martes, 10 de agosto de 2010

Biologia del Cancer


Chapter 187 – BIOLOGY OF CANCER
Jeffrey A. Moscow,
Kenneth H. Cowan
GENETIC CANCER BIOLOGY

Cancer is an acquired genetic disease (Chapter 186). Spontaneous genetic changes overwhelm the mechanisms that maintain normal cellular homeostasis, disrupt the normal tight control of cell division and death, and result in the malignant phenotype.
The genetic damage that results in cancer can occur in several ways: translocations of genes can juxtapose two genes in ways that cause dysregulation of their function; mutations can activate cancer-causing genes or deactivate cancer-preventing genes; and epigenetic modifications of proteins that associate with DNA can alter the expression of critical genes (Chapter 186). Sometimes, the first step is the mutation of genes that normally prevent mutations in other genes—cells then quickly gain additional mutations that, through a morbid natural selection, ultimately produce the mutant clone that gives rise to a malignancy.
The very processes that generate malignant transformations also present obstacles to their treatment. The genetic plasticity that can manufacture cancer-causing mutations can also generate mutations that result in resistance to anticancer drugs.
Tumor formation after malignant transformation requires changes in cell biology that, in the processes of invasion and metastases, favor propagation of the malignant cells. Because individual cells, like organisms, are programmed to die, cancer cells must learn to evade the intricate systems of apoptosis that ensure cell death. Cancer cells must also enable the formation of new blood vessels to provide nutrition for the growing mass and develop strategies to escape the immune surveillance that suppresses tumor formation. These processes that give rise to malignant tumors can again raise barriers to successful therapies; for example, cells with impaired apoptosis may be resistant to anticancer drugs that kill cells through activation of apoptosis. However, the distinctive biology of malignancies can also provide opportunities for directed therapies.
Specific Mutations, Targeted Therapies
All cancer cells contain some genomic damage. In most cases, several abnormalities must occur, but sometimes even a single mutation appears sufficient to produce malignant transformation. The latter examples demonstrate clearly that cancer is a genetic disease of dysregulated growth, and they demonstrate that identification of a genetic cause can lead to a specific targeted therapy aimed at the product of the damaged gene.
In the case of acute promyelocytic leukemia (APL; Chapter 194), the characteristic t(15;17) translocation is the only identified genetic lesion. This genetic mishap splices the retinoic acid receptor (RAR) gene to another gene called PML, and the resulting hybrid RAR-PML protein does not function properly. In normal cells, RAR binds to its ligand, vitamin A, which causes the RAR to disassociate with a complex of proteins that repress transcription of certain genes and recruit a different complex of proteins that activate transcription of those genes. In APL, the hybrid RAR-PML protein has lost the capacity to activate gene transcription in response to normal levels of vitamin A. However, the functional defect of the RAR-PML hybrid protein product can be overcome with pharmacologic doses of a vitamin A analogue, all-trans-retinoic acid (ATRA). The addition of ATRA to chemotherapy for APL doubles disease-free survival, from approximately 40% to approximately 80%.
Cancers driven by single mutations appear to be restricted to specific tissue types. The t(15;17) creates malignancy in promyelocytes, but this translocation does not lead to cancer when it occurs in other tissues. Similarly, the accidental genetic recombination that creates the BCR/ABL hybrid protein results in malignant transformation of myeloid cells (Chapters 186 and 195), but this genetic alteration is found only in a limited number of nonhematologic tissues (Chapter 202). Thus, genetic alterations that lead to malignant transformation are also dependent on the cellular context in which they occur.
The Multistage Evolution of Malignancy
Most cancers do not have a single cause but rather are the products of a progression of genetic lesions that can be years in the making, as evidenced by their multiple and complex genetic alterations. The classical model of two stages of cancer development, initiation and promotion, has been replaced by a more dynamic and multistep model in which accumulated genetic damage leads to dysregulation of cell division and the disarming of the mechanisms of cell death.
Often the first step in the creation of a tumor is the development of genomic instability. During each cell division, some 3 billion nucleotide pairs must be faithfully copied to produce exact replicas in each daughter cell. The process of cancer can begin with alterations in any one of a number of factors that influence the accuracy of this process of genetic replication. Two major mechanisms create this acquired genetic damage: spontaneous mutations can disable the machinery that edits DNA replication and removes damaged genes, or, more frequently, cells are exposed to carcinogens that directly damage DNA, and the imprecise repair of damaged DNA results in an increase in spontaneous mutations.
The evidence for the loss of genomic integrity in cancer is exemplified by studies that examine the stability of DNA sequences scattered throughout the genome, that is, microsatellite repeats. These sequences have no apparent role in gene expression or regulation, but they can be viewed as a bellwether for the fidelity of genomic replication. In most tumors, the increase in microsatellite instability, or an increase in the variation of the length of these DNA sequences in tumor tissue versus normal tissues, demonstrates the loss of the ability to replicate the genome faithfully in malignant cells.
Heritable conditions that impair the proteins that ensure genomic fidelity cause a predisposition to a variety of types of cancer. For example, defects in genes that repair DNA damage (Table 187-1) cause disorders such as xeroderma pigmentosum (Chapter 462) and ataxia-telangiectasia (Chapter 271) and are associated with an increased risk of cancer. The colon cancer predisposition syndrome, hereditary nonpolyposis colon cancer (Chapter 203), is caused by inherited defects in a number of related genes that repair DNA mismatches (MSH2, MSH6, MLH1, and PMS2).


