New Assay Test Predicts That 50% of Ovarian Cancers Will Respond To In Vitro PARP Inhibition

U.K. researchers develop a new test that could be used to select ovarian cancer patients who will benefit from a new class of drugs called “PARP inhibitors.”

U.K. researchers have developed a new test that could be used to select which patients with ovarian cancer will benefit from a new class of drugs called “PARP (poly (ADP-ribose) polymerase) inhibitors,” according to preclinical research presented at the National Cancer Research Institute (NCRI) Cancer Conference held in Liverpool on November 8th.  According to the test results, approximately 50 percent of all patients with ovarian cancer may benefit from PARP inhibitors.

Dr. Asima Mukhopadhyay Discusses Her Research Into A More Tailored Treatment For Ovarian Cancer

PARP Inhibition & BRCA Gene Mutations: Exploiting Ovarian Cancer’s Inherent Defects

  • Genetics 101

DNA (deoxyribonucleic acid) is the genetic material that contains the instructions used in the development and functioning of our cells. DNA is generally stored in the nucleus of our cells. The primary purpose of DNA molecules is the long-term storage of information. Often compared to a recipe or a code, DNA is a set of blueprints that contains the instructions our cells require to construct other cell components, such as proteins and RNA (ribonucleic acid) molecules. The DNA segments that carry this genetic information are called “genes.”

A gene is essentially a sentence made up of the bases A (adenine), T (thymine), G (guanine), and C (cytosine) that describes how to make a protein. Any change in the sequence of bases — and therefore in the protein instructions — is a mutation. Just like changing a letter in a sentence can change the sentence’s meaning, a mutation can change the instruction contained in the gene. Any changes to those instructions can alter the gene’s meaning and change the protein that is made, or how or when a cell makes that protein.

Gene mutations can (i) result in a protein that cannot carry out its normal function in the cell, (ii) prevent the protein from being made at all, or (iii) cause too much or too little of a normal protein to be made.

  • Targeting DNA Repair Through PARP Inhibition

Targeting DNA repair through PARP inhibition in BRCA gene-mutated cancer cells. "DSB" stands for DNA "Double Stand Break." (Photo Credit: AstraZeneca Oncology)

Normally functioning BRCA1 and BRCA2 genes are necessary for DNA repair through a process known as “homologous recombination” (HR).  HR is a form of genetic recombination in which two similar DNA strands exchange genetic material. This process is critical to a cell’s ability to repair its DNA in the event that it becomes damaged, so the cell can continue to function.

A cell’s DNA structure can be damaged by a wide variety of intentional (i.e., select cancer treatments) or unintentional (ultraviolet light, ionizing radiation, man-made chemicals, etc.) factors.  For example, chemotherapy regimens used in the treatment of cancer, including alkylating agents, topoisomerase inhibitors, and platinum drugs, are designed to damage DNA and prevent cancer cells from reproducing.

In approximately 10 percent of inherited ovarian cancers, the BRCA 1 or BRCA2 gene is damaged or mutated.  When the BRCA1 or BRCA2 gene is mutated, a backup type of DNA repair mechanism called “base-excision repair” usually compensates for the lack of DNA repair by HR.  Base-excision repair represents a DNA “emergency repair kit.” DNA repair enzymes such as PARP, whose activity and expression are upregulated in tumor cells, are believed to dampen the intended effect of chemotherapy and generate drug resistance.

When the PARP1 protein – which is necessary for base-excision repair – is inhibited in ovarian cancer cells possessing a BRCA gene mutation, DNA repair is drastically reduced, and the cancer cell dies through so-called “synthetic lethality.”  In sum, PARP inhibitors enhance the potential of chemotherapy (and radiation therapy) to induce cell death.  Healthy cells are unaffected if PARP is blocked because they either contain one or two working BRCA1 or BRCA2 genes which do an effective DNA repair job through use of HR.

  • PARP Inhibitors: A New Class of Targeted Therapy

PARP inhibitors represent a new, targeted approach to treating certain types of cancers. PARP inhibition has the potential to overwhelm cancer cells with lethal DNA damage by exploiting impaired DNA repair function inherent in some cancers, including breast and ovarian cancers with defects in the BRCA1 gene or BRCA 2 gene, and other DNA repair molecules. Inhibition of PARP leads to the cell’s failure to repair single strand DNA breaks, which, in turn, causes double strand DNA breaks. These effects are particularly detrimental to cancer cells that are deficient in repairing double strand DNA breaks and ultimately lead to cancer cell death.

