Archive for the ‘Oncology Research’ Category

Scintillation Proximity Assay technology (SPA) provides a homogeneous assay format that is useful for receptor ligand binding assays. The homogeneous nature eliminates the separation steps necessary for filtration assays and enables optimization for automated high-throughput drug screening. Applying this technology to 384-well plate formats has allowed researchers to further increase assay throughput. However, not every assay adapts well to higher density plate formats, so another means of increasing assay throughput is required. In these cases we propose that screening with compound mixtures, or ‘compressed plate screening’, is a useful alternative. Compressed plates are not new to the screening community, but the complexity of data analyzis and cumbersome follow-up investigations of primary hits have kept many researchers from using them^(1,2). This article addresses these limitations including the issues of false positives and the preservation of assay sensitivity. Additionally, compressed plate generation using compression algorithms and the process of deconvolution for the purpose for data analyzis are discussed. We propose that compressed plate screening is feasible when primary hit rates are low (<1%) and when screening at low compound concentrations (1μM). Results indicated that more than 7-fold savings in time, money, and reagents after follow-up analyzis has been completed.

Compressed Plates

Testing mixtures of compounds in a well increases assay throughput, but a method for identifying active compounds from active mixtures is needed⁽³⁾. Using an algorithm to ‘compress’ 96-well microtiter plates in a ‘self-deconvoluting’ fashion, generated compressed plates in an 8:1 format. The 8:1 compressed plate contained 720 unique test compounds (18 or 20 compounds per well) and is prepared from eight 96- well plates containing 90 compounds each.

To reduce the imbalance in the number of compounds per well on the destination plate, the eight 96-well compound plates are reformatted into 9 x 10 matrices. These matrices are aligned to make two 18 x 20 matrices, or two ‘source plates’ (one source plate is shown in Figure 1).

Figure 1. One source plate (18 x 20 matrix).

Compounds in each 18 x 20 matrix are then combined to make row mixtures and column mixtures; each destination plate would have 36 row mixtures and 40 column mixtures containing 18 or 20 compounds per well. Row mixtures from source plates 1 and 2 (S1 and S2) were dispensed into the upper half of the destination plate (18 row mixtures each: S1R1–S1R18 and S2R1-S2R18). S1 and S2 column mixtures were dispensed into the bottom half of the destination plate (20 column mixtures each: S1C1-S1C20 and S2C1-S2C20). Wells H7-H12 are reserved for controls (Figure 2).

Figure 2. 96-well destination plate containing compound mixtures.

Each compound was present on the destination plate in two mixtures: one row and one column mixture. Both mixtures are otherwise unique, so that a positive assay result in two such wells identified one and only one compound. For example, if mixtures in wells A3 and E7 in the assay plate above were identified as active, the deconvoluting algorithm identifies the source plate or 18 x 20 matrix in which the compound resides. The algorithm also identified the original compound plate and the putatively active compound (identified by the intersection of the row 3 column 7 mixtures, Figure 3). Deconvolution of hits using the matrix layout was time consuming and error prone, so software was developed to automate the process.

Figure 3. Intersection of active column and row mixtures identifies compound.

An active row mixture and column mixture from a source plate was needed to identify an active compound; the intersection of row and column ‘uniquely’ identifies it. When more than one row and column mixture on a plate are active an individual compound has not been identified (Figure 4a). Assume compounds B11 and C10 were true actives. Compound C9 was falsely declared active because it was in column 3 (an active mixture because of compound B11) and in row 4 (an active mixture because of compound C10). A similar scenario exists for compound B12. The deconvolution program reported that all 4 mixtures (R3, R4, C3, and C4) were active in the assay, but could not decipher which combination of active mixtures constituted this activity. Therefore there was a configuration artifact which generated false positives. All four compounds had to be retested and only half (compounds in B11 and C10) were confirmed. This scenario becomes more complex as hit rates increased (Figure 4b). For effective screening, primary hit rates for 8:1 compressed plates should be <1% and the plate to plate hit rate variability should also be low.

Figure 4a. Configuration dilemma leading to false positives.

Figure 4b. Computer simulation deriving the % of false positives generated with increasing hit rate in compressed plates.

