Chemosensitivity prediction

[Rich66]

Target Now, molecular profiling:
http://www.carislifesciences.com/oncology-target-now

Target Now helps patients and their treating physicians create a cancer treatment plan based on the tumor tested. By comparing the tumor's information with data from published clinical studies by thousands of the world's leading cancer researchers, Caris can help determine which treatments are likely to be most effective and, just as important, which treatments are likely to be ineffective.
The Target Now test is performed after a cancer diagnosis has been established and the patient has exhausted standard of care therapies or if questions in therapeutic management exists. Using tumor samples obtained from a biopsy, the tumor is examined to identify biomarkers that may have an influence on therapy. Using this information, Target Now provides valuable information on the drugs that will be more likely to produce a positive response. Target Now can be used with any solid cancer such as lung cancer, breast cancer, and prostate cancer.

What does my doctor need from me to perform the test?
Target Now is performed on tissue that is obtained during the surgical removal or biopsy of your tumor. Even if your doctor doesn’t order Target Now testing at this time, the hospital where your biopsy was performed will typically store some of your tissue as standard procedure. Now or in the future, your doctor can request that Target Now testing be run and coordinate with the hospital to have your sample sent to us.

Is Target Now reimbursed?
The Target Now is typically reimbursed by Medicare and other third party payers. Other than co-payments or deductibles required by the patient's plan, there are normally no out-of-pocket costs for the patient

RESEARCH:
Prediction of breast cancer sensitivity to neoadjuvant chemotherapy based on status of DNA damage repair proteins

Breast Cancer Research 2010, 12:R17 doi:10.1186/bcr2486
Hideki Asakawa (hiddie_vr4@hotmail.com)
Hirotaka Koizumi (koizumi@marianna-u.ac.jp)
Ayaka Koike (a2koike@marianna-u.ac.jp)
Makiko Takahashi (takamaki@marianna-u.ac.jp)
Wenwen Wu (wuwenwen@marianna-u.ac.jp)
Hirotaka Iwase (hiwase@kumamoto-u.ac.jp)
Mamoru Fukuda (m2fukuda@marianna-u.ac.jp)
Tomohiko Ohta (to@marianna-u.ac.jp)
ISSN 1465-5411
Article type Research article
Submission date 18 September 2009
Acceptance date 5 March 2010
Publication date 5 March 2010
Article URL http://breast-cancer-research.com/content/12/2/R17
http://breast-cancer-research.com/co...df/bcr2486.pdf

Abstract
Introduction: Various agents used in breast cancer chemotherapy provoke DNA double-strand breaks (DSBs). DSB repair competence determines the sensitivity of cells to these agents whereby aberrations in the repair machinery leads to apoptosis. Proteins required for this pathway can be detected as nuclear foci at sites of DNA damage when the pathway is intact. Here we investigate whether focus formation of repair proteins can predict chemosensitivity of breast cancer.

Methods: Core needle biopsy specimens were obtained from sixty cases of primary breast cancer before and 18-24 hours after the first cycle of neoadjuvant epirubicin plus cyclophosphamide (EC) treatment. Nuclear focus formation of DNA damage repair proteins was immunohistochemically analyzed and compared with tumor response to chemotherapy.

Results: EC treatment induced nuclear foci of H2AX, conjugated ubiquitin, and Rad51 in a substantial amount of cases. In contrast, BRCA1 foci were observed
before treatment in the majority of the cases and only decreased after EC in
thirteen cases. The presence of BRCA1-, H2AX-, or Rad51-foci before treatment
or the presence of Rad51-foci after treatment was inversely correlated with tumor response to chemotherapy. DNA damage response (DDR) competence was further evaluated by considering all four repair indicators together. A high DDR score significantly correlated with low tumor response to EC and EC + docetaxel whereas other clinicopathological factors analyzed did not.

Conclusions: High performing DDR focus formation resulted in tumor resistance to DNA damage-inducing chemotherapy. Our results suggested an importance of evaluation of DDR competence to predict breast cancer chemosensitivity, and merits further studying into its usefulness in exclusion of non-responder patients.

