of finding treatments for individual cancers.

The CRISPR-Cas9 technology is still in the research stages and has not yet reached clinical use, but work is moving ahead at a rapid pace. In June, a proposal to use CRISP technology for the first-in-human clinical trial was approved by an NIH panel.

 Plans for the complex therapy call for taking NY-ESO-1, a peptide-based form of adoptive immunotherapy already in clinical trials, and disrupting the activity of the 

 gene that encodes PD-1 as well as the TRAC and TRBC genes, which control T-cell receptor (TCR) alpha and beta, respectively.

The FDA must approve the protocol for the trial to proceed. The research team, led by investigators from the University of Pennsylvania, would seek to enroll 18 patients with multiple myeloma, sarcoma subtypes, or melanoma who have progressive, refractory, or metastatic disease.

Meanwhile, researchers in China already have gained approval to proceed with a clinical trial in which 10 patients with non—small cell lung cancer would receive a therapy in which CRISPR technology is used to knock out the PD-1 gene.

Scientists in the Cancer Program at the Broad Institute of MIT and Harvard are using CRISPR in their research to try to understand what genes are essential for cancer cell growth. “You can imagine that if you have a cancer cell line and you use CRISPR to eliminate the function of a gene and, as a result, the cells die, that what you can conclude is that these cancer cells are normally dependent in some way on the function of that gene or that protein,” Brett Tomson, PhD, explained in an interview. “A really great thing about this technology is that you can use it at an enhanced scale to assess thousands of different genes and cancer cell lines to reveal the dependencies of every type of human cancer.”

These types of experiments give researchers large data sets that allow them to compare and contrast across different contexts, define what is critical for cancer cell survival, and try to exploit these “Achilles heels” of cancer to combat the disease in patients.

“Additionally, the Broad’s Cancer Program is using CRISPR with cellular and animal models to create and recapitulate critical gene mutations clinically observed in cancer patients, which will allow a better understanding of how they cause cancer,” said Tomson, who is manager of Broad Institute of MIT and Harvard’s Cancer Outreach Solutions.

Researchers first worked with CRISPR researching single-gene disorders, such as muscular dystrophy and sickle cell anemia. In 2015, researchers from Duke University reported successfully treating muscular dystrophy in an adult mouse model.

Doudna believes that there will be a clinical application of the technology within the next 10 years. “I think that it’s likely that we will see clinical trials and possibly even approved therapies within that time, which is a very exciting thing to think about,” she said in her TED talk.

The market potential for CRISPR technology is huge, with substantial predicted growth in the industry. According to one analysis, the global genome editing market is expected to reach more than $3.5 billion by 2019.9

“There are several companies that have been started based on CRISPR technology; I know of at least three and I am sure that there are many more,” Jacks explained. “And, while the companies do have cancer programs, they are not focused on cancer alone in looking at short-term clinical applications. Instead, they are focusing on genetic diseases that could be corrected with CRISPR technology, such as sickle cell disease or cystic fibrosis.

“The likelihood is that many, if not all, pharmaceutical companies who are interested in cancer will be using this technology in the future,” Jacks said. “In cancer, you often know what the defective gene is. CRISPR could help you correct that gene defect more efficiently and effectively than existing technologies. There are many other diseases that, in theory, could be corrected by CRISPR, and I think that those companies are focused in these areas.”

As with many scientific advances, CRISPR is not without some ethical controversies. “Any technology that involves manipulation of the DNA has certain risks,” Jacks said. “I think it’s too early to say how risky this technology is.”

The safety issues involve the off-targeting of genes, which means that the process could affect genes other than the ones being targeted.

Several researchers are investigating this aspect of CRISPR technology. Currently, use of CRISPR-Cas9 in human cells has shown a high frequency of off-target effects, but fewer in cells from mice and zebrafish.

 In 2015, Chinese researchers attempted to modify the gene responsible for beta-thalassemia in nonviable embryos.

This raised concern in the scientific community.

 In July 2016, Haeussler et al published a study that evaluated CRIPSR-Cas9 predictions to determine off-target and on-target scoring.12 They established a new website (

) for researchers that predicts off-targets, and a program that helps design, evaluate, and clone guide sequences for the CRISPR/Cas9 system.

