The impact and indications for Oncotype DX on adjuvant treatment recommendations when third-party funding is unavailable.

OBJECTIVES: Industry-supported decision impact studies demonstrate that Oncotype Dx (ODX) changes treatment recommendations (TR) in 24-40% of hormone receptor+/HER2- patients. ODX is not reimbursed by third-party payers in Australia, potentially resulting in more selective use. We sought to evaluate the impact of self-funded ODX on TRs. METHODS: Data collected included demographics, tumor characteristics, indication for ODX and pre- and post-recurrence score (RS) TR. Primary endpoint was frequency of TR change and associations with TR change were sought. RESULTS: Eighteen physicians contributed 382 patients (median age 54). A total of 232 (61%) of tumors were T1 and were grade 1, 2 and 3 in 49 (13%), 252 (66%) and 79 (21%). A total of 257 (67%) were node negative. Assay indications were: confirm need for chemotherapy (CT) (36%), confirm omission of CT (40%) and genuine equipoise (24%). RS was low (≤17) in 55%, intermediate (18-31) in 36% and high (≥32) in 9%. Thirty-eight percent of patients had TR change post-ODX. Sixty-five percent of patients recommended CT pre-ODX changed to hormone therapy alone (HT)-more likely if lower grade and if ER and/or PR > 10%. Fourteen percent of patients with pre-ODX TR for HT added CT-more likely if ER and/or PR ≤10% and if Ki67 > 15% Overall, TR for CT decreased from 47% to 24%. CONCLUSION: Patient-funded ODX changed TRs in 38% of patients, de-escalating 65% from CT to HT and adding CT to 14% of those recommended HT. These changes were greater than an industry-funded study suggesting that physicians can identify situations where the assay may influence decisions.

A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Critical review of the management of early-stage breast cancer in elderly women.

The treatment of elderly women (> or =70 years) with early-stage breast cancer is an emerging clinical problem in the setting of an ageing population. There is a lack of clinical trial evidence to formulate clinical guidelines for management because of the small number of elderly women included in previous clinical trials of adjuvant therapy. This often results in elderly patients being denied standard management based on age alone. The often-complex interaction between age, comorbid conditions and function complicate the planning and outcomes of surgery and can have an effect on the delivery of postoperative adjuvant therapy. A comprehensive assessment of the elderly patient is essential to determine overall prognosis and morbidity risk from treatments; however, a simple comorbidity scale for use in routine clinical practice remains elusive. Thus, treatment decisions should be tailored to the individual to ensure that therapies are not unduly withheld and are appropriate for the patient's overall condition. The assessment of the elderly patient with breast cancer requires the involvement of a multidisciplinary team. The evidence for efficacy, safety and potential risks of surgery and adjuvant therapies (including radiotherapy, hormone therapy and chemotherapy) in the elderly population is discussed in this review and the role of comprehensive geriatric assessment is outlined.

Nonmyeloablative allogeneic stem cell transplantation: early promise and limitations.

Allogeneic stem cell transplantation is used to treat a variety of malignant and nonmalignant hematologic diseases. Conventionally, high-dose chemoradiotherapy-based preparative regimens were considered essential both for tumor eradication and facilitation of donor stem cell engraftment. It is now apparent that an immune-mediated graft-versus-tumor effect has a pivotal role in the curative potential of allogeneic stem cell transplantation. This has prompted the development of less toxic, nonmyeloablative but profoundly immunosuppressive preparative regimens, often fludarabine- or radiation-based. Full donor engraftment can be achieved; however, a significant number of patients achieve a mixed chimeric state. Mixed hematopoietic chimerism provides a platform for the use of adoptive immunotherapy using donor lymphocyte infusions to maximize the immune-mediated antitumor effect, but the optimal usage has yet to be determined. Immediate procedure-related mortality with nonmyeloablative regimens has been low, but graft-versus-host disease remains a major clinical concern and treatment challenge. Major tumor responses have been seen in many hematologic malignancies primarily including patients with highly chemorefractory disease. Follow-up data have been short and additional time is needed to determine the efficacy and toxicities of this immunotherapy. This approach has potential for widespread clinical application including HLA mismatched and matched unrelated donor transplantation, exploration of a graft-versus-solid tumor effect, and correction of phenotypic expression in nonmalignant disorders.

Update on adjuvant chemotherapy for early breast cancer.

Adjuvant chemotherapy represents a significant advance in the management of early-stage breast cancer and, as such, has saved many lives. Worldwide, adjuvant chemotherapy has benefitted all groups tested, including pre- and postmenopausal women, those with node-negative and node-positive disease, and those with estrogen-receptor (ER)-positive and ER-negative disease. However, the significant number of women who relapse despite adjuvant therapy provides the impetus to develop more efficacious regimens. Results from large randomized clinical trials, which will mature over the next few years, will clarify the potential benefits of the taxanes in the adjuvant setting, provide answers as to the efficacy of a dose-dense approach, and define a role, if any, for high-dose chemotherapy. A shift toward targeted therapies has also begun, with the incorporation of trastuzumab (Herceptin) into the adjuvant setting. Minimizing the long-term toxicity of adjuvant therapy for the large number of women who survive their disease is paramount. This article highlights the need to develop predictive factors to help tailor individual therapy.

The regulation of vascular smooth muscle cell apoptosis.

Apoptosis describes the morphological changes that identify a specific form of regulated cell death. Over recent years, the importance of either aberrant onset or suppression of apoptosis within tissues has become apparent and is associated with the development of several terminal diseases. Here we describe the relevance of apoptosis to the maintenance of vascular homeostasis. Specifically, we address the role of vascular smooth muscle cell death, how this may be regulated at the molecular level and whether any of these molecular mediators will provide targets for intervention in diseases such as atherosclerosis.