PARP inhibitors are the first targeted treatment to be developed for women with inherited forms of breast and ovarian cancer carrying faults or mutations in a BRCA gene. Early results from clinical trials are showing promise for patients with the rare inherited forms of these cancers.

Study Hypothesis: PARP Inhibitors May Be Effective Against a Large Proportion of Non-Inherited Ovarian Cancers

As noted above, PARP inhibitors selectively target HR–defective cells and have shown good clinical activity in hereditary breast and ovarian cancers associated with BRCA1 or BRCA2 mutations. The U.K. researchers hypothesized that a high proportion (up to 50%) of sporadic (non-inherited) epithelial ovarian cancers could be deficient in HR due to genetic or epigenetic inactivation of the BRCA1, BRCA2, or other HR-related genes, which occur during a woman’s lifetime. Therefore, PARP inhibitors could prove beneficial to a larger group of ovarian cancer patients, assuming a patient’s HR status can be properly identified.

To test this hypothesis, the U.K. researchers developed a functional assay to test the HR status of primary ovarian cancer cultures derived from patients’ ascitic fluid. The test, referred to as the “RAD51 assay,” scans the cancer cells and identifies which tumor samples contain defective DNA repair ability (i.e., HR-deficient) which can be targeted by the PARP inhibitor. The researchers tested the HR status of each culture, and then subjected each one to in vitro cytotoxicity testing using the potent PARP inhibitor PF-01367338 (formerly known as AG-14699).

Study Results: 90% of HR-Deficient Ovarian Cancer Cultures Respond to PARP Inhibition

Upon testing completion, the U.K. researchers discovered that out of 50 primary cultures evaluated for HR status and cytotoxicity to the PARP inhibitor, approximately 40% of the cultures evidenced normal HR activity, while 60 percent of the cultures evidenced deficient HR activity. Cytotoxicity to PARP inhibitors was observed in approximately 90 percent of the HR deficient cultures, while no cytotoxicity was seen in the cultures that evidenced normal HR activity. Specifically, the PARP inhibitor PF-01367338 was found to selectively block the spread of ovarian tumor cells with low RAD51 expression.


Based upon the findings above, the U.K. researchers concluded that HR-deficient status can be determined in primary ovarian cancer, and that such status correlates with in vitro response to PARP inhibition.  Accordingly, the researchers concluded that potentially 50 to 60 percent of ovarian cancers could benefit from PARP inhibitors, but they note that use of the RAD51 assay as a biomarker requires additional clinical trial testing.  Although the RAD51 assay test that was used by the U.K. researchers to examine tumor samples in the laboratory is not yet suitable for routine clinical practice, the U.K. research team hopes to refine it for use in patients.

Upon presentation of the testing results, Dr. Asima Mukhopadhyay said:

“Our results show that this new test is almost 100 percent effective in identifying which ovarian cancer patients could benefit from these promising new drugs.  We have only been able to carry out this work because of the great team we have here which includes both doctors and scientists.”

The team based at Queen Elizabeth Hospital, Gateshead and the Newcastle Cancer Centre at the NICR, Newcastle University collaborated with Pfizer to develop the new assay to test tumor samples taken from ovarian cancer patients when they had surgery.

Dr. Mukhopadhyay added:

“Now we hope to hone the test to be used directly with patients and then carry out clinical trials. If the trials are successful we hope it will help doctors treat patients in a personalised and targeted way based on their individual tumour. It is also now hoped that PARP inhibitors will be useful for a broad range of cancers and we hope this test can be extended to other cancer types.”

Dr. Lesley Walker, Cancer Research UK’s director of cancer information, said:

“It’s exciting to see the development of promising new ‘smart’ drugs such as PARP inhibitors. But equally important is the need to identify exactly which sub-groups of patients will benefit from these new treatments. Tests like this will become invaluable in helping doctors get the most effective treatments quickly to patients, sparing them from unnecessary treatments and side effects.”