In addition to false positives generated with increasing hit rates, the presence of several compounds in a mixture also increased the likelihood of additive, synergistic, or antagonistic interactions^(2,3). Such interactions (in addition to pipetting errors) resulted in spurious hits, where a single row or column hit on a plate occurred without the corresponding column or row hit. In the absence of a correspondingly active mixture, this spuriously active well would not be flagged as a hit and would not need to be reconfirmed. The presence of several compounds in a mixture may reduce assay sensitivity, or the ability to identify an active compound within a mixture. Assay sensitivity was tested using 76 mixtures from an 8:1 compressed plate library using a receptor binding assay for serotonergic 5-HT_2C ligands (Figure 5). Hits were identified as compounds producing >50% inhibition of specific radioligand binding⁽⁴⁾. However, each of the 76 mixtures gave <50% inhibition in the assay and would not have been flagged as active in a screen. Mixtures were then spiked with 5-HT_2C ligands of varying affinity; MK212, mCPP, or metergoline⁽⁵⁾. In each of the inactive compound mixtures, these ligands were detectable, indicating that unknown mixtures do not adversely affect assay sensitivity. Additionally, the assay was able to distinguish between low, moderate, and high responses with compounds of varying affinity. Similar results were obtained using serotonergic 5-HT₇ and dopaminergic D₄ receptor binding assays (data not shown).

Figure 5. Measure of SPA 5-HT_2C receptor binding assay sensitivity.

Following compressed plate SPA assay development and validation, a screen was conducted for serotonergic 5-HT_2C ligands. Hits were identified as compounds producing >50% inhibition of specific radioligand binding. Fresh samples of these putative actives were obtained and tested individually. Confirmed active compounds were further evaluated for potency and receptor selectivity.

Results

Primary Screen

• 107,644 compounds were screened
• 88,744 compounds were screened in 8:1 compressed plates
• 18,900 compounds were screened in singleton plates (new compound acquisitions were not available in compressed plate format) Primary/Putative Hits
• 862 primary actives were identified (0.8% primary hit rate)
• 752 primary actives from compressed plates (0.8% compressed plate primary hit rate)
• 110 primary actives from singleton plates (0.6% singleton plate primary hit rate)

Confirmed Hits

• 369 compounds confirmed as actives (43% confirmation rate)
• 269 confirmed compressed plate hits (36% compressed plate confirmation rate)
• 100 confirmed singleton plate hits (91% singleton plate confirmation rate)

Final Hit Rate

• Overall confirmed hit rate for the screen: 0.3%e
• Overall compressed plate hit rate: 0.3%
• Overall singleton plate hit rate: 0.5%

The 36% confirmation rate for primary actives in compressed plates seems remarkably low in comparison to the 91% confirmation rate for hits identified in singleton plates. Later screens for serotonergic 5-HT₇ ligands and dopaminergic D₄ ligands revealed similar hit rates for compressed and singleton plate sets.

The low compressed plate confirmation rates resulted from the complexity of the plate configuration and the deconvolution process. The computer simulation in Figure 4b illustrated the relationship between hit rates and false positives; the 0.8% primary hit rate in the 5-HT_2C screen generated the predicted number of false positives.

Although 862 putative actives had to be retested in order to confirm 369 active compounds, compressed plates still provided considerable savings in time and money. The primary screening and confirmation of hits from the 88,744 compounds tested in compressed plates required 164 assay plates. In singleton plate format, 986 plates would have been required to assay the same compounds. This reflects better than 7-fold savings in time, reagents, plates, and compounds for assays using 8:1 compressed plates.

With primary hit rates of <1%, follow-up and data analyzis were manageable. Compounds discovered in the screen can not yet be disclosed, the identification of several known commercial 5-HT_2C ligands in this assay (Table 1) further validates the feasibility of screening successfully with compressed plates.

Table 1. Known compounds identified in the 5-HT_2C SPA screen.

Conclusions

Testing compounds as mixtures in SPA receptor binding assays can dramatically reduce screening effort.