TABLE 187-1   -- GENOMIC INSTABILITY GENES ASSOCIATED WITH INCREASED HEREDITARY CANCER RISK
Genes
Syndrome
Hereditary Tumor Types
MUTYH
Attenuated polyposis
Colon
ATM
Ataxia-telangiectasia
Leukemias, lymphomas, brain
BLM
Bloom's
Leukemias, lymphomas, skin
BRCA1, BRCA2
Hereditary breast cancer
Breast, ovary
FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG
Fanconi's anemia A, C, D2, E, F, and G
Leukemias
NBS1
Nijmegen breakage
Lymphomas, brain
MSH2, MLH1, MSH6, PMS2
Hereditary nonpolyposis colon cancer
Colon
XPA, XPC, ERCC2, ERCC3, ERCC4, ERCC5, DDB2
Xeroderma pigmentosum
Skin


Carcinogens are substances that create genomic alterations. As cancer is a genetic disease, carcinogens are genomic toxins that damage DNA and create mutations. The DNA damage from cigarette smoke (Chapter 30) results from exposure to dangerous hydrocarbon products that bind DNA and disrupt faithful replication. Ultraviolet irradiation causes characteristic DNA damage in the skin that can lead to melanoma and other skin cancers (Chapter 214). Ionizing radiation (Chapter 18) from diagnostic and therapeutic radiation can also cause cancer.
One key regulator of genomic integrity is the p53 protein, the product of a tumor suppressor gene that is commonly referred to as the “guardian of the genome.” The p53 protein can sense DNA damage and direct the cell either to cell cycle arrest by increased expression of the cyclin kinase inhibitor p21 (Fig. 187-1), which provides the cell with the opportunity to repair genetic damage prior to cell division, or down the path of programmed cell death (apoptosis) by increased expression of bax, a proapoptotic protein. Inactivating mutations in p53 are among the most common abnormalities observed in cancer, emphasizing the critical role of unrepaired genetic damage in the etiology of cancer. Individuals with inherited mutations of the p53 gene have the Li-Fraumeni syndrome (Chapter 186), with increased susceptibility to specific types of cancer. The retinoblastoma (RB) gene is another tumor suppressor gene whose normal function is to regulate cell growth (see Fig. 187-1). Mutations in a number of other tumor suppressor genes are also associated with specific cancer syndromes (Table 187-2).

FIGURE 187-1  P53 and DNA damage response. Following DNA damage, p53 expression induces the cyclin kinase inhibitor p21, resulting in cell cycle arrest, and bax, which induces apoptosis. Rb, retinoblastoma
TABLE 187-2   -- TUMOR SUPPRESSOR GENES ASSOCIATED WITH INCREASED HEREDITARY CANCER RISK
Genes
Syndrome
Hereditary Tumor Types
P53
Li-Fraumeni
Breast, sarcoma, adrenal, brain,
APC
Familial adenomatous polyposis
Colon, thyroid, stomach, intestine
CDHI
Familial gastric carcinoma
Stomach
VHL
von Hippel–Lindau
Kidney
WT1
Familial Wilms' tumor
Wilms'
PTEN
Cowden's
Hamartoma, glioma, uterus
CDKN2A
Familial malignant melanoma
Melanoma, pancreas
CDK4
Familial malignant melanoma
Melanoma
RB1
Hereditary retinoblastoma
Eye
NF1
Neurofibromatosis
Neurofibroma
MEN1
Multiple endocrine neoplasia type 1
Parathyroid, pituitary, islet cell,
NF2
Neurofibromatosis
Meningioma, acoustic neuromas