The ethical aspect involves the potential for CRIPSR to repair defective genes in an embryo or in the germline of an individual, or germ-line manipulation. Jacks pointed out that in some situations, it may be very reasonable to consider this approach. For example, in a family with 

 mutation that predisposes to breast and ovarian cancer, why would the technology not be used to fix this gene if it were feasible to do so? “That seems quite reasonable and I think that most people are comfortable with that notion,” he said. “The concern, however, is that once you open the door to the germline genetic alteration, what goes through that door? When do you stop?”

The NIH issued a statement last year, outlining its stance on genomic editing in human embryos: “[The] NIH will not fund any use of gene-editing technologies in human embryos. The concept of altering the human germline in embryos for clinical purposes has been debated over many years from many different perspectives, and has been viewed almost universally as a line that should not be crossed.”

The NIH will continue to support research, but “in a fashion that reflects well-established scientific and ethical principles.”13 Patent Fight Over CRISPR The significance of CRISPR became apparent in other ways as several academic investigators made important discoveries at around the same time. Doudna and Emmanuelle Charpentier, PhD, first reported in 2012 that they had been able to cut isolated DNA strands using CRISPR technology.14 They filed a patent in 2013.

However, Feng Zhang, PhD, of the Broad Institute and the Massachusetts Institute of Technology, filed for a patent in 2013, which was granted in 2014. He was the first to engineer the CRISPR system for use in human cells, demonstrating targeted genome cleavage in human and mouse cells. “There are some well-publicized disputes involving a few universities and others around the world who have filed patents roughly simultaneously,” Jacks said. “I think the courts will have their hands full sorting out who did what when, and who deserves the credit.”

Ironing out who should be granted patents is vital given the enormous market potential of CRISPR. Some companies have raised money in developing gene therapy using the technology.

Current research efforts using CRISPR complement other areas of longtime wide scientific interest, such as understanding how to block the function of genes that are critically involved in many cancer types.

As one example, the RAS family of proteins, which includes the gene 

, are mutated in many human cancers, including approximately 35% of lung cancers, 45% of colorectal cancers, and 95% of pancreatic cancers.15

 “We’re actually measuring the impact of every gene on growth of KRAS-mutant cancer cell lines,” Tina Yuan, PhD, a senior staff scientist at the Broad Institute said in an interview with with OncologyLive.

“By knocking down every gene in the genome in many different KRAS-mutant cancer cell lines, we can identify which genes will kill KRAS-mutant cancers, and those genes would thus represent promising candidates for drug development,” she said.

In the longer term, if researchers could deliver the CRIPSR components to a cancer cell in a patient—for example, a cancer cell with a KRAS mutation&mdash;perhaps the RAS gene could be inactivated by CRISPR.

“You could do an in vivo gene deletion as a possible cancer therapy,” Jacks said. “We’re not there yet, but there is the prospect of doing that in the future.”

Unfortunately, patients with cancer often develop resistance to certain targeted therapies. This relapse occurs because their cancer cells find a way to evade sensitivity to a treatment, often by acquiring specific key gene mutations.

“There’s a big research effort around the world in understanding that kind of acquired drug resistance. Here at the Broad Cancer program, we are conducting research utilizing CRISPR technology to very precisely determine which genes and mutations are influencing the development of resistance to clinical therapies,” Tomson explained.

“That, in turn, can allow us to think about ways to stop cancer cells from becoming drug resistant in the first place. If many cancer cells in a given cancer type are becoming resistant to a therapy due to the modification of one gene or pathway, maybe it’s worth thinking about a combination approach to target that resistance pathway right at the onset of treatment so that patients’ long-term responses to therapy could be improved.”

Scientists know that this is just the beginning of CRISPR technology. One of the first Cas9-like enzymes has been identified, but many labs are working to isolate other versions of Cas9, which can modify DNA in different ways for different applications. For example, CRISPR-Cpf1, which differs from CRISPR-Cas9, is expected to be even more exact in its ability to alter genes.16 “This conversation could be different in a year or two because as this technology continues to evolve that will completely change the realities of what we can accomplish in the lab using CRISPR. Clearly, CRISPR technology has an exciting future,” Tomson said.

