There are two main clinicopathologic variants of endo-etrial carcinoma (EC) (Tables 1 and 2). According to our
urrent knowledge about EC, type I tumors are low-gradend estrogen-related endometrioid carcinomas (EEC) (Fig-re 1) that usually develop in perimenopausal women andoexist or are preceded by complex and atypical endome-rial hyperplasia. In contrast, type II tumors are non-ndometrioid (mainly papillary serous) carcinomas (NEEC)Figure 2), very aggressive tumors, unrelated to estrogentimulation, arising occasionally in endometrial polyps orrom precancerous lesions developing in atrophic endome-rium, that mainly occur in older women. cDNA analysislearly shows that EEC and NEEC exhibit different expres-ion profiles.1
The molecular alterations involved in the development ofEC (type I) carcinomas are different from those of NEEC
Address reprint requests and correspondence: Xavier Matias–Guiu,epartment of Pathology and Molecular Genetics, Hospital Universitarirnau de Vilanova, Av Alcalde Rovira Roure 80, 25198 Lleida, Spain.
type II) carcinomas.2–4 Whereas EEC shows microsatellitenstability (MI) and mutations in the PTEN, PIK3CA, k-AS, and �-catenin (CTNNB1) genes,5–9 NEEC exhibitlterations of p53, loss of heterozygosity (LOH) on severalhromosomes, as well as other molecular alterationsSTK15, p16, E-cadherin and C-erb B2).10,11 Some of theenes that are abnormal in the two forms of EC may beood targets for therapeutic approaches.
argeted therapies in endometrial carcinoma
The PI3K/AKT pathway is the most frequent abnormalignaling pathway in EEC, often resulting from mutations inhe tumor suppressor gene PTEN, and activating mutationsn PIK3CA.6,7,12–14 The tumor suppressor gene termedTEN, located on chromosome 10q23.3, is frequently ab-ormal in endometrial carcinomas. LOH at chromosome0q23 occurs in 40% of EC. Somatic PTEN mutations arelso common in EC, and they are almost exclusively re-tricted to EEC, occurring in 37% to 66% of them. PTENntagonizes the PI3K/AKT pathway by dephosphorylatingIP3, leading to a decreased translocation of AKT to cel-
ular membranes, and subsequent downregulation of AKThosphorylation and