Additionally 8:1 compressed plate formats can further reduce screening effort by more than 7-fold when screening at low compound concentrations, when compound mixtures do not adversely affect assay sensitivity, and when hit rates are low. The deconvolution program identified false positives due to complexity of the compressed plate format, therefore all putative hits from mixtures had to be confirmed as singletons. It worked well when hit rates and plate to plate variability are low, such that the complexity of analyzis and follow-up were manageable. Although confirmation rates are likely to be much lower for compressed plate hits than those identified in singleton plates, the savings achieved via combination of SPA technology and compressed plate formats are substantial. Compressed plates can therefore provide an excellent format for increasing assay throughput when assays are not easily adaptable to higher density plate formats.

Source: las.perkinelmer.com

There are a lot of tales told about skin health. One of the most damaging is that sun exposure causes skin cancer. As you’ll see in a moment, this is simply not true.
Melanoma is the form of skin cancer the media likes to refer to when they want to scare the dickens out of the public about the dangers of sun exposure. There are a number of reports of the fact that melanoma has been steadily increasing over the last 20 years. Most dermatologists will say this increase is due to the fact that more people are getting far too much sun exposure in their younger years.
A closer look at the matter, however, reveals a far different story. Skin cancer awareness programs have been effective at increasing the number of people undergoing full-body screening exams, and the result is a huge increase in the number of skin biopsies being performed. It seems that even with biopsies there is still considerable confusion and disagreement among pathologists when it comes to identifying melanoma. It’s apparently not a cut-and-dried diagnosis.
Looking at the same tissue, one pathologist will see a benign lesion while another will see it as melanoma. Thus, the dramatic increase in biopsies has led to more melanoma diagnoses, many of which are false, as a new study shows.
The study, conducted by doctors at Dartmouth Medical School, found that there has been a 250% increase in skin biopsies since 1986-which just happens to be roughly the same percentage increase in the number of people diagnosed with early-stage melanoma. These researchers became skeptical about the rise in melanoma after they noticed that over that time there hasn’t been any increase in deaths from melanoma or any increase in the number of advanced cases of the disease. (BM] 05;331(7518):698)
Plain and simple, there has not been an actual increase in the overall incidence of melanoma. The apparent increase is due merely to improved detection because of the increased number of screening procedures and subsequent biopsies, which by the way, hasn’t led to any increase in survival or cure rates.
Much like cancers of the prostate, breast, and lung, the more doctors look for cancer, the more likely they will find it and the number of false diagnoses will also increase.
If you or someone you know is diagnosed with melanoma, I would definitely suggest getting a second or possibly even a third opinion.
Obviously, excessive exposure that results in sunburn isn’t a benefit at all. However, moderate amounts of sunlight, along with a varied diet containing nature’s natural protective anti- oxidants, vitamins, and fatty acids (omega-3s) is actually beneficial and has been shown to help prevent many forms of cancer-including skin cancer.
Lifetime sun exposure was actually shown to result in a lower risk of developing melanoma. (I Invest Dermatol 03;120(6):1087-1093) Past studies have shown that individuals who utilize sun exposure reasonably have a lower incidence of colon and breast cancer, prostate cancer, multiple sclerosis, osteoporosis, hip and vertebra fractures, et cetera.
Over 20 years ago it was discovered that vitamin D has an “anti-proliferative” effect on cells. In other words, vitamin D can stop cells from multiplying out of control (i.e., from developing into cancer). The body has only two sources for vitamin D. The first is from oily foods (vitamin D is fat-soluble) such as oily fish, organ meats, and eggs. The second is from your own skin cells, which use the same “cancer-causing” UV rays from the sun to convert a form of cholesterol into vitamin D.
Not surprisingly, those who consume more fish and omega- 3 foods have a reduced incidence of melanoma, while those consuming more of the omega-6 oils (the vegetable oils that are now so pervasive throughout our food supply) have increased rates of melanoma and other skin cancers.
A couple of other chemicals that your skin makes when it has adequate exposure to the UV rays of the sun. The function of these two vitamin D-related compounds, lumisterol and tachysterol, isn’t yet fully understood. It’s possible that they’re associated with helping prevent blood sugar problems and obesity.
Avoiding sunlight puts you at a far greater health risk than exposing yourself moderately. Dr. William Grant, one of the top researchers on this subject, has studied the relationship between sunlight and health for years. He’s found that every year 47,000 individuals in this country die from 16 different types of cancer due to insufficient vitamin D, whereas 8,000 die of melanoma and another 2,000 die from other skin cancers.
Furthermore, pale skin, numerous moles, smoking, a diet high in fat and low in fruits and veg- etables, and frequent sunburns are all stronger predictors of later skin cancer than UV exposure. As with most things, moderation is the watchword. Enjoy your time in the sun every day and prepare your body with an adequate intake of the right fatty acids.