Additional Information:


About The Researchers

Dr. Asima Mukhopadhyay is a doctor and clinical research fellow working at the Queen Elizabeth Hospital, Gateshead and the Northern Institute for Cancer Research at Newcastle University. Queen Elizabeth Hospital is run by Gateshead Health NHS Foundation Trust and is the home for gynecological oncology for the North East of England and Cumbria. She received a bursary to attend the conference, which was awarded on the merit of her work.

Key researchers on the study included Dr. Richard Edmondson, who was funded by the NHS, and Professor Nicola Curtin, who was funded by the Higher Education Funding Council. Dr Asima Mukhopadhyay is funded by the NHS.

Dr Richard Edmondson is a consultant gynecological oncologist at the Northern Gynaecological Oncology Centre, Gateshead and a Senior Lecturer at the Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, and is a member of the research team.

Nicola Curtin is Professor of Experimental Cancer Therapeutics at Newcastle University and is the principal investigator of this project.

Current and future work involves working closely with Pfizer. Pfizer developed one of the PARP inhibitors and supported this project.

About The Newcastle Cancer Centre

The Newcastle Cancer Centre at the Northern Institute for Cancer Research is jointly funded by three charities: Cancer Research UK, Leukaemia and Lymphoma Research, and the North of England Children’s Cancer Research Fund.  Launched in July 2009, the Centre is based at the Northern Institute for Cancer Research at Newcastle University.  The Centre brings together some of the world’s leading figures in cancer research and drug development. They play a crucial role in delivering the new generation of cancer treatments for children and adults by identifying new drug targets, developing new drugs and verifying the effectiveness and safety of new treatments. This collaborative approach makes it easier for researchers to work alongside doctors treating patients, allowing promising new treatments to reach patients quickly.

About the NCRI Cancer Conference

The National Cancer Research Institute (NCRI) Cancer Conference is the UK’s major forum for showcasing the best British and international cancer research. The Conference offers unique opportunities for networking and sharing knowledge by bringing together world leading experts from all cancer research disciplines. The sixth annual NCRI Cancer Conference was held from November 7-10, 2010 at the BT Convention Centre in Liverpool. For more information visit

About the NCRI

The National Cancer Research Institute (NCRI) was established in April 2001. It is a UK-wide partnership between the government, charity and industry which promotes cooperation in cancer research among the 21 member organizations for the benefit of patients, the public and the scientific community. For more information visit

NCRI members include: the Association of the British Pharmaceutical Industry (ABPI); Association for International Cancer Research; Biotechnology and Biological Sciences Research Council; Breakthrough Breast Cancer; Breast Cancer Campaign; CancerResearch UK; CHILDREN with LEUKAEMIA, Department of Health; Economic and Social Research Council; Leukaemia & Lymphoma Research; Ludwig Institute for Cancer Research; Macmillan Cancer Support; Marie Curie Cancer Care; Medical Research Council; Northern Ireland Health and Social Care (Research & Development Office); Roy Castle Lung Cancer Foundation; Scottish Government Health Directorates (Chief Scientist Office);Tenovus; Welsh Assembly Government (Wales Office of Research and Development for Health & Social Care); The Wellcome Trust; and Yorkshire Cancer Research.

Novel Targeted Gene Therapies Use Diphtheria Toxin To Fight Ovarian Cancer; One Clinical Trial Underway

Two separate research teams reported promising results last week based upon preclinical studies involving the use of diphtheria toxin to fight ovarian cancer. … A targeted gene therapy was utilized in both studies, wherein a gene fragment capable of producing diptheria toxin was combined with a nanoparticle which was targeted against a unique or overexpressed genetic characteristic of the ovarian cancer tumor cells. Both research teams reported significant reduction in ovarian cancer tumor mass and extended survival for the treated mice. Based upon these findings, one research team already announced the opening of a Phase I/II clinical trial which will test the novel therapy on patients with advanced stage ovarian cancer.