National Academies of Sciences, Engineering, and Medicine. Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington, DC: The National Academies Press. 2016. doi:10.17226/23405.

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Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. 

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. 2016;7:266. doi:10.4172/2155-9627.1000266.

Liang P, Xu, Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. 

Haeussler M, Schönig K, Eckert H, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. 

 2016;17(1):148. doi:10.1186/ s13059-016-1012-2.

National Institutes of Health. Statement on NIH funding of research using gene-editing technologies in human embryos. https://goo.gl/ yhoc86. Published April 29, 2015. Accessed August 9, 2016.

Ledford H. Battle over CRISPR patent heats up. 

National Cancer Institute. The RAS initiative: the problem. http://goo. gl/OtxzW4. Updated September 24, 2014. Accessed August 3, 2016.

In CRISPR genome editing, Cpf1, proved its specificity and produced a mutant mouse. 

. http://goo.gl/x5z1tP. Published June 6, 2016. Accessed August 3, 2016.

Although researchers have been exploring gene editing for more than 40 years, scientists say the CRISPR technology offers game-changing methods for anticancer research as well as a host of other applications.

Why CRISPR Gene-Editing Technology Is Captivating Research Field

: What are the main limitations of the current treatment paradigm for ovarian cancer?

What are the major somatic molecular drivers of ovarian cancer?

What have genome-wide studies taught us about ovarian cancer?

Has this been translated to any therapeutic advances in the clinic?

What are the most significant unanswered questions or hurdles to our understanding of the genomic background of ovarian cancer?

Christina M. Annunziata, MD, PhD, has been instrumental in the genomic profiling of ovarian cancers and in elucidating the molecular pathways that are central to this disease. She is principal investigator and clinical director as well as head of the Translational Genomics Section at the Women’s Malignancies Branch of the National Cancer Institute in Bethesda, Maryland.

: The current standard of care for women with newly diagnosed ovarian cancer is surgery and chemotherapy with platinum and taxane. This approach is applied to all women regardless of age, family history, histology, or other molecular diagnoses. There are no tools to predict who will remain in remission and who will relapse soon after completing therapy.

There are currently no true “drivers” of ovarian cancer that have been identified. Several consistent genetic aberrations have been associated with particular types of ovarian cancer, such as p53 mutations or deletions in high-grade serous cancer, ARID1A loss in clear cell or endometrioid cancer, KRAS or BRAF mutations in low-grade serous cancer. These may all be instrumental in the development of cancers, but are not yet proven to be independent “drivers” of cancer growth and spread.

Genome-wide studies have taught us important aspects of the biology of ovarian cancer. For example, we have learned that the high-grade serous type of ovarian cancer has a lot of genomic instability, and this causes cells to have high or low numbers of particular areas of the chromosomes. This could include regions of genes that become overexpressed, such as cyclin E. We also learned that certain types of cancers have hallmark mutations.

These findings have resulted in the development of focused clinical trials that intend to target these genetic aberrations. For example, PARP inhibitors are being tested in women with high-grade serous ovarian cancer in attempt to selectively kill the cancer cells that have high genomic instability. Diagnostic tests are under development to identify the cancers with high genomic instability and are incorporated into some of these clinical trials.

The genomic instability of ovarian cancer presents several hurdles to both understanding the genomic background and treating the disease. It appears that the cancer is not only different between individual women, but can be different depending on the anatomical site of the tumor; that is, tumor growing in the ovary, versus tumor spread to the lymph node, versus tumor spread to the liver, etc.

Moreover, the cells on the outside of a tumor can be different from those on the inside of the same tumor. And, the composition of the tumors may change over time, especially after exposure to different chemotherapies. This presents a challenge in both understanding what genomic alterations are driving the cancer, what treatments would best treat the cancer, and when to use the treatments.

Christina M. Annunziata, MD, PhD, has been instrumental in the genomic profiling of ovarian cancers and in elucidating the molecular pathways that are central to this disease.