The relative impact of heritable predispositions and environmental exposures depends on the frequency of the genetic abnormality and its penetrance. Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 (Chapter 208) are rare, so relatively few women inherit abnormal copies of these gene alleles from their parents, and only approximately 10% of breast cancers can be attributed to BRCA1 and BRCA2 mutations. The mechanisms involved in carcinogenesis induced by BRCA1 and BRCA2 mutations are not clear. BRCA1 is involved in the regulation of gene expression, whereas both BRCA1 and BRCA2 associate with many intracellular proteins that are involved in DNA repair, including rad50, rad51, ATM, and p53 (Fig. 187-2). Cells defective in BRCA1 or BRCA2 display defects both in the response to DNA and in DNA repair.

FIGURE 187-2  Protein interactions with BRCA1 and BRCA2. BRCA1 and BRCA2 act as molecular scaffolds and promote assembly of protein complexes involved in cell cycle regulation in response to DNA damage as well as DNA repair

The penetrance of BRCA1 is high, so that an affected woman has a 60 to 85% lifetime risk for breast cancer. By comparison, other cancer susceptibility genes are both more common and less penetrant, and the interactions among these genes are complex. For example, studies in monozygotic twins estimate that inherited genes may account for almost a third of breast, colorectal, and prostate cancers, with the remaining attributable risk thought to be environmental.
DNA damage, whether from heritable conditions, from unfortunate somatic mutations in the DNA repair mechanisms themselves, or from carcinogen exposure, increases the rate of spontaneous mutations in cells and sets the stage for the natural selection of malignant clones. Although mutations of most of the approximately 25,000 genes in the genome do not result in malignancy, mutations or other disruptions of a few critical genes, which confer a selective proliferative or survival advantage, are frequently altered in many types of cancers.
Disease of the Messenger Proteins
In health, cells respond to external stimuli with complex and redundant protein networks that interact with external stimuli and transmit appropriate signals to the nucleus. The proteins involved in signal transduction include cell surface receptors, second messenger systems, and multiple transcription factors that directly regulate gene expression. Each of these elements of the signal transduction network is controlled by multiple proteins that tightly regulate the activation state of each element of the network. Disruption of any of the genes that encode the proteins involved in signal transduction—proteins that relay the external stimulus to the nucleus—occurs frequently in cancer.
Cell surface receptors for external stimuli involve families of receptor tyrosine kinases, proteins that become activated after binding to specific ligands, often as a result of homodimerization or heterodimerization and subsequent phosphorylation of cytoplasmic proteins. For example, the epidermal growth factor (ERBB) family receptor kinases are often amplified or found activated by mutations in breast, ovary, gastric, and lung cancers. Identification of genetic abnormalities in cancer cells has provided valuable targets for selective therapies. Thus, trastuzumab, a monoclonal antibody directed against the ERBB2/Her2neu protein, which is amplified and overexpressed in 25% of breast cancers, has proved to be very useful in improving disease-free survival and overall survival when given in combination with chemotherapy (Chapter 208). Erlotinib, an inhibitor of ERBB1 (epidermal growth factor [EGF]) receptor, may be effective in treatment of lung cancers that contain mutations in the ERBB1 (EGF) receptor (Chapter 201).
Unregulated growth can also result from mutations that affect downstream signal transduction networks (Fig. 187-3). Receptor tyrosine kinases transmit their signals through biochemical pathways and ultimately drive gene transcription. In some pathways, receptor tyrosine kinases activate Ras proteins, which act as second messengers that amplify and direct signals from receptor tyrosine kinases to other intracellular proteins, which then ultimately generate the cellular response to the stimulus. Ras proteins are guanosine triphosphate binding proteins that sit in the cell membrane, and their activation is tightly regulated in normal cells. Ras mutations, which are frequently found in cancer, typically result in the Ras protein being stuck in the “on,” or activated, position; instead of switching on and off in response to a stimulus, activated (mutated) Ras provides a constant, unregulated stimulus to downstream proteins, which in turn creates a cascading effect that stimulates cell growth. Although mutations in other genes are also commonly found in tumors that harbor Ras mutations, mutation of Ras alone has been shown to transform normal cells into malignant cells. Activating mutations of Ras family proteins can frequently be found in melanoma, myeloid leukemias, and cancers of the colon, pancreas, and lung. Genetic abnormalities are also frequently found in the other signaling pathways that transmit signals from receptor tyrosine kinases, including the PIP3/PDK1/Akt pathway, as well as in the nonreceptor tyrosine kinases signaling pathways, including the Jak/STAT and the c-Src pathways.