Targeted Gene Therapy In the Fight Against Ovarian Cancer

The peritoneal cavity is a common site of ovarian cancer and accompanying ascites caused by the disease. Ascites is an abnormal buildup of fluid in the peritoneal cavity that causes swelling.  Malignant tumor cells may be found in the ascites fluid in connection with late stage ovarian cancer.  Massive ascites and the related abdominal distention can cause anorexia, nausea, vomiting and respiratory difficulties, and negatively impact the patient’s quality of life. Ovarian cancer patients frequently experience disease involvement of the pelvic and retroperitoneal lymph nodes as well. The standard primary treatment of patients with advanced stage ovarian cancer is cytoreductive surgery followed by platinum drug and taxane drug doublet chemotherapy. Despite this aggressive approach, there is a high rate of disease recurrence. Although discovery of several other active nonplatinum cytotoxic agents has improved outcome, long-term survival rates are low. Success of traditional chemotherapy has been limited by drug resistance and lack of specificity with respect to disease formation and progression. Thus, novel “targeted” ovarian cancer therapies that achieve improved long-term disease control with lower toxicity are desperately needed.

A so-called “targeted therapy” utilizes drugs or other medically manufactured substances (e.g., small molecule drugs or monoclonal antibodies) to block the growth and spread of cancer by interfering with specific molecules involved in cancer tumor growth and progression.  By identifying and selectively focusing upon molecular and cellular changes or unique genetic characteristics that are specific to cancer, targeted cancer therapies may be more effective than other types of treatment, including chemotherapy, and less harmful to normal cells.

It is possible for a targeted therapy to incorporate a gene therapy. Gene therapy is an experimental treatment that involves the introduction of genetic material (DNA or RNA) into a human cell to fight a disease such as cancer.  When both therapeutic approaches are combined by researchers, a “targeted gene therapy” is the result.  A targeted gene therapy is an attractive approach to controlling or killing human cancer cells only if the therapy can selectively identify and exploit the genetic and epigenetic alterations in cancer cells, without harming normal cells that do not possess such alternations.

Two separate research groups reported promising results last week based upon preclinical studies involving the use of diphtheria toxin to fight ovarian cancer.  The toxin is produced by a deadly bacterium (Corynebacterium diphtheriae).  A targeted gene therapy was utilized in both studies, wherein a gene fragment capable of producing diptheria toxin was combined with a nanoparticle which was targeted against a unique or overexpressed genetic characteristic of the ovarian cancer tumor cells.  Both research teams reported significant reduction in ovarian cancer tumor mass and extended survival for the treated mice. Based upon these findings, one research team already announced the opening of a Phase I/II clinical trial which will test the novel therapy on patients with advanced stage ovarian cancer.

MIT-Lankenau Institute Researchers Use Diphtheria Toxin Gene Therapy To Target Overexpression Of The MSLN & HE4 Ovarian Cancer Genes.


Daniel Anderson, Ph.D., Research Associate, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

The first study, which appears in the August 1 issue of the journal Cancer Research, was conducted by a team of researchers from the Massachusetts Institute of Technology (MIT) and the Lankenau Institute of Medical Research (Lankenau Institute). In this study, the researchers used a nanoparticle as a delivery vehicle (or vector) for DNA that encodes a diphtheria toxin suicide protein (DT-A).  The novel nanoparticles are made with positively charged, biodegradable polymers known as poly(beta-amino esters). When mixed together, these polymers can spontaneously assemble with DNA to form nanoparticles. The polymer-DNA nanoparticle can deliver functional DNA when injected into or near the targeted tissue.

The nanoparticle carrying the DT-A is designed to target overexpression of two genes (mesothelin (MSLN) and HE4 (or WFDC2)) that are highly active in ovarian tumor cells, but not in normal cells. Once inside an ovarian cancer tumor cell, the DT-A disrupts the tumor cell’s ability to manufacture critical life sustaining proteins, thereby causing cell death.  Accordingly, the choice of the DT-A fragment of a diptheria toxin gene ensures high ovarian cancer cell killing activity.  It also avoids unintended toxicity to normal cells because the DT-A released from destroyed ovarian cancer cells is not able to enter normal neighboring tissue cells in the absence of the DT-B fragment which was excluded from the original nanoparticle delivery system or vector.

As part of this study, researchers administered DT-A nanoparticles directly into the peritoneal cavity – which encases abdominal organs such as the stomach, liver, spleen, ovaries and uterus – of mice xenografted with primary and metastatic ovarian tumors.  Ovarian cancer is known to initially spread throughout the peritoneal cavity, and current therapeutic approaches in humans include direct injection into the peritoneal space, thereby targeting the therapy to the ovaries and nearby tissues where tumors may have spread.

