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Novel strategies and therapeutics for the treatment of prostate carcinoma

2000; Wiley; Volume: 89; Issue: 6 Linguagem: Inglês

10.1002/1097-0142(20000915)89

1097-0142

Michael J. Morris, Howard I. Scher,

Ubiquitin and proteasome pathways

AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract BACKGROUND An increased understanding of the biology of prostate carcinoma has led to the clinical evaluation of mechanism-based and targeted therapies. Modulating the immune system has been pursued through the use of both active and passive immunity as well as the ex vivo genetic manipulation of effector cells. A variety of gene therapies has been proposed not only to replace defective genes but to localize activation of prodrugs. Angiogenesis and tumor invasion also have been targeted, as have cell cycling and signal transduction. Strategies promoting apoptosis and augmenting differentiation are also under study. METHODS This study is a review of current clinical strategies using biologic, immunologic, and genetic approaches for the treatment of prostate carcinoma. RESULTS The clinical development of therapy targeting differentiation, apoptosis, cell signaling, angiogenesis, metastasis, immune surveillance, and others are in various stages of clinical development. A disease states model is used to discuss treatment groups, outcome measures, and other trial design elements in relation to specific therapeutic strategies. CONCLUSIONS Development of novel agents requires consideration of where in the natural history of the disease they should be applied. In addition, understanding the genetic and molecular alterations that occur as the disease progresses from a localized to a metastatic state, and from androgen dependence to independence, is necessary. Clinical trial design will require consideration of cytostatic and cytotoxic effects, the status of pathways not directly targeted, and potentially unexpected influences on prostate specific antigen expression by these agents. Cancer 2000;89:1329–48. © 2000 American Cancer Society. The outlook for patients with prostate carcinoma is changing. No longer are drug development efforts focused exclusively on hormonal approaches that are unlikely to alter the survival equation significantly. It is now recognized that androgen-independent prostate carcinomas are not as resistant to therapy as previously believed, and many groups are reporting response proportions in excess of 50% on a consistent basis by using combination chemotherapy.1-4 The definitive trials to assess the value of these approaches are ongoing. Through focused efforts on the biology of the disease, we now have available approaches designed to modulate the immune system, inhibit the metastatic cascade, block angiogenesis, promote apoptosis, alter signal transduction, and induce tumor differentiation. At issue is how best to prioritize these approaches and to determine at what point in the natural history of disease they are best evaluated. Cancer is a dynamic and evolving process. To begin to characterize this evolution, we have proposed a model of clinical states that considers the natural and treated history of the disease from a hormone-naive primary to lethal androgen-independent metastases.5 In this schema, illustrated in Figure 1, specific targets may be present at different frequencies within states or across states. Central to management is the direct characterization of tumors representing the point in the history where a specific therapy is considered. For this reason, target selection must be made in conjunction with genetic and biologic profiling of the tumor in each clinical state. Furthermore, because many of these strategies may slow tumor growth without inducing tumor regression in the classic sense, the endpoints required to demonstrate “efficacy” differ from those used in the evaluation of conventional cytotoxic drugs. The ambiguities in establishing endpoints complicate the judgement of when a treatment is good enough to justify large-scale Phase III comparisons. Figure 1Open in figure viewerPowerPoint The clinical states model illustrates how any given patient can represent a shifting target for the purposes of developing novel therapeutics. PSA: prostate specific antigen; Rx: therapy. IMMUNOLOGIC APPROACHES The metastatic process involves invasion of the basement membrane, detachment from the primary tumor, survival in the circulation, adhesion to an endothelial cell in a distant site, invasion, and proliferation (Figure 2). For a tumor to grow beyond a certain size, establishing a vascular supply is also necessary. In prostate carcinoma, as in other cancers, metastases spread via the lymphatics or directly through the vasculature. This micrometastatic phase is most commonly manifest as a rising prostate specific antigen (PSA) in the absence