BOSTON–The ability to make, test, and map the atomic structure of new anti-cancer agents has enabled a team of Dana-Farber Cancer Institute scientists to discover a compound capable of halting a common type of drug-resistant lung cancer.

In a study to be published in the December 24/31 issue of the journal Nature, the researchers report that non-small cell lung cancers that had become invulnerable to the drugs Iressaâ and Tarcevaâ were stymied by a compound designed and formulated in a Dana-Farber lab. The compound, whose basic chemical framework is different from that of other cancer drugs, acts against a protein — known as an epidermal growth factor receptor (EGFR) kinase — that carries a specific structural defect.

“This type of drug discovery, in which an agent is developed for a specific gene or protein target, and then screened against cancer cells as well as in laboratory models, is rare in academic medicine,” says the study’s senior author Pasi A. Jänne, MD,PhD, of Dana-Farber and Brigham and Women’s Hospital (BWH). “This requires contributions from researchers in multiple disciplines and a coordinated approach to planning experiments and sharing results. That we accomplished this is evidence of the contribution academic medical centers can make to the quest for new cancer treatments.”

The study also illustrates how rapidly lung cancer research and treatment are advancing. It was less than five years ago that investigators at Dana-Farber and elsewhere traced some non-small cell lung cancers (NSCLCs) to mutations in the EGFR gene and discovered that Iressa and Tarceva slowed such tumors’ growth by targeting the abnormal EGFR protein. While the discovery has extended the lives of thousands of NSCLC patients around the world, EGFR blockers are only temporarily effective: after about eight months of treatment, the tumors begin to grow back. And because the drugs target normal EGFR protein as well as abnormal, many patients have severe side effects such as skin rashes and diarrhea.

All current EGFR inhibitors have a structural “backbone” known as a quinazoline core. They lodge in a notch on EGFR normally reserved for a molecule known as ATP, which delivers chemical energy to the cell. By blocking ATP from binding to EGFR, the inhibitors prevent EGFR from sending signals that are essential to keep the tumor cells growing.

Over time, however, the tumor cells develop additional abnormalities in EGFR, enabling them to recommence their growth, even in the presence of Iressa or Tarceva. The most common of these abnormalities — present in about 50 percent of patients with drug-resistant tumors ? is known as EGFR T790M.

Dana-Farber investigators hypothesized that current agents lose their potency because they don’t bind as tightly or fully to the EGFR T790M protein as they ideally should. To improve the fit, researchers led by chemical biologist Nathanael Gray, PhD, prepared a group of inhibitors with a different structural scaffold, known as a pyrimidine core, which, it was thought, would mesh more thoroughly. They lab-tested the agents in NSCLC cells with EGFR T90M and found several that were up to 100 times more potent than quinazolines in restricting cell growth. As an unexpected bonus, these compounds were nearly 100 times less powerful at slowing the growth of cells with normal EGFR, suggesting they would be less likely to produce side effects than current drugs. The agent which performed the best is the pyrimidine WZ4002.

“This work provides a possible therapeutic chapter to a longstanding record of validating EGFR as a drug target,” says Gray. “This has involved the identification of activating mutations in EGFR as a predictor of drug response, the discovery of multiple drug resistance mechanisms, and the elucidation of how these mutations work at an atomic level.”

In follow-up experiments, Dana-Farber and BWH’s Kwok-Kin Wong, MD, PhD, screened the pyrimidine agents in mice with Iressa- and Tarceva-resistant NSCLC tumors driven by EGFR T790M, and found them to be highly effective at impeding tumor growth. Dana-Farber’s Michael Eck, MD, PhD, conducted crystallography studies to determine the molecular structure of the pyrimidines, providing a better picture of why they are so potent and how they target EGFR T790M cells so precisely.