“… [The researchers] discovered that the intraperitoneal (IP) administration of DT-A nanoparticles resulted in a significant reduction in ovarian tumor mass and extended survival for the treated mice.  The researchers also found that the targeted gene-therapy treatment was as effective, and in some cases more effective, than the traditional chemotherapy combination of cisplatin and paclitaxel. …”


Robert S. Langer is the David H. Koch Institute for Integrative Cancer Research Professor (there are 14 Institute Professors at MIT; being an Institute Professor is the highest honor that can be awarded to a faculty member). Dr. Langer has written approximately 1,050 articles. He also has approximately 750 issued and pending patents worldwide. Dr. Langer’s patents have been licensed or sublicensed to over 220 pharmaceutical, chemical, biotechnology and medical device companies. He is the most cited engineer in history.


Janet Sawicki, Ph.D., Professor, Lankenau Institute of Medical Research. Dr. Sawicki also serves as an Associate Professor at the Kimmel Cancer Center of Thomas Jefferson University. Her ovarian cancer research is funded by the National Institutes of Health, the U.S. Department of Defense, the Sandy Rollman Foundation, the Teal Ribbon Ovarian Cancer Foundation, and the Kaleidoscope of Hope Foundation.

Daniel Anderson, Ph.D., research associate in the David H. Koch Institute for Integrative Cancer Research at MIT and a senior author of the paper, and others from MIT, including Institute Professor Robert Langer, along with researchers from the Lankenau Institute, led by Professor Janet Sawicki, discovered that the intraperitoneal (IP) administration of DT-A nanoparticles resulted in a significant reduction in ovarian tumor mass and extended survival for the treated mice.  The researchers also found that the targeted gene-therapy treatment was as effective, and in some cases more effective, than the traditional chemotherapy combination of cisplatin and paclitaxel. Furthermore, the novel therapy did not have the toxic side effects of chemotherapy because the diptheria toxin gene is engineered to function in ovarian cells but is inactive in normal cell types.

Based upon these finding, the MIT and Lankenau Institute researchers concluded that IP administration of DT-A nanoparticles, combined with designed targeting of those nanoparticles against ovarian tumor cell gene (MSLN & HE4) expression, holds promise as an effective therapy for advanced-stage ovarian cancer. According to Anderson, human clinical trials could start, after some additional preclinical studies, in about 1 to 2 years.  Currently ovarian cancer patients undergo surgery followed by chemotherapy. In many cases, the cancer returns after treatment.  Disease recurrence is problematic because there are no curative therapies for advanced-stage tumors.

For several years, the MIT-Lankenau Institute team worked to develop the DT-A nanoparticles as an alternative to viruses, which are associated with safety risks. In addition to ovarian cancer, these nanoparticles have demonstrated treatment potential for a variety of diseases, including prostate cancer and viral infection. “I’m so pleased that our research on drug delivery and novel materials can potentially contribute to the treatment of ovarian cancer,” Langer said. In future studies, the team plans to examine the effectiveness of nanoparticle-delivered diphtheria toxin genes in other types of cancer, including brain, lung and liver cancers.

Other MIT authors of the paper are recent MIT Ph.D. recipients Gregory Zugates and Jordan Green (now a professor at John’s Hopkins University), and technician Naushad Hossain. The research was funded by the Department of Defense and the National Institutes of Health.

Israeli Researchers Use Diphtheria Toxin Gene Therapy To Target Overexpression Of The H19 Ovarian Cancer Gene.

The second study was conducted by Israeli researchers and was published August 6 online ahead of print in the Journal of Translational Medicine.

In the provisional study report, the researchers note that based upon earlier studies from their team and others, the H19 gene has emerged as a candidate for cancer gene therapy. The H19 gene is expressed at substantial levels in ovarian cancer tumor cells, but is nearly undetectable in surrounding normal tissue cells.  Although the Israeli research team acknowledges that the exact function of H19 is the subject of past debate, it notes that recent data suggests a role for H19 in promoting cancer progression, angiogenesis and metastasis.

As a first step, Israeli researchers tested H19 gene expression in ovarian cancer cells obtained from the ascites fluid of 24 patients, and established that H19 expression levels were detected in 90% of the tested patients. Of those patients with positive H19 expression, 76% showed a moderate or high level of expression, while 24% showed a low level of expression.