Introduction
Recent advances in chemotherapy have significantly improved the prognosis of breast cancer patients. However, prediction of tumor sensitivity to chemotherapy
has not reached a high level of confidence, whereas determining sensitivity to hormone therapy or trastuzumab is relatively more established. Estrogen and
progesterone receptors (ER and PR) and HER2/ErbB2 are practical benchmarks
to exclude non-responding patients, and tailoring treatment based on gene status significantly optimizes the response rate of hormone therapy and trastuzumab, respectively. Prediction of chemosensitivity with equivalent accuracy is currently anticipated to further improve breast cancer prognosis.
Anthracycline-based regimen, such as epirubicin plus cyclophosphamide (EC), and taxanes represent the major chemotherapeutic agents used in the breast cancer field[1, 2]. Of these, anthracycline-based chemotherapy induces DNA double-strand breaks (DSBs)[3, 4], the most cytotoxic DNA lesion, that leads cells into apoptosis especially when relevant repair pathways (represented by homologous recombination (HR) repair) are perturbed[5]. It is important to note that DNA damage repair competence varies among individual breast tumors and closely correlates with chemosensitivity. For example, secondary mutations of BRCA1 or 2 (essential factors in the HR pathway) caused by chemotherapy using cisplatin or poly(ADP-ribose) polymerase (PARP) inhibitor in BRCA1/2-mutated cancers restore the wild-type reading frame and, therefore, the tumor acquires resistance to these drugs [6-8].
These facts indicate that chemosensitivity of BRCA-associated cancers could be
strongly affected by DNA damage repair capability. Based on this evidence it has
been suggested that HR competence could be a potential biomarker for chemosensitivity [9]. Rad51, a protein that plays a direct role in HR, especially
reflects the HR-competence of cells. Therefore, knowing its status is likely
valuable when assessing HR-competence in tumor cells in order to instruct therapeutic decisions [9].
The HR pathway for DSB repair is executed by sequential recruitment of repair proteins to chromatin around DNA lesions. Accumulation of the proteins is regulated by complex mechanisms that utilize phosphorylation and ubiquitination
modifications mediated by kinases, including ATM, and at least three ubiquitin
E3 ligases, RNF8, RNF168, Rad18, and BRCA1 [10-17]. The Mre11-Rad50-Nbs1 complex first recognizes DSBs and recruits ATM. ATM then phosphorylates the histone variant H2AX (H2AX) [18, 19] that triggers accumulation of the downstream E3 ligases RNF8 [11-13, 20] and RNF168 [14,15]. Lysine 63 (K63)–linked polyubiquitin chains built at the sites of DNA damage by these E3 ligases next recruits the BRCA1-Abraxas-RAP80 complex through the RAP80 component, a protein that contains UIM (ubiquitin interacting motif) domains [21-23]. BRCA1 is then essential to recruit repair effector proteins, including Rad51, that perform HR through sister chromatid exchange [24, 25].
Depletion of any one of these proteins results in HR deficiency accompanied by
loss of Rad51 focus formation, causing cells to become hypersensitive to
DSB-inducing agents.
In this study we attempt to clarify the value of HR-competence for prediction of breast cancer chemosensitivity. One contention is that nuclear focus formation of repair proteins in baseline breast cancer tissues is a response to spontaneous DNA damage during cell proliferation and, in turn, may represent a marker of HR-competence of cells to exogenous DNA damage. Therefore, it may predict tumor response to DNA damage-inducing chemotherapy such as EC. Also, the focus formation after chemotherapy could provide us additional information regarding the DNA damage response capacity. To verify in vivo whether focus formation of repair proteins actually occurs in response to DNA damage-inducing chemotherapy and whether it correlates with tumor fates after chemotherapy, we analyzed foci in core needle biopsy specimens from breast cancer before and after neoadjuvant EC treatment.
Materials and methods
Patients and tumors: Sixty patients with primary breast cancer (2 cm or larger) who
consecutively underwent neoadjuvant chemotherapy with epirubicin plus cyclophosphamide (EC) followed by docetaxel (DOC) at the Division of Breast
and Endocrine Surgery, St. Marianna University School of Medicine, Japan, were enrolled in the present study from August 2005 to July 2007. Tumor specimens were obtained by core needle biopsy prior to starting therapy and 18 to 24 hours after the first cycle of EC treatment.