of detectable disease using conventional imaging studies. Such a scenario is unique in oncology because it represents a state of minimal tumor burden in healthy patients in whom the disease can be monitored. The utility of standard hormonal therapy early in the disease course remains controversial. Any potential benefits must be balanced against the side effects of treatment, especially in patients who are otherwise asymptomatic. This clinical state provides an ideal setting in which to explore immunologic approaches, and several are under evaluation (Table 1). The targets vary. Some, such as prostate specific membrane antigen (PSMA), PSA, or prostate stem cell antigen (PSCA) are relatively prostate specific. Others are oncogenic proteins, altered tumor suppressor gene products, and differentiation antigens that are not cancer or organ site specific. For the latter, relative cancer specificity is achieved because normal sites of expression, typically the luminal surface of the gut, are not accessible to the immune system, limiting the potential consequences for autoimmune phenomena. Some targets are therapeutically appropriate only within specific disease states whereas others are valid across various phases of the disease. For example, targets associated with metastatic disease, such as BCL-2, may have narrower applications than those expressed in a variety of disease states, such as PSMA. Figure 2Open in figure viewerPowerPoint Noncytolytic treatments are now available in the therapeutic armamentarium. However, the issue of when in a patient's disease course agents of various mechanisms should be applied has yet to be determined. Table 1. Immunologic and DNA-Based Strategies for the Treatment of Prostate Carcinoma Strategy Target KLH + QS-21 conjugates vaccine MUC1, sTn, Globo H, GM2, TF6, 13 T-cell activation PSMA180 Monoclonal antibodies STn, PSMA, TAG72 Tumor cell vaccines transfected with cytokines Expression of GM-CSF, IL-2, gamma interferon20-23 Pulsed dendritic cells PSMA24 Replacement of faulty genes p5333 Activation of prodrugs HSVtk/ganciclovir27 Viral (vaccinia) induction of antibody response PSA180 Genetically engineered replication-competent herpes virus Viral induced oncolysis181 KLH: keyhole limpet hemocyanin; PSMA: prostate specific membrane antigen; GM-CSF: granulocyte macrophage–colony stimulating factor; HSVtk: herpes simplex thymidine kinase gene; PSA: prostate specific antigen. Active and passive immune approaches directed to all of these targets are ongoing. The strategies include synthetically produced and chemically modified cell surface differentiation antigens, infusions of “cold” and radiolabeled monoclonal antibodies, genetically modified T cells and/or dendritic cells, and other gene therapy approaches. Active Immunity Strategies that induce active immunity are predicated on stimulating the patient's own immune system to recognize cancerous cells that have otherwise escaped immune surveillance. A variety of surface molecules, including glycoproteins (MUC1 and PSMA) and some carbohydrates (Globo H, sTn, and Ley), reside on the cell surface of prostate carcinomas and other cells.6 MUC1 is a glycoprotein that is highly expressed on the apical membranes of the bronchus, breast, salivary gland, pancreas, prostate, and uterus. It also is expressed, to a lesser degree, on gastric, gallbladder, small intestine, and colonic epithelium. In many adenocarcinomas, mucins are expressed aberrantly, exposing different portions of the peptide core.7 MUC1 was isolated and synthetically produced, but when administered as a 32-peptide fragment, was not immunogenic.8, 9 Linking the modified antigen to keyhole limpet hemocyanin (KLH) and injecting the conjugate with the saponin fraction QS-21 as an immune adjuvant consistently enhances immunogenicity and induces a specific antibody response.10, 11 Pilot trials are ongoing at Memorial Sloan-Kettering Cancer Center (MSKCC) to test the safety and the specificity of the immune response, and to understand the timing of immune response in relation to changes in PSA as an initial clinical endpoint. Patient eligibility is limited to those who have undergone definitive local therapy (surgery or radiation) with rising PSA levels and no clinical evidence of disease on imaging studies. Our preliminary results have shown that a prostate carcinoma specific immune response can be elicited with few side effects, and that the rate of rise of PSA can be modulated favorably in some patients.12, 13 The clinical significance of this effect on the PSA is not known. Phase II trials are ongoing, and definitive Phase III trials using a multiple antigen vaccine are planned. Passive Immunity Passive immune strategies that have been evaluated include the mouse monoclonal antibody CC49 that recognizes the tumor-associated glycoprotein TAG72, which also is expressed on a range of glandular