Next, the researchers created a DT-A nanoparticle similar to the one created by the MIT/Lankenau research team as described above, except the Israeli nanoparticle was designed to target H19 overexpression within ovarian cancer cells.  The therapeutic effect of the DT-A/H19 nanoparticles was first tested in vitro against various ovarian cancer cell lines and cells obtained from patient ascites fluid.  The researchers determined that the DT-A/H19 nanoparticle therapy caused ovarian cancer cell death.  The therapeutic effect of the DT-A nanoparticles was tested in vivo by injecting the DT-A nanoparticles into mice xenografted with ovarian cancer tumors. The researchers estimate that the DT-A nanoparticle therapy reduced ovarian cancer tumor growth in the treated mice by 40%.

Based upon these finding, the researchers note that although the study report issued is provisonal, it is their working hypothesis that intraperitoneal administration of DT-A/H19 nanoparticles holds the potential to (1) reach ascites tumor cells, (2) deliver its intracellular toxin without targeting normal tissue cells, and (3) reduce tumor burden & fluid accumulation; and therefore, improve the patient’s quality of life, and hopefully, prolong her survival.

  • DT-A/H19 Nanoparticle Therapy Administered To An Israeli Patient On A Compassionate Use Trial Basis

In the provisional study report, the researchers state that the targeted gene therapy was administered to an Israeli patient with advanced, recurrent ovarian cancer, who qualified for compassionate use treatment under Israeli regulatory rules.  Specifically, the patient’s intraperitoneal ovarian cancer metastases and ascites were treated with the DT-A/H19 nanoparticle therapy after the failure of conventional chemotherapy. The results of the single patient compassionate use trial suggest that the drug caused no serious adverse events at any drug dosage level.  Moreover, the patient experienced (1) a 50% decrease in serum cancer marker protein CA-125, (2) a significant decrease in the number of cancerous cells in the ascites, and (3) a clinical improvement as reported by her doctors.  It is reported that the patient’s quality of life increased during the course of treatment and her condition continues to be stable, with no new cancerous growths.

  • Phase I/II Clinical Trial To Test DT-A/H19 Nanoparticle Therapy (BC-819) In the U.S. & Israel

The DT-A/H19 nanoparticle therapy is being developed commercially by BioCancell Therapeutics, Inc (BioCancell) Recently, BioCancell announced the opening of a clinical trial to test the DT-A/H19 nanoparticle therapy (also referred to as BC-819) in patients with advanced stage ovarian cancer.  The clinical trial is entitled, Phase 1/2a, Dose-Escalation, Safety, Pharmacokinetic, and Preliminary Efficacy Study of Intraperitoneal Administration of DTA-H19 in Subjects With Advanced Stage Ovarian Cancer, and the trial investigators are recruiting patients in the U.S. and Israel as indicated below.

University of Pennsylvania Medical Center [Abramson Cancer Center] (Recruiting)
Philadelphia, Pennsylvania, United States, 19104-6142
Contact: Lana E. Kandalaft, Pharm.D, PhD – 215-537-4782 (
Principal Investigator: George Coukos, M.D., Ph.D.

Massey Cancer Center (Not yet recruiting)
Richmond, Virginia, United States, 23298-0037
Contact: Jane W. Baggett, RN 804-628-2360 (
Principal Investigator: Cecelia H. Boardman, M.D.

The Edith Wolfson Medical Center (Recruiting)
Holon, Israel
Contact: Pnina Nir (972)-52-8445143 (
Principal Investigator: Tally Levy, M.D.

Hadassah University Hospital (Recruiting)
Jerusalem, Israel
Contact: Zoya Bezalel (972)-2-6776725 (
Principal Investigator: David Edelman, MD

Meir Hospital (Recruiting)
Kfar Saba, Israel
Contact: Tal Naderi 09-7472213 (
Principal Investigator: Ami Fishman, MD

In the provisional study report, the Israeli researchers discuss the importance of collecting data regarding the correlation between the level of ovarian cancer cell H19 expression and the efficacy of the treatment as part of the clinical trial discussed above.  Based upon accrued future clinical trial data, the researchers believe that they will be able to identify in advance patients that will respond to this novel therapy, as well as non-responders who are resistant to all known therapies, thereby avoiding treatment failure and unnecessary suffering and cost.