We found that foci of BRCA1, H2AX and Rad51 prior to treatment and EC-induced foci of Rad51 correlated with tumor response when compared either with the mean tumor volume reduction or the tumor response rate. Upon incorporating these four factors into one DDR score, a significant correlation was observed with mean tumor volume reduction after EC, whereas no other factors correlated with the mean tumor volume reduction (Table 4, and Figure 3a). Although it was not statistically significant the similar correlation was also observed between DDR score and the tumor response rate (Table 3). These correlations became more significant after EC+DOC treatment (Table 3, 4, 5, and Figure 3b) and the DDR score was independent predictive factor of other factors including tumor subtype when evaluated with volume reduction using 50% of the PR/SD border (Table 6). Recent studies suggested that luminal tumors have low response rate to neoadjuvant chemotherapy, while basal-like and HER2+ tumors have higher response rates. For example it has been reported that clinical response rate (CR and PR) to anthracyclin-based chemotherapy of luminal A was 39% whereas that of basal-like, which has been implicated with BRCA1 dysfunction[44, 45], was 85%[46]. The response rates to EC treatment of luminal A (15/37 cases, 40.5%) and basal-like (4/6 cases, 66.7%) subtypes in the current study were not very different from the previous report. However, we could not find any correlation between subtype and DDR sore while DDR score independently predicted the chemosensitivity. The result may reflect the fact that luminal A tumors also include DNA damage-sensitive tumors with defective HR pathway that can be counted by the DDR score.
Supporting this it has been shown that tumors caused by BRCA2 deficiency mainly become luminal A tumor[45, 47, 48].
The reason why the correlation between the DDR score and tumor response after EC+DOC treatment became more significant than that after EC is not clear at present. Because DOC does not induce DNA double-strand breaks, the observed effect is not likely due to the sensitivity to DNA damage in those tumors. DOC might be more toxic for the cells with gross genomic aberration caused by the pretreatment with EC under the condition with less HR competent. Alternatively it is possible that time length after EC treatment enhanced the difference of the outcome.
Interestingly, DDR score group 4 consisted of cases with poor tumor responses to chemotherapy when evaluated for both mean tumor volume reduction (Figure 3) and tumor response rate (Table 3). This result may lead to the possibility of using DDR status in the clinic to predict and exclude non-responders to EC treatment. It is noteworthy to point out that the HR repair cascade for DSB contains many essential proteins other than that tested in this study. By including select subsets of proteins for analysis, it may be possible to identify non-responders in order to avoid unnecessary chemotherapy. Ideally in such cases, the levels of baseline foci present prior to treatment would provide enough information to determine appropriate treatment, preventing the need for additional core needle biopsy after chemotherapy.
Conclusions
In conclusion, our results suggest the importance of evaluating DDR competence to predict breast cancer chemosensitivity and warrant further investigation into its effectiveness as a way to exclude non-responding patients.

9/2010

24-hour Test Predicts Breast Cancer's Likely Response To Chemotherapy

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A new test has been developed which can predict whether a breast cancer patient will respond to chemotherapy [adriamycin]within 24-hours of starting treatment, thus sparing her unnecessary treatment and side effects, according to a study published in the medical journal Clinical Cancer Research...

More...


Abstract

Low RAD51 score was strongly predictive of pathological complete response to chemotherapy, with 33% low RAD51 score cancers achieving pathological complete response compared to 3% of other cancers (p=0.011).

[gdpawel]

Showing movies in 3-D has produced a box-office bonanza in recent months. Could viewing cell behavior in three dimensions lead to important advances in cancer research? A new study led by Johns Hopkins University engineers indicates it may happen. Looking at cells in 3-D, the team members concluded, yields more accurate information that could help develop drugs to prevent cancer's spread.

The study, a collaboration with researchers at Washington University in St. Louis, appears in the June issue of Nature Cell Biology.

"Finding out how cells move and stick to surfaces is critical to our understanding of cancer and other diseases. But most of what we know about these behaviors has been learned in the 2-D environment of Petri dishes," said Denis Wirtz, director of the Johns Hopkins Engineering in Oncology Center and principal investigator of the study. "Our study demonstrates for the first time that the way cells move inside a three-dimensional environment, such as the human body, is fundamentally different from the behavior we've seen in conventional flat lab dishes. It's both qualitatively and quantitatively different."

One implication of this discovery is that the results produced by a common high-speed method of screening drugs to prevent cell migration on flat substrates are, at best, misleading, said Wirtz, who also is the Theophilus H. Smoot Professor of Chemical and Biomolecular Engineering at Johns Hopkins. This is important because cell movement is related to the spread of cancer, Wirtz said. "Our study identified possible targets to dramatically slow down cell invasion in a three-dimensional matrix."