tumors. A pilot study using radiolabeled 131I-CC49 in patients with colon carcinoma showed good tumor localization, but most patients developed a human anti-mouse antibody (HAMA) response, which limited repeat dosing.14 A Phase I trial in patients with prostate carcinoma also showed good tumor localization and palliation of pain.15 At MSKCC, 16 androgen-independent patients were pretreated with interferon gamma for 1 week to enhance antigen expression, followed by treatment with CC49. Myelosuppression was dose limiting. Although no major responses were observed, overall radiation delivery was estimated to be subtherapeutic. An important observation in this trial was a mismatch between the images obtained from the antibody scan relative to conventional bone scanning. In several cases, a bone marrow phase of the disease was identified, showing the importance of direct tumor targeting relative to approaches that are directed exclusively at the bone tumor interface or the bone stroma. Repeat dosing was again precluded by the development of a HAMA response.16 Monoclonal antibodies to PSMA, a 100-kilodalton cell surface glycoprotein expressed most commonly on prostate epithelial cells, are also under study. A monoclonal antibody, 7E11, raised against an intracellular epitope of PSMA forms the basis of the ProstaScint scan, which is Food and Drug Administration (FDA) approved to detect occult prostate carcinoma metastases.17 Several anti-PSMA antibodies have been developed against both intra- and extracellular epitopes, shown in Table 2. Liu et al. reported on the first antibodies to target external epitopes of the PSMA molecule.18 These antibodies bind to LNCaP cells in vitro as well as to prostate epithelial cells. In addition, they strongly react with vascular endothelium in a variety of tumors, including lung, colon, and breast. There is no reactivity with normal endothelium. J591 is one of the several monoclonal antibodies that bind to the extracellular domain of PSMA18 and is in clinical trials both as a “cold” antibody and as a conjugate with 131I. Table 2. Antibodies Targeting PSMA Ab Domain 7E11 Intracellular J591 Extracellular J415 Extracellular J533 Extracellular E99 Extracellular PEQ226.5 Extracellular PM2J004.5 Intracellular From Chang et al.182 PSMA: prostate specific membrane antigen; Ab: antibody. Gene Therapy The proliferation of malignant prostate epithelial cells at low tumor volumes suggests that these cells can escape immune surveillance, or that they are not immunogenic. This may be attributed in part to the finding that prostate carcinoma cells have impaired production of major histocompatibility class (MHC) I molecules.19 Genetic based immunizations are being explored as a means of enhancing the immunologic profile of the tumor by “vaccinating” patients with tumor cells that contain sequences encoding cytokines and other immunostimulants. The feasibility of the approach was demonstrated in the Dunning rat prostate carcinoma model using irradiated prostate carcinoma cells transduced with DNA encoding granulocyte macrophage–colony stimulating factor (GM-CSF), interleukin (IL)-2, or gamma interferon.20, 21 Rats vaccinated with IL-2 secreting tumor cells had the greatest prolongation of survival and cure rate after receiving the vaccine.21 Similarly, a clinical trial is ongoing at MSKCC using allogeneic tumor cells transduced with sequences encoding IL-2 and gamma interferon.21, 22 Using a different vector system, investigators at Johns Hopkins have been treating men found to have metastatic disease while undergoing prostatectomy. These patients were treated with irradiated GM-CSF–secreting allogeneic prostate carcinoma cells transduced ex vivo with the MFG-S–GM-CSF vector. Both T-cell and B-cell immune responses were elicited against prostate carcinoma antigens.23 Other groups have focused on ex vivo activation of immune effector cells that have been stimulated to recognize specific sequences of PSA and PSMA. One used autologous dendritic cells that were pulsed with histocompatibility antigen A0201 specific PSMA peptides. Treated patients included those with local recurrence after local therapy and patients with hormone-refractory metastatic disease. Thirty percent of the 62 patients responded, for a mean of 149 days for patients with local disease and 187 days for those with metastatic disease.24 GENE THERAPY General Principles Gene therapy is an umbrella term that describes a range of strategies. Approaches include replacing deleted or mutated genes, altering protein transcription, modifying conversion of prodrugs to active drugs by enzymatic activation, and reinfusing attenuated tumor cells that express cytokines (described earlier). Prostate carcinoma is particularly amenable to gene therapy because the tumor is easily accessible for local treatments, and specific proteins—namely, PSA and