FIGURE 187-3  Growth factor–mediated signal transduction. Upon binding to specific receptor ligands, receptor tyrosine kinases activate downstream signal transduction cascades that result in changes in gene expression and enhanced cell growth. Alterations in various steps in the signal transduction cascade are frequently observed in cancer. MAP, mitogen-activated protein.

Disruption of the genes that encode the proteins that regulate gene transcription, which are the downstream targets of signal transduction cascades, can also create malignancy. For example, amplification of the Myc family of transcription factors is frequently observed in neuroblastoma and in cancers of the lung, bladder, breast, stomach, and colon. Certain acute leukemias (Chapter 194) arise when the normal gene recombination process that produces diverse immune responses goes awry and an immunoglobulin or T-cell receptor locus is accidentally spliced onto a gene encoding a transcription factor, resulting in loss of regulation of the transcription factor activity. Several pathognomonic oncogenic chromosomal translocations commonly found in sarcomas also involve genes for transcription factors; for example, the t(11;22) of Ewing's sarcoma (Chapter 212) creates the Fli1-EWS hybrid transcription factor, and the characteristic t(2;13) of alveolar rhabdomyosarcoma creates the hybrid Pax3/FKHR DNA binding protein.
Suppression of Tumor Suppressors
Normal cells contain proteins that prevent malignant transformation. Inactivation of the tumor suppressor genes that encode these proteins also leads to cancer. Because a loss of function is required for a malignant effect, both copies of the tumor suppressor gene must be affected. In almost all cases, patients have a physical loss of one copy of a gene and an acquired mutation of the other. The spontaneous deletion of genetic material, called loss of heterozygosity, is a frequently observed genetic abnormality in tumors.
Some familial cancer predisposition syndromes are based on the inheritance of one damaged copy of a tumor suppressor gene (Chapter 186). Hereditary retinoblastoma, which results from inheritance of a mutated RB gene, and the Li-Fraumeni syndrome (Chapter 186), which results from inheritance of a mutant p53 gene, predispose affected individuals to cancers. Both the RB and p53 proteins play critical roles in regulating the progression of proliferating cells through the cell cycle, and the loss of this checkpoint regulation can contribute to uncontrolled cell growth and cancer.
The importance of the p53 and RB signaling pathways in tumor suppression is further revealed by the mechanism by which infection with the human papillomavirus (HPV; Chapter 396) in cervical epithelial cells leads to cervical cancer (Chapter 209). HPV proteins E6 and E7 inactivate p53 and RB proteins, respectively, thereby creating a virally induced premalignant state in which the machinery that prevents damaged cells from proliferating has been turned off. For this reason, vaccination against certain HPV serotypes holds the promise of preventing most cases of cervical cancer.
DISTINCTIVE CANCER BIOLOGY
Cancer cells do not behave like normal cells. The alterations in the regulation of cell growth, differentiation, and death that give rise to cancer also produce common abnormal biologic characteristics, which are shared by tumors that arise from different cells of origin. These common features conspire to allow the transformed cell to grow into a tumor.
Normal cells are programmed to differentiate and ultimately to die, and this programming is regulated through enzymatic pathways that lead to terminal differentiation, senescence, or apoptosis. Cancer cells evade the mechanisms that are designed to steer cells toward terminal differentiation and senescence by altering the function of telomerase. As primitive cells divide and differentiate, the ends of chromosomes, called telomeres, progressively shorten and ultimately lead to a growth arrest that is termed replicative senescence. An enzyme called telomerase adds length back to the telomeres and reverses the process of replicative senescence. Telomerase is usually expressed at significant levels only in stem cells. However, telomerase is highly expressed in most malignant tissues, demonstrating a common alteration of cell biology that is necessary for creation and maintenance of the malignant phenotype.
In addition to bypassing senescence, cancer cells disable the pathways that lead to apoptosis. Because apoptosis is literally a life-and-death decision for the cell, it must be tightly controlled by intricate pathways of regulatory proteins. Apoptosis can be triggered through either external or internal pathways that converge to activate a family of enzymes, called caspases, that systematically degrade cellular proteins and DNA in a characteristic pattern resulting in cell death. Cancer cells contain many common aberrations in the machinery that regulates apoptosis, including increased activity of antiapoptotic proteins, such as Bcl-2 and Mcl-1, or increased levels of inhibitors of apoptosis, such as the protein survivin, which inhibits caspase activity.