When cells are grown in two dimensions, Wirtz said, certain proteins help to form long-lived attachments called focal adhesions on surfaces. Under these 2-D conditions, these adhesions can last several seconds to several minutes. The cell also develops a broad, fan-shaped protrusion called a lamella along its leading edges, which helps move it forward. "In 3-D, the shape is completely different," Wirtz said. "It is more spindlelike with two pointed protrusions at opposite ends. Focal adhesions, if they exist at all, are so tiny and so short-lived they cannot be resolved with microscopy."

The study's lead author, Stephanie Fraley, a Johns Hopkins doctoral student in Chemical and Biomolecular Engineering, said that the shape and mode of movement for cells in 2-D are merely an "artifact of their environment," which could produce misleading results when testing the effect of different drugs. "It is much more difficult to do 3-D cell culture than it is to do 2-D cell culture," Fraley said. "Typically, any kind of drug study that you do is conducted in 2D cell cultures before it is carried over into animal models. Sometimes, drug study results don't resemble the outcomes of clinical studies. This may be one of the keys to understanding why things don't always match up."

Fraley's faculty supervisor, Wirtz, suggested that part of the reason for the disconnect could be that even in studies that are called 3-D, the top of the cells are still located above the matrix. "Most of the work has been for cells only partially embedded in a matrix, which we call 2.5-D," he said. "Our paper shows the fundamental difference between 3-D and 2.5-D: Focal adhesions disappear, and the role of focal adhesion proteins in regulating cell motility becomes different."

Wirtz added that "because loss of adhesion and enhanced cell movement are hallmarks of cancer," his team's findings should radically alter the way cells are cultured for drug studies. For example, the team found that in a 3-D environment, cells possessing the protein zyxin would move in a random way, exploring their local environment. But when the gene for zyxin was disabled, the cells traveled in a rapid and persistent, almost one-dimensional pathway far from their place of origin.

Fraley said such cells might even travel back down the same pathways they had already explored. "It turns out that zyxin is misregulated in many cancers," Fraley said. Therefore, she added, an understanding of the function of proteins like zyxin in a 3-D cell culture is critical to understanding how cancer spreads, or metastasizes. "Of course tumor growth is important, but what kills most cancer patients is metastasis," she said.

To study cells in 3-D, the team coated a glass slide with layers of collagen-enriched gel several millimeters thick. Collagen, the most abundant protein in the body, forms a network in the gel of cross-linked fibers similar to the natural extracellular matrix scaffold upon which cells grow in the body. The researchers then mixed cells into the gel before it set. Next, they used an inverted confocal microscope to view from below the cells traveling within the gel matrix. The displacement of tiny beads embedded in the gel was used to show movement of the collagen fibers as the cells extended protrusions in both directions and then pulled inward before releasing one fiber and propelling themselves forward.

Fraley compared the movement of the cells to a person trying to maneuver through an obstacle course crisscrossed with bungee cords. "Cells move by extending one protrusion forward and another backward, contracting inward, and then releasing one of the contacts before releasing the other," she said. Ultimately, the cell moves in the direction of the contact released last.

When a cell moves along on a 2-D surface, the underside of the cell is in constant contact with a surface, where it can form many large and long-lasting focal adhesions. Cells moving in 3-D environments, however, only make brief contacts with the network of collagen fibers surrounding them - "We think the same focal adhesion proteins identified in 2-D situations play a role in 3-D motility, but their role in 3-D is completely different and unknown," Wirtz said. "There is more we need to discover."

Fraley said her future research will be focused specifically on the role of mechanosensory proteins like zyxin on motility, as well as how factors such as gel matrix pore size and stiffness affect cell migration in 3-D.

Notes:

Co-investigators on this research from Washington University in St. Louis were Gregory D. Longmore, a professor of medicine, and his postdoctoral fellow Yunfeng Feng, both of whom are affiliated with the university's BRIGHT Institute. Longmore and Wirtz lead one of three core projects that are the focus of the Johns Hopkins Engineering in Oncology Center, a National Cancer Institute-funded Physical Sciences in Oncology Center. Additional Johns Hopkins authors, all from the Department of Chemical and Biomolecular Engineering, were Alfredo Celedon, a recent doctoral recipient; Ranjini Krishnamurthy, a recent bachelor's degree recipient; and Dong-Hwee Kim, a current doctoral student.

