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Programmed Cell Death and Cancer Therapy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Programmed Death: A Cat and Mouse Game

 http://www.cancernetwork.com/blog/programmed-death-cat-and-mouse-game

Prologue: The world of cancer care has been shaken up by the news that patients with hard-to-treat tumors benefit from a new type of immunotherapy, called checkpoint inhibition. A key receptor, called programmed death 1 (PD-1), is charged with suppressing the ability of activated T cells and other immune cells to destroy cancer cells, all in the name of preventing damage to normal tissue via autoimmunity. When PD-1 receptors on T cells bind with PD-L1 and PD-L2, complimentary receptors expressed on tumor cells, the immune response (call it the assassination of the cell) is checked and the tumor lives on. The anti PD-1 monoclonal antibodies nivolumab and pembrolizumab keep PD-L1 from turning off T cells, which has produced durable responses in several tumor types including melanoma, lung cancer, and renal cell carcinoma and represents a new hope for many.

Oncologists are excited to relay this news to patients, but is there a way to explain this without putting everyone in the room to sleep? Well, I like to use analogies to make seemingly complicated mechanisms easier to understand and the PD-1/PD-L1 relationship has inspired several colorful examples, to wit:

“Think of T cells as killers that use photographs to identify individual bad guys. Their weakness is that they will not act if the intended victim shakes their hand first. The bad guys used to be born without arms, but over time they evolved to grow arms and hands, thus avoiding elimination. The antibodies are boxing gloves that cover the hands of the T cells. Goodbye, bad guys.”

“Think of T cells as cats specially trained to eliminate mice wherever they hide. Their only weakness is if they smell catnip they will roll over and purr like idiots instead of doing their job. The mice then develop special glands that secrete catnip, thus pacifying the kitties. Solution: plug up the cats’ noses with nivolumab or pembrolizumab. Sayonara, Mr. Mouse.”

“Think of T cells as a fire sprinkler system designed to activate when a metal plug is heated to its melting point, releasing water from a pipe. The fire then emits a toxin that coats the fusible metal, keeping it below its melting point. By fitting a protective shield around the plug we block the toxic molecules and allow the plug to melt in a fire. The shield is the monoclonal antibody against PD-1 and thus the fire is successfully extinguished.”

This is getting exhausting, so I think I will stop, but don’t you agree that the concept of checkpoint inhibition lends itself to a plethora of metaphors? Now for the next lesson: how to explain chimeric antigen receptor T-cell therapy to patients. Hold on—I think I need to explain it to myself first.

 

As an clinical immunologist, i agree with the concept, looks pretty straightforward but definitely much more complex as our immune system work like a network. I would appreciate clinical trial data with statistical significance.

 

Much more confusing. Most people understand simplified concepts.

“Some cancer cells turn off your immune systems ability to recognise them. These drugs ramp up the immune system and prevent the cancer cells from hiding. This allows your cells to attack and kill cancer cells”

If i think patient seem to have better ability to understand I say “the drugs block the “off switch” that cancer cells use to escape their detection. This turns your immune systems ability to attack and kill cancer cells back on”

I haven’t had one patient that has looked confused since.

 

HDAC Inhibitors Enhance Immunotherapy Efficacy in Lung Cancer

http://www.oncotherapynetwork.com/lung-cancer-targets/hdac-inhibitors-enhance-immunotherapy-efficacy-lung-cancer

Histone deacetylase (HDAC) inhibitors like romidepsin might improve the efficacy of programmed cell death-1 (PD-1) blockade in lung cancer, suggest preclinical findings reported in the journal Clinical Cancer Research.

Most lung cancer patients’ tumors do not respond to immune checkpoint blockade agents like those that target PD-1. One possible mechanism underlying tumor resistance to PD-1 blockade is the failure of sufficient numbers of T cells to infiltrate tumor tissue.

Hypothesizing that upregulating T-cell chemokine expression and thereby T-cell infiltration of tumors would improve PD-1 blockade’s efficacy against lung tumors, the research team went hunting for FDA-approved oncology agents that induce chemokine expression. Screening 97 approved agents, they found one class that did: HDAC inhibitors.

The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor infiltration and impacted lung tumor growth in mouse models, the team reported—and when romidepsin was subsequently combined with PD-1 blockade in several lung tumor models, the combination showed greater antitumor activity than either agent on its own.

“These results suggest that combination of HDAC inhibitors with PD-1 blockade represent a promising strategy for lung cancer treatment,” said senior study author Amer A. Beg, PhD, of the Moffitt Cancer Center’s Immunology Program, in a news release.

Romidepsin and other HDAC inhibitors have already been approved by the FDA for use against lymphoma and other hematologic cancers, Dr. Beg noted.

The combination will next be tested in several clinical trials, including a study of patients diagnosed with non-small cell lung cancer (NSCLC) at Moffitt Cancer Center.