"True" Genomic Drivers of Ovarian Cancer Not Yet Identified, NCI Researcher Says

The armamentarium for advanced melanoma was sparse in March 2011, when ipilimumab (Yervoy) became the first drug in 13 years to earn approval FDA approval for the disease. Since then, the armamentarium has rapidly expanded.

“In the last 5 years, there have been FDA approvals for 12 new agents and combination therapies for patients with advanced melanoma,” said Keith T. Flaherty, MD, who moderated an 

 Peer Exchange panel titled “Treatment of Advanced Melanoma and Basal Cell Carcinoma.”

Novel melanoma treatments fall broadly into 2 categories: agents that target MAPK pathway signaling or agents that target immune system proteins. Several more melanoma treatments are poised to emerge from the pipeline. Data on the optimal way to incorporate these novel drugs into practice lag far behind the breakneck pace of approvals.

What sets melanoma apart from other malignancies is its high mutational burden and immunogenicity. Jeffrey S. Weber, MD, PhD, explained how the interplay between these 2 factors makes melanoma highly susceptible to targeted therapies. “If you look at average mutational load, melanoma is No. 1 of all tumors, with a median of about 600 to 700 individual non-synonymous mutations,” he said.

Non-synonymous means the mutation is found only in tumor cell DNA, where it encodes peptides and protein fragments not expressed by normal cells. This is important because melanoma derives from transformed melanocytes, and one function of melanocytes is to present antigens to immune cells. When the melanoma melanocytes present the “alien” mutation-encoded antigens to T cells, it triggers an immune response.

“A lot of mutations means the cells grow out of control and make cancers, [but the cell] contains within it the potential seed of its own destruction… by the immune system,” Weber said.

Georgina Long, PhD, MBBS, identified the 3 most common genetic drivers of melanoma as 

 alterations. Approximately 40% of patients with cutaneous melanoma have a BRAF mutation, up to 25% have an NRAS mutation, and about 12% have an 

 mutations are the most critical aberrations to test for when evaluating patients for treatment. Long’s institution also routinely tests for RAS, NF1, and KIT aberrations in case patients are eligible for a clinical trial.

The frequency of driver mutations varies according to melanoma site and sun damage. As Jason J. Luke, MD, explained, “There are melanocytic melanomas that can rise in other parts of the body, including acral lentiginous melanomas or mucosal melanomas, as well as uveal melanomas, and we see a different mutational spectrum in those types of tumors.”

For example, 40% of patients with cutaneous melanoma have a 

 mutation compared with 5% of patients with mucosal melanoma. Conversely, 20% of patients with mucosal melanoma have a KIT mutation compared with only 2% of patients with cutaneous melanoma.

 Clinical trials are evaluating therapies in melanoma patients that target mutations other than 

. “We haven’t cured melanoma, and there’s a lot of work still to be done,” Luke said.

Circling back to the interplay between mutational burden and immunogenicity, Luke said he and his colleagues are investigating whether certain genetic aberrations compromise the immune system’s ability to attack melanoma cells through pathway disruption. The findings could lead to novel targeted therapies that hone in on signaling pathways instead of driver mutations.

“The toughest issue right now in melanoma medical oncology [is] what to do with stage III patients,” Luke said. These are patients whose melanoma has spread to regional lymph nodes or lymphatic vessels. The first approach is excision of the primary tumor with wide margins and removal of regional lymph nodes.

With several more melanoma treatments poised to emerge from the pipeline, data on the optimal way to incorporate these novel drugs into practice lag far behind the breakneck pace of approvals.

Vol. 17/No. 16 | Oncology Live®

Excitement Continues to Build for Advances in Neuroendocrine Tumor Field

Genome Sequencing Is Mapping a Path to Personalized Treatment for Ovarian Cancer

Why CRISPR Gene-Editing Technology Is Captivating Research Field

"True" Genomic Drivers of Ovarian Cancer Not Yet Identified, NCI Researcher Says

Treatment Options for Melanoma Outpace Data on How to Use Them

Models Pave the Way for Improved Outcomes in Medulloblastoma

The Future Is Calling

What Is a Patient Who Refuses Chemotherapy Really Saying?