PSMA—can be targeted for systemic treatments.25 The means of transferring the genetic material into the tumor cells is variable. Nonviral means include plasmid–liposomal complexes or a gene gun, although the effects are transient and transfer efficiency needs to be enhanced. Alternatively, several viral vectors, including retroviruses, adenoviruses, and poxviruses, have been used.26 In the neoadjuvant setting, gene therapy has been applied in an attempt to maximize definitive local therapy.27 Others have evaluated the approach in patients who have failed radiation therapy. These local interventions are being assessed primarily for effects on the primary site. Ultimately, more effective means of systemic targeting will be required. Gene Replacement p53 has a variety of functions in a normal cell, including the mediation of G1 arrest, induction of p21-induced cyclin, and inhibition of cyclin-dependent kinases.28 Studies of primary and metastatic tumors in patients with prostate carcinoma reveal that p53 mutations are late events in prostate carcinoma development and progression and are associated with a poorly differentiated phenotype, metastatic disease, and androgen independence.29 Although primary carcinomas contain heterogeneous p53 mutations, there appears to be clonal selection during metastatic progression.30 Preclinical studies show that cells harboring mutant p53 undergo apoptosis after transfection with wild-type p53 in a recombinant adenoviral vector.31, 32 This approach is being evaluated clinically by Logothetis et al. who are delivering Ad5CMV-p53 by intraprostatic injection to patients with localized prostate carcinoma. Of the 14 patients who completed therapy, 3 had a reduction in tumor size that exceeded 25% as measured by transrectal sonography. Confirmation of apoptosis and pathologic correlations are pending.33 Gene Therapy to Direct Prodrug Activation Another use of gene therapy is the direct activation of a prodrug to its activated cytolytic state, as illustrated in Figure 3. The most studied model is the herpes simplex thymidine kinase gene (HSVtk). HSVtk converts the prodrug ganciclovir to ganciclovir monophosphate, which is then further phosphorylated to the active drug ganciclovir triphosphate. Not only does the transfected cell die, but surrounding cells then also undergo apoptosis as a result of the bystander effect, most likely mediated by cell–cell interactions. A Phase I trial using this approach recently has been completed at the Baylor College of Medicine in Houston, Texas. Using an adenoviral vector, investigators introduced the HSVtk gene into prostatic tissue with intratumor injections. Eighteen patients, all with rising PSAs without measurable disease after radiation therapy, were treated. Ganciclovir then was administered intravenously for 2 weeks. Three patients had an objective response of a decline in PSA of greater than 50%. The therapy was well tolerated.27 Figure 3Open in figure viewerPowerPoint Gene therapy can be used to induce cells to convert cytotoxic prodrugs to their active form. The resulting cell death induces neighboring cells to also die through cell–cell interactions, although they do not contain the active gene. TARGETING TUMOR GROWTH AND INVASION Recently, investigators have started using antimetastatic and antiangiogenic agents in clinical settings, as shown in Table 3. Unfortunately, despite significant media coverage, there has been little focus on the complexity of the trials necessary to show favorable treatment effects. This is because most of the preclinical studies have shown a “slowing of the rate of progression” without stabilization or regression of established disease. Diminution of growth rate or tumor invasiveness is a difficult endpoint to assess. The identification of relevant and appropriate intermediate endpoints is therefore an area of active study. It is possible that safety will be an easier quality to define than biologic activity and clinical efficacy. As such, many of these agents are being developed in combination with other “standard” approaches such as hormonal therapy and chemotherapy. In fact, cytotoxic drugs used to treat prostate carcinoma, such as paclitaxel and estramustine, have independent antiangiogenic and antimetastatic activity.34-39 The class of drugs to be used in combination with the angiogenesis inhibitors and antimetastatic agents will need to be defined, as will the correct sequencing, appropriate dosing, and duration of therapy. Finally, although there is a presumption that these drugs will be nontoxic relative to traditional chemotherapy, it is unknown what effect inhibition of angiogenesis will have on an aging male population whose primary cause of death is cardiovascular disease. These patients may be reliant on neovascularization for long term survival. Table 3. Drugs under Development that Act against Either