 

HDAC inhibitors enhance T cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma

 

Cancer Cell Survival Driven by Novel Metabolic Pathway

http://www.genengnews.com/gen-news-highlights/cancer-cell-survival-driven-by-novel-metabolic-pathway/81252584/

Researchers have identified a novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells. [National Cancer Institute, NIH] http://www.genengnews.com/Media/images/GENHighlight/thumb_28216_large1422477191.jpg

Being attached to the extracellular matrix (ECM) provides cells with numerous advantages for survival, for instance, receiving much needed growth stimuli. However, for malignant cells to function, they must overcome their anchorage-dependent growth—a scenario that is associated with increased production of reactive oxygen species (ROS) and altered glucose metabolism.

Now, researchers at the Children’s Medical Center Research Institute at UT Southwestern (CRI) believe they have uncovered a novel metabolic pathway that helps cancer cells thrive in conditions that would otherwise be lethal to healthy cells.

“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” explained senior study author Ralph DeBerardinis, M.D., Ph.D.,  associate professor, and director of CRI’s Genetic and Metabolic Disease Program. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

This new study describes an alternate version of two well-known metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle, to defend against ROS that destroy cells via oxidative stress.

The findings from this study were published recently in Nature in an article entitled “Reductive Carboxylation Supports Redox Homeostasis During Anchorage-Independent Growth.”

Previous work from Dr. DeBerardinis’ laboratory found that the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis also noted that cells “are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival.” Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells. Yet, cancer cells seem to have evolved workaround.

A landmark study from 2009 elucidated that healthy cells were destroyed when detached from the ECM. Moreover, in the same study, investigators found that inserting an oncogene into a normal cell caused it to behave like a cancer cell and survive detachment.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

Dr. DeBerardinis and his team worked from the premise that the two findings were pieces of the same puzzle and that a crucial part of the picture seemed to be missing.

It had been well known that the PPP was an important source of nicotine adenine dinucleotide phosphate (NADPH), which provides a source of reducing electrons to scavenge ROS; however, the PPP produces NADPH in the cytosol, whereas the ROS are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis noted.  But how could NADPH from the PPP help deal with the stress of ROS produced in an entirely different part of the cell? “What we did was to discover how this happens.”

The CRI team was able to demonstrate that cancer cells use a “piggybacking” system to carry the reducing electron from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle.The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing electrons to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells. But we had no idea that they were linked in this unusual fashion,” Dr. DeBerardinis stated. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

The researchers stressed that their findings were based on cultured cell models and more research will be necessary to test the role of the pathway in living organisms.

“We are particularly excited to test whether this pathway is required for metastasis because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” Dr. DeBerardinis concluded.

 

Reductive carboxylation supports redox homeostasis during anchorage-independent growth

Lei Jiang, Alexander A. Shestov, Pamela Swain, Chendong Yang, …., Brian P. Dranka, Benjamin Schwartz & Ralph J. DeBerardinis

Nature(2016)      http://dx.doi.org:/10.1038/nature17393

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)¹. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells². Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism². Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

 

Liquid Biopsy Accurately Detects Mutations in Advanced NSCLC

http://www.oncotherapynetwork.com/lung-cancer/liquid-biopsy-accurately-detects-mutations-advanced-nsclc#sthash.bEPxRkAq.dpuf

Droplet digital polymerase chain reaction (ddPCR)-based plasma genotyping—referred to as liquid biopsy—exhibited perfect specificity in identifying EGFR and KRAS mutations in patients with advanced non–small-cell lung cancer (NSCLC), according to the results of a study published in JAMA Oncology.

“We see plasma genotyping as having enormous potential as a clinical test, or assay—a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said senior author, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital, in a press release. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients.”

According to the press release, the test proved so reliable in the study that the Dana-Farber/Brigham and Women’s Cancer Center this week became the first medical facility in the country to offer it to all patients with NSCLC, either at the time of first diagnosis or of relapse following previous treatment.

Oxnard and colleagues enrolled 180 patients with advanced NSCLC. Patients were either newly diagnosed with the disease (n = 120) or had acquired resistance to prior EGFR kinase inhibitors (n = 60) and were planned for rebiopsy. Patients underwent initial blood sampling and immediate plasma ddPCR screening for EGFR exon 19 deletion, L858R, the EGFR T790M acquired resistance mutation, or KRAS G12X. In addition, patients underwent biopsy for tissue genotyping used to compare the accuracy of ddPCR.

Among the enrolled patients, 80 had EGFR exon19/L858R mutations, 35 had T790M mutations and 25 had KRAS G12X mutations. The median test turnaround time for liquid biopsy was 3 days. In comparison, the median turnaround time for tissue genotyping was 12 days for newly diagnosed patients and 27 days for patients with acquired EGFR inhibitor resistance.

“This long turnaround time is due largely to the practical reality that many patients with newly diagnosed NSCLC require a repeat biopsy to obtain tissue for genotyping, as do all patients with acquired resistance,” the researchers noted.

The liquid biopsy showed 100% positive predictive value for detecting EGFR 19 deletion, L858R, and KRAS mutations. However, it had only a positive predictive value of 79% for T790M mutations. The sensitivity of the test was lower. ddPCR had a sensitivity of 82% for EGFR 19 deletion, 74% for L858R, 77% for T790M, and 64% for KRAS.