Funding for the research was provided by the National Cancer Institute.

Source: Johns Hopkins University

The whole concept of proper genetic markers (molecular profiling) is not to put patients in the position of having to receive toxic cancer drugs if they're not going to do any good. However, genomics is far too limited in scope to encompass the vagaries and complexities of human cancer biology.

Trying to find tumor mutations to predict chemo success is still a "trial-and-error" approach to therapy. Testing for the EGFR mutation may be able to tell you whether or not your cells are "potentially" susceptible to this mechanism of attack. It cannot tell you if a "targeted" drug will work for "your" individual cancer cells. They don't even test your tumor cells against the EGFR-inhibitor drug.

The situation with Erbitux and Vectibix for colon cancer, Iressa and Tarceva for lung cancer, and Herceptin for breast cancer is that all the mutation or amplication studies can tell us is whether or not the cancer cells are potentially susceptible to this mechanism of attack.

They don't tell you if one drug or the other is worse or better than some other drug which may target this. The cell is a system, an integrated, interacting network of genes, proteins and other cellular constituents that produce functions.

No genetic profile can discriminate differing levels of anti-tumor activity occurring among different targeted therapy drugs. Nor can it identify situations in which it is advantageous to combine a targeted drug with other types of conventional cancer drugs.

"Targeted" drugs are poorly-predicted by measuring the ostansible "target," but can be well-predicted by measuring the effect of a drug on the function of live cells, the net effect of all processes, not just the individual molecular targets.

The benefits of newer targeted therapies are marginal. These targeted therapies may impart a clinical benefit by stabilizing tumors, rather than shrinking them (substituting shrinkage for stabilization).

I would not want to be denied treatment with any targeted therapy because of a gene mutation or amplication. Genetic testing (molecular profiling) is not a clear predictor of a lack of benefit.

BTW. The validaton standard that private insurance companies are accepting from molecular profiling tests is accuracy and not efficacy. The "bar" had been instantly lowered. No longer will it be essential to prove that the use of a diagnostic test improves clinical outcomes, all they have to do for these molecular profiling tests is prove that the test has a useful degree of accuarcy. However, the validation standard wanted for functional tumor cell profiling is efficacy. What's good for the goose is good for the gander.

[gdpawel]

Cell-based chemoresponse assays test fresh "live" cells in their three dimensional (3D), floating clusters (in their natural state), not passaged cells (cell-lines). Established cell-line is not reflective of the behavior of "fresh" tumor cells in primary culture in the lab, much less in the patient. Solid tumor specimens are cultured in concical polypropylene microwells for 96 hours to increase the proportion of tumor cells, relative to normal cells.

Polypropylene is a slippery material which prevents the attachment of fibroblasts and epithelial cells and encourages the tumor cells to remain in the form of three dimensional (3D), floating clusters. Real life 3D analysis makes chemoresponse assays indicative of what will happen in the body.

One of the problems with genetic tests is in evaluating the data which exists to validate the predictive accuracy of them. Generally, a large number of archival specimens are batch processed together, within a very narrow time frame, by the same research team, so all the technical variables are minimized, which makes it much easier to get good results than in a "real world" setting, where specimens are tested over a period of weeks, months, years, by different people, with different laboratory reagents, as occurs in the "real world."

Evaluating "real world" data, requires specimens that are tested as they are logged into the lab in question, in "real time." No one is publishing "real world" studies, except private laboratories performing cell-based chemoresponse assays, which can only do "real world" studies, because their studies require fresh, viable specimen, which must be accessioned and tested in "real time," under "real world" conditions.

The "cell-death" assays are not growing anything. They are testing a drug or combinations of drugs with cells that are in their natural state (live or fresh). Three dimensional tumor cell clusters. Clusters maintain natural cell-cell interactions. This makes the assays indicative of what will happen in the body. The protocol takes "fresh" patient tumor cells and floats them in newer 3D cell suspensions.

As the researchers at Johns Hopkins and Washington University have found out, our body is 3D, not 2D in form, undoubtedly, this novel step better replicates that of the human body. Traditionally, in-vitro (in lab) cell-lines have been studied in 2 dimensions (2D) which has inherent limitations in applicability to real life 3D in-vivo (in body) states. Recently, other researchers have pointed to the limitations of 2D cell line study and chemotherapy to more correctly reflect the human body.

Literature Citation:
Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007
Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)