Angiogenesis or Tumor Invasion Mechanism Drug Sponsor Mechanism Direct inhibition of endothelial cells TNP-470 TAP Pharmaceutical, Deerfield, IL Synthetic analog of fumagillin; inhibits endothelial cell growth Thalidomide Calgene, Warren, NJ Unknown Squalamine Magainin Pharmaceuticals, Plymouth Meeting, PA Extract from dogfish shark liver; inhibits sodium-hydrogen exchanger, NHE3 Endostatin EntreMed, Rockville, MD Inhibition of endothelial cells Angiogenesis inhibitors Marimastat British Biotech, Annapolis, MD Synthetic inhibitor of MMPs AG3340 Agouron; LaJolla, CA Synthetic MMP inhibitor COL-3 Collagenex, Newton, PA/NCI Synthetic MMP inhibitor and tetracycline derivative Inhibition of activators of angiogenesis Anti-VEGF antibody Genentech, South San Francisco, CA Monoclonal antibody to VEGF PTK787/ZK Novartis; East Hanover, NJ Block VEGF receptor signaling Adapted from Angiogenesis Inhibitors in Clinical Trials, National Cancer Institute Cancer Trials web site, (http://CancerTrials.nci.nih.gov). MMP: matrix metalloproteinase; VEGF: Vascular endothelial growth factor. Antimetastatic Agents For a malignant cell to acquire invasive potential, it must break tissue boundaries, circulate, interact with the vascular endothelium, and proliferate to form distant colonies. These processes all involve extensive interactions both between cells and between the tumor and the extracellular matrix (Fig. 4) that are carefully orchestrated. In a nonmalignant state, such interactions protect tissue integrity and boundaries. In a malignant state, this process is dysregulated, as is the balance of tissue proteases and inhibitors. Through a series of discrete steps involving the release of proenzymes that are activated, and changes in cell adhesion and cell migration, tumor invasion is achieved.40 Integral to this process are plasminogen activators and members of the matrix metalloproteinase (MMP) superfamily. Figure 4Open in figure viewerPowerPoint To achieve invasive growth and metastasis, the cell must encroach on the basement membrane and extracellular matrix. This process involves attachment, proteolysis, and migration, which are mediated by integrins, cadherins, and other molecules. Plasminogen activators and MMPs have been implicated in the invasive potential of prostate carcinoma in both animal and human studies. Extracellular matrix degradation and urokinase-type plasminogen activator activity in cell lines derived from the Dunning rat prostate model correlate with in vivo metastatic behavior.41 In addition, production of MMP-2, MMP-9, and urokinase correlate with invasive capacity in several prostate carcinoma cell lines.42 Studies of prostate carcinoma patients reveal significant disruptions in the normal physiologic levels of collagenase. In a study by Baker et al., sera from 40 patients with prostate carcinoma (22 with metastatic and 18 with localized disease) were analyzed for collagenase and compared with controls. Those with prostate carcinoma had significantly higher collagenase levels than those without, as did those with metastatic disease compared with those with localized disease. TIMP-2, an MMP inhibitor, was suppressed in the cancer patients also.43 Marimistat (British Biotech, Annapolis, MD) is the first oral agent to undergo testing in clinical trials. A dose finding study has been completed in hormone refractory patients. Eighty-eight men were treated with escalating doses. The drug was well tolerated, with arthralgias as the most common toxicity, none higher than Grade 2. There was a statistically significant decrease in the rate of rise of the patients' PSA while undergoing treatment.44 Batimistat (British Biotech, Annapolis) is another MMP inhibitor that has shown promise in the laboratory.45 Although the MMP inhibitors appear to warrant further clinical development, other strategies to inhibit metastasis are under investigation. Citrus pectin is a plant fiber derived from citrus fruit. When its pH is modified, the polysaccharide, rich in galactosyl residues, is cleaved into smaller residues. Modified citrus pectin appears to inhibit cell–cell interactions that are mediated by galectins, which are carbohydrate binding proteins. Pienta et al. have shown that oral administration of modified citrus pectin reduced the metastatic spread of cancer in the Dunning rat prostate carcinoma model. Local growth was not affected.46 A wholly different approach to preventing metastatic disease focuses not on the abrogation of the extracellular matrix, but on downstream events. Endothelin-1 is a vasoconstrictor found in high concentrations in semen and in prostatic epithelium that enhances tumor growth in prostate carcinoma cell lines, binds to receptors on osteoblasts and induces bone matrix formation, and may play a role in the pathogenesis of metastatic prostate carcinoma with bony disease and symptoms.47 The tolerability and