Epigenetic changes in gene expression are also hallmarks of malignancy (Chapter 186). In normal cells, gene expression is controlled by epigenetic processes that limit the physical accessibility of genes to transcription factors. Gene expression can be silenced by processes that methylate specific DNA sequences on chromosomes, called CpG islands. In cancer, tumor suppressor genes are frequently found to be abnormally methylated, leading to a loss of their expression and function. The drug 5-azacytidine and the newer agent decitabine reverse methylation and have activity in the myelodysplastic syndrome (Chapter 193) and leukemia. However, drug resistance genes may also be methylated, and strategies to reverse global methylation may have uncertain effects on cellular sensitivity to chemotherapy. Gene expression is also controlled by histone acetylases, which influence how tightly genomic DNA is spooled around large complex nuclear proteins called histones and alter the interaction of chromatin proteins with DNA. A novel group of drugs called histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid and depsipeptide, are currently under development as novel anticancer agents.
Cancer cells also demonstrate characteristic alterations in glucose metabolism, known as the Warburg effect. Malignant cells tend preferentially to shunt glucose into the glycolytic pathway, taking up excessive amounts of glucose and metabolizing glucose into lactate instead of channeling glucose into the typical aerobic pathway that more efficiently captures energy and creates the end products of carbon dioxide and water. This disruption of normal cellular energy metabolism is thought to be due to dysregulation of a gene that is also involved in the regulation of apoptosis, Akt, and its downstream effectors. This abnormal metabolism of glucose in cancer cells is also the principle behind the use of positron emission tomographic scans to image tumors. Inherited inactivating mutations of the phosphatase and tensin homologue (PTEN), which inactivates Akt, are also responsible for Cowden's syndrome (Chapter 186), in which the susceptibility to breast and thyroid cancers is increased.
Normal cells have tightly regulated mechanisms to degrade and recycle proteins by attaching one or more ubiquitin molecules to a protein. Ubiquitinylation can serve as a signal that a protein should be trafficked to and degraded by lysosomes or the proteasome. Ubiquitinylation plays an important role in the regulation of receptor tyrosine kinases, in cell cycle progression, and in repair of DNA damage. This regulatory mechanism is altered in many types of cancer. The first anticancer drug targeted at this abnormality in cancer cells is bortezomib, which inhibits proteasome activity and is effective in the treatment of multiple myeloma (Chapter 198).
Because tumors must find mechanisms to feed themselves as they grow, the malignant transformation must include the ability to stimulate new blood vessel formation, or angiogenesis. Malignant cells have in common the ability to stimulate the formation of endothelial cells and the breakdown of extracellular membranes, often by secreting vasculature endothelial growth factor (VEGF). The resulting tumor vasculature, although functional, does not have the vessel architecture or endothelial wall characteristics of a normal vascular bed. The realization that tumor cells have unique angiogenesis has led to the novel therapeutic approach of targeting tumor vessel formation, and not the malignant cell itself, such as with the monoclonal anti-VEGF antibody bevacizumab in metastatic colorectal cancer (Chapter 203).
Tumors also develop strategies to evade immune surveillance. The T cells and natural killer cells of the immune system play a role in protecting the host against malignancy, just as they also protect against infectious agents. Tumors can grow unimpeded after malignant cells have been selected with properties that disarm the host's immune response to tumors; these mechanisms include down-regulation of costimulatory and major histocompatibility complex molecules, as well as secretion of cytokines that inhibit the immune system, such as transforming growth factor β, interleukin-10, and signal transducer and activator of transcription 3 (STAT3). The development of therapies designed to harness the immune system for cancer treatment, including tumor antigen vaccines, has been hindered by properties that allow tumors to escape immune destruction.