The opposing roles of the Akt and c-Myc signalling pathways in survival from CD95-mediated apoptosis.

Expression of the proto-oncogene c-myc stimulates cell proliferation in the presence of the appropriate survival factors and triggers apoptosis in their absence; this dual capacity ensures that cell growth is restricted to the correct paracrine environment and is thereby strictly controlled. Recently our laboratory demonstrated that c-Myc-induced apoptosis requires the CD95 death receptor pathway and that insulin-like growth factor (IGF-1) signalling suppresses this killing. To investigate further the links between c-Myc and IGF-1 pathways in CD95-induced apoptosis, we examined the effects of c-Myc and a downstream IGF-1 survival kinase, Akt, on killing mediated by CD95 and its recruited effector proteins (FADD and caspase-8). Here, we show that c-Myc activation does not exacerbate killing induced by FADD or pro-caspase-8, which narrows the point at which c-Myc exerts its action downstream of the interaction of CD95 with its ligand and upstream of FADD. We show further that activated Akt suppresses CD95-induced apoptosis and that Akt exerts its activity at a point downstream of FADD but upstream of caspase-8. These results restrict the possible mechanisms by which CD95-induced apoptosis is modulated by death signals and survival factors.

drICE is an essential caspase required for apoptotic activity in Drosophila cells.

Caspases are involved in the execution of cell death in all multicellular organisms so far studied, including the nematode worm, fruit fly and vertebrates. While Caenorhabditis elegans has only a single identified caspase, CED-3, whose activity is absolutely required for all developmental programmed cell deaths, most mammalian cell types express multiple caspases with varying specificities. The fruit fly Drosophila melanogaster is genetically tractable, less complex than vertebrates and possesses two known caspases, DCP-1 and drICE. The fly may therefore provide a good model system for examining the hierarchy and relative roles of individual caspases in the execution of apoptosis. We have examined the role of drICE in in vitro apoptosis of the D.melanogaster cell line S2. We show that cytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper (rpr) expression or cycloheximide treatment contain a caspase activity with DEVD specificity which can cleave p35, lamin DmO, drICE and DCP-1 in vitro, and which can trigger chromatin condensation in isolated nuclei. Using antibodies specific to drICE, we show that immunodepletion of drICE from these lysates is sufficient to remove most measurable in vitro apoptotic activity, and that re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. We conclude that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis.

Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode "death" gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage-induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing.

The Epstein-Barr virus gene BHRF1, a homologue of the cellular oncogene Bcl-2, inhibits apoptosis induced by gamma radiation and chemotherapeutic drugs.

Analysis of apoptosis, active and controllable cell death, has demonstrated that the size of a cell population can be regulated by changes in the cell death rate as well as in the rates of proliferation and differentiation. Factors which alter the rate of cell death, such as expression of the proto-oncogene bcl-2, can therefore directly affect the number of cells within a population. Bcl-2 has been shown to suppress apoptosis in response to a variety of stimuli and to act as a complementary survival signal for the random acquisition of other oncogenic mutations, such as deregulated c-myc. The Epstein Barr virus (EBV) gene BHRF1 was the first of a family of bcl-2 homologues now being identified. BHRF1 and bcl-2 share 25% primary amino acid sequence homology. Here we show that gamma radiation and several cytotoxic anticancer agents induce apoptosis in Burkitt's lymphoma (BL) cell lines, as has been found in several other systems. Using gene transfection studies we have also shown that expression of either BHRF1 or bcl-2 in BL cell lines significantly suppresses apoptosis in response to a variety of anticancer treatment. This has confirmed that BHRF1 is functionally homologous to bcl-2 in B-cells and suggests that BHRF1 may act to prevent apoptosis during EBV infection, maximising virus particle production, as has been suggested for other human and insect viral genes. Suppression of chemotherapeutic drug induced cell death by bcl-2 and BHRF1 as demonstrated in this cell system, results in resistance to a variety of different agents and may represent an alternative mechanism by which multidrug resistance arises during chemotherapy.

Apoptosis in the development of the immune system: growth factors, clonal selection and bcl-2.

The mammalian immune system is essential for surviving challenge infections with a great range of potential pathogens. The protective effect produced is dependent on many different types of cells which require flexible and independent production and regulation. In particular, many important responses are carried out by lymphocytes, which recognise foreign antigen through exquisitely specific receptors: i.e. surface immunoglobulin (sIg) on B lymphocytes and the T cell receptor (TCR) on T lymphocytes. Each lymphocyte displays receptors with a single specificity, allowing cells with particular specificities to be regulated independently. Since millions of different Igs and TCRs are expressed, the precise selection and regulation of each T and B cell population to produce a useful self-tolerant repertoire is a very complex process. Control of cell populations can, in theory, be exercised at a number of levels, including modulation of active cell death by apoptosis. Recent research has demonstrated that regulation of apoptosis is indeed a crucial element in the control of the immune system in general, and in the development of the TCR and Ig repertoires in particular. The molecular analysis of apoptosis now takes a high priority and the proto-oncogene bcl-2 appears to be responsible for specific suppression of apoptosis in several important situations. It is also clear that malfunctions affecting apoptosis, and in particular bcl-2, can result in significant progression towards malignancy.

Apoptosis: final control point in cell biology.

The discovery of apoptosis, a widespread and morphologically distinct form of physiological cell death, has had an extraordinary impact on cell biology. The importance of apoptosis stems from its active nature and its potential for controlling biological systems. The growing appreciation of the significance of this process has stimulated intense investigation into the molecular mechanisms involved and into its fundamental implications for developmental biology, immunology and oncology.