preliminary efficacy of ABT-627, an antagonist for the endothelin receptor A, was tested in a Phase I trial for patients with androgen-independent prostate carcinoma. The drug was well tolerated, and 19 of 26 patients treated had either a decline in PSA or radiographically stable disease.48 Antiangiogenic Therapy Angiogenesis describes a process of new vessel formation necessary for a tumor to grow larger than 2–3 mm49 or approximately 1 million cells.50 To exceed this critical mass, defined as the “angiogenic window,” the tumor must establish new vessels. The role of angiogenesis in tumor growth and metastasis has been under study for almost 3 decades,50 but it is only now that drugs are beginning to enter into clinical trials. The angiogenic factors vascular endothelial growth factor (VEGF), IL-8, and fibroblast growth factor (FGF) have all been implicated in the growth of prostate carcinoma.51-54 Preclinical data demonstrate that antiangiogenic treatments may have activity against prostate carcinoma. The angiogenesis inhibitor TNP-470 acts on the cyclin-dependent kinases and retinoblastoma gene product and inhibits endothelial cell proliferation.55 As a single agent, it inhibits prostate carcinoma growth in a dose-dependent manner in mice xenografted with PC-3 cells, and synergy was observed when TNP-470 was combined with cisplatin.56 In patients with androgen-independent disease, TNP-470 was well tolerated with no significant toxicities. There was a suggestion of activity in that several patients had a plateau of the slope of their PSA and 1 patient had a PSA decline that exceeded 50%.57 Another strategy to inhibit angiogenesis is to target proangiogenic factors such as VEGF. A recent study demonstrated that antibodies that neutralize VEGF had no effect on the cell growth of DU-145 cells in vitro. However, when DU-145 cells were injected into severe combined immunodeficiency mice, growth of established primary tumors and the progression of metastatic disease were inhibited.58 Phase II trials of anti-VEGF neutralizing antibody are ongoing. In a recent study reported in abstract form, patients with hormone refractory prostate carcinoma were treated with humanized monoclonal anti-VEGF antibodies at a dosage of 10 mg/kg every 2 weeks. Although the therapy was well tolerated, there was little evidence of clinical benefit.59 More potent inhibitors of endothelial cell proliferation such as endostatin and angiostatin have entered clinical testing. The mechanism of action of both these drugs has yet to be established, but preclinical testing has shown regression of a range of established xenografts including prostate, colon, breast, lung, and sarcoma.60 Some traditionally cytotoxic drugs, particularly those that act on microtubules, also inhibit angiogenesis.37-39 These agents will require clinical testing both alone and with other biologic agents to maximize their cytotoxic and antiangiogenic properties. ALTERING CHEMORESISTANCE AND ANDROGEN INDEPENDENCE Recent work has shown that among the mechanisms associated with the development of androgen independence are continued signaling through a functional but altered androgen receptor, ligand-independent activation of the receptor by receptor tyrosine kinase signaling molecules, inhibition of apoptosis, and increased expression of markers of drug resistance. An essential component of the therapeutic approaches currently under evaluation is an understanding of the molecular and phenotypic differences between a tumor that is proliferating in the primary versus a metastatic site. This involves multiple genetic events. In parallel with the evolution from a localized to a metastatic tumor is the proliferation of cells that are resistant to androgen withdrawal. To characterize these events, attention must be paid to the material studied. Not finding a particular genetic alteration in an untreated primary tumor does not rule out its importance in the progression of the disease to a metastatic site or in the evolution of hormone independence. Most reports have evaluated cell lines, primary tumors, or regional lymph nodes removed during the performance of a radical prostatectomy; few studies evaluate distant metastases either before or after androgen deprivation. Several genetic alterations are known to be associated with metastatic disease and androgen independence. p53 alterations have a higher frequency in androgen-independent metastases compared with hormone-naive primary tumors.61 Similarly, progression to androgen independence is associated with an increase in the proportion of cells that express BCL-2.62 Preliminary data show that the frequency of androgen-receptor mutations increases as prostate carcinomas become resistant to androgen ablation.63-67 These results suggest that functionally significant mutations can be identified in human material and that the specific alter