Drug Resistance
Genomic instability sets the stage for the natural selection of cells with acquired genetic alterations that permit dysregulated growth. The same genetic plasticity also allows cancer cells access to the repertoire of the human genome and its capability to express and mutate any of its genes—sometimes resulting in resistance to anticancer therapy.
Cancer cells can specifically alter the target to become resistant. In the case of imatinib mesylate, the targeted therapy for chronic myeloid leukemia (Chapter 195), cells can become resistant by mutating the binding site of the drug to the already mutant BCR/ABL protein. Cancer cellular resistance to older targeted drugs such as methotrexate, which targets the enzyme dihydrofolate reductase, can be mediated by multiple steps in the folate metabolic pathway.
Cancer cells can also reach into their genomes and call upon more general mechanisms of protection from stress. Normal cells can upregulate genes to protect against environmental toxins. In cancer, malignant cells use the same proteins to evade chemotherapy. For example, when exposed to lipid-soluble anticancer drugs that diffuse through the cell membrane, cancer cells respond by increasing the expression of cell membrane proteins that can pump a wide variety of anticancer drugs out of the cell. These ATP-dependent efflux drug pumps, which create resistance to multiple drugs, include MDR1 (for multiple drug resistance) and the MRP (for multidrug resistance–related protein) family of transmembrane proteins. Cancer cells also use other detoxification pathways, such as those involving glutathione, for protection against chemotherapy.
For every anticancer drug developed, a cancer cell has found a way to circumvent it. Also, because cancer cells recruit many different physiologic mechanisms that are also used by different tissues within the host, clinical approaches to overcoming drug resistance have largely been disappointing, as drug resistance reversal strategies also reverse the protective mechanisms in normal tissues and lead to increased toxicity. In this way, the genomic plasticity of cancer cells and the rich human genomic repertoire, the very properties essential for malignant transformation, are also properties that make cancer a difficult therapeutic challenge.
Cancer Stem Cells
Tissues are composed of a vast majority of cells that are irreversibly committed toward terminal differentiation. Hidden within tissues are also a very small minority of primitive, seemingly nondescript cells that are capable of repopulating the tissue with new cells and have the potential for self-renewal, that is, the ability to divide without differentiating. Cells that possess both the ability to produce the different, specialized cells of a tissue and the additional capacity for self-renewal are called stem cells (Chapter 160). For example, a very small population of hematopoietic stem cells in the bone marrow gives rise to more committed erythroid, myeloid, and lymphocytic lineage progenitor cells that ultimately undergo terminal differentiation to produce the formed elements in blood and have the ability to repopulate the marrow, such as in a stem cell transplantation (Chapter 184).
The view of cancer as a homogeneous mass of clonally derived malignant cells has been replaced by a view that cancer cell division is organized more like a tissue in that most of the cells of a tumor do not have the capacity for self-renewal but rather are the progeny of a minute population of cancer stem cells. In hematopoietic malignancies and other tumors, flow cytometric techniques have demonstrated small, unique populations of cancer cells within tumors that have the ability to re-form the tumor, whereas the vast majority of cells in a malignancy do not have this potential.
This concept of cancer, in which tumors contain small numbers of malignant stem cells and a vast majority of malignant but biologically differentiated cells, has enormous consequences for the study and treatment of cancer. Most important, all previous studies that have examined the overall expression of genes in a malignancy may not reveal critical and unique characteristics of the cancer stem cells from which the tumor has arisen. The pattern of treatment response followed by treatment relapse may be due not only to the acquisition of drug resistance but also to a failure of the therapy to treat the distinctive biology of the cancer stem cell. Isolation and characterization of cancer stem cells derived from different malignancies may reveal new cancer stem cell–specific targets that distinguish them from both their malignant progeny and normal stem cells. Specific therapeutics aimed at cancer stem cells hold the potential for markedly improving cancer therapy.

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