The researchers pointed out that “a key limitation of plasma ddPCR is that although this method is adept at rapidly detecting specific targetable mutations, it cannot easily detect copy number alterations and rearrangements. The ddPCR panel assessed in this study thus cannot currently detect targetable alterations in either ALK or ROS1,” two other common mutations in NSCLC.

In an editorial that accompanied the article, P. Mickey Williams, PhD, of Frederick National Laboratory for Cancer Research, and Barbara A. Conley, MD, from the National Cancer Institute, questioned whether or not these results, and the rapid turnaround time for liquid biopsy, could be replicated widely by other institutions.

“However, if this performance were generally applicable, this would indeed be an advance in clinical care, reducing the proportion of patients requiring biopsy, at least in the resistance setting,” Williams and Conley wrote.

“This study is a step in the right direction of preparing needed clinical validation for the use of ctDNA for detection and serial monitoring of clinically relevant tumor mutations. Owing to low sensitivity and high positive predictive value and specificity, this approach is probably best suited for detection of resistance mutations and for serial plasma testing to assess treatment response, and should not replace tumor biopsy assessment for initial treatment decision-making,” they concluded.

 

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer

Adrian G. Sacher, MD^1,2; Cloud Paweletz, PhD³; Suzanne E. Dahlberg, PhD^4,5; Ryan S. Alden, BSc¹; Allison O’Connell, BSc³; Nora Feeney, BSc³; Stacy L. Mach, BA¹; Pasi A. Jänne, MD, PhD^1,2,3; Geoffrey R. Oxnard, MD^1,2

JAMA Oncol. Published online April 07, 2016.    http://dx.doi.org:/10.1001/jamaoncol.2016.0173

Importance  Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.

Objective  To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.

Design, Setting, and Participants  Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.

Main Outcomes and Measures  Plasma ddPCR assay sensitivity, specificity, and TAT.

Results  Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.

Conclusions and Relevance  Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

CYP3A7*1C Allele Associated With Poor Outcomes in CLL, Breast, and Lung Cancer

 http://www.oncotherapynetwork.com/breast-cancer-targets/cyp3a71c-allele-associated-poor-outcomes-cll-breast-and-lung-cancer#sthash.7uiD8XFD.dpuf

Patients with the CYP3A7*1C allele suffer higher rates of cancer progression and mortality, possibly because of worse outcomes among patients treated with chemotherapy drugs that are broken down by the enzyme encoded by CYP3A7, according to authors of a retrospective study published in the journal Cancer Research.

“We found that individuals with breast cancer, lung cancer, or CLL [chronic lymphocytic leukemia] who carry one or more copy of the CYP3A7*1C allele tend to have worse outcomes,” said Olivia Fletcher, PhD, a senior investigator at the Breast Cancer Now Toby Robins Research Centre at the Institute of Cancer Research in London, England, in an American Association for Cancer Research (AACR) news release.

Approximately 8% of cancer patients harbor the CYP3A7*1C allele, the coauthors noted. For these patients, it is possible that standard chemotherapy with CYP3A substrates “may not be optimal,” they cautioned.

The team analyzed DNA samples from 1,008 patients with breast cancer, 1,128 patients with lung cancer, and 347 patients with CLL. They found that the CYP3A7*1C-associated single nucleotide polymorphism (SNP) rs45446698 is associated with increased breast cancer mortality (hazard ratio [HR] 1.74; P = .03), all-cause mortality among patients with lung cancer (HR 1.43; P = .009), and progression of CLL (HR 1.62; P = .03). The rs45446698 SNP is one of seven SNPs that form the CYP3A7*1C allele.

The CYP3A7*1C allele is expressed in adults, whereas other variants of CYP3A7 are expressed during fetal development. CYP3A7 encodes an enzyme that degrades estrogen and testosterone, and some anticancer drugs.

“We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (P = .06),” they noted.

“Even though we did not see a statistically-significant difference when stratifying patients by treatment with a CYP3A7 substrate, the fact that we see the same effect in three very different cancer types suggests to me that it is more likely to be something to do with treatment than the disease itself,” commented Dr. Fletcher. “However, we are looking at ways of replicating these results in additional cohorts of patients and types of cancer, as well as overcoming the limitations of this study.”

Limitations included the retrospective nature of the study and the absence of data on how quickly individual patients metabolized chemotherapeutic agents, she said.

 

Cytochrome P450 AlleleCYP3A7*1C Associates with Adverse Outcomes in Chronic Lymphocytic Leukemia, Breast, and Lung Cancer

 

 

Liquid Biopsy for NSCLC

‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.

http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276

A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations.

The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment.

…….

“Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.”

The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies.

The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients.

Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies.

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFRand KRAS Mutations in Advanced Lung Cancer

Adrian G. Sacher, MD^1,2; Cloud Paweletz, PhD³; Suzanne E. Dahlberg, PhD, et al.       JAMA Oncol. Published online April 07, 2016.  http://dx.doi.org::/10.1001/jamaoncol.2016.0173

Importance  Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.

Objective  To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.

Design, Setting, and Participants  Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.

Main Outcomes and Measures  Plasma ddPCR assay sensitivity, specificity, and TAT.

Results  Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.

Conclusions and Relevance  Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.