“Not only did we determine that the compound WZ4002 could slow tumor growth, we also demonstrated that it is possible to selectively target the drug-resistant mutant EGFR in tumors, with relatively less effect on the normal EGFR in healthy tissues,” says Wong.

Much work remains to determine if WZ4002 and its chemical cousins will be effective therapies, the authors caution, but the discovery demonstrates the power of screening specially designed compounds against cancers with certain genetic quirks.

“Obviously these are very early days with respect to the possible use of these compounds in patients ? we still have much to learn about their possible liabilities,” Eck remarks. “But I am optimistic that our approach is correct and that it will lead to an effective treatment for the thousands of non-small cell lung cancer patients worldwide who development resistance to Iressa and Tarceva every year.”

Other contributors to the study include lead author Wenjun Zhou, PhD, and co-first authors Dalia Ercan, Liang Chen, PhD, Cai-Hong Yun, PhD, as well as Danan Li, PhD, Marzia Capelletti, PhD, Alexis Cortot, MD, all of Dana-Farber; Lucian Chirieac, MD, and Robert Padera, MD, of Brigham and Women’s Hospital; and Roxana Iacob, PhD, and John Engen, PhD, of Northeastern University.

The study was supported by grants from the National Institutes of Health, the Cecily and Robert Harris Foundation, Uniting Against Lung Cancer, the Flight Attendant Medical Research Institute, the Hazel and Samuel Bellin research fund, and the Damon Runyon Foundation.

Dana-Farber Cancer Institute (www.dana-farber.org) is a principal teaching affiliate of the Harvard Medical School and is among the leading cancer research and care centers in the United States. It is a founding member of the Dana-Farber/Harvard Cancer Center (DF/HCC), designated a comprehensive cancer center by the National Cancer Institute.

(Nanowerk News) Scientists from A*STAR in Singapore and the USA have developed a fast bioluminescence imaging technique that may greatly assist in the search for drugs that target mobile—or metastatic—cancer cells (“A screening platform for glioma growth and invasion using bioluminescence imaging Laboratory investigation”).

Chemotherapy treatments for this type of cancer using ‘anti-migratory’ drugs are important because some of the most mobile cells that cause metastasis can resist conventional cancer drugs. This is a problem because patients tend to be at greater risk of developing metastases over the extended survival periods associated with modern cancer therapies. Using zebrafish as a model organism, researchers could spot as few as eight cells undergoing metastasis from glioblastoma multiforme (GBM)—the most common and aggressive type of brain tumor.

Embryos of Zebrafish could provide important insights into the spread of cancer throughout the body.

The team, including Beng-Ti Ang from A*STAR’s Singapore Institute for Clinical Sciences and co-workers at the University of Singapore and the Methodist Hospital, Cornell University, USA, used a method called gene transfection to develop GBM cells that express a gene from fireflies, causing them to emit light in a process known as bioluminescence. They assessed the ‘invasiveness’ of the cells by measuring how quickly they moved through a three-dimensional matrix, and found that the most invasive cells express a gene that makes them more mobile. The same gene has also been correlated previously with reduced patient survival.

The researchers then injected the GBM cells into zebrafish embryos, and observed tumors in the embryos a few days later. By placing the embryos under a charge-coupled device camera, they were able to watch the bioluminescent tumor cells growing and moving around the body, invading other organs.

“The advantages of the zebrafish are that it is transparent under microscopy imaging, has a fast development cycle (major features are seen within 24 hours after birth), and it is a vertebrate animal,” explains Stephen Wong of the Methodist Hospital. Furthermore, the zebrafish tumors have genomes and development processes very similar to human cancers.

This new bioluminescence screening platform represents a unique real-time method for observing small numbers of cancer cells in a live animal. It is cheaper, easier and far more sensitive than existing imaging methods such as positron emission or computed tomography scanning, or magnetic resonance or fluorescent imaging. Furthermore, the discovery of a genetic subset of highly invasive GBM cells could help greatly in the development of drugs that target tumor-initiating cells.

The team plans to use the platform to screen anti-migration and invasion candidate compounds for GBM treatment and extend the platform for drug screening in other invasive tumors and for drug combination studies.

Source: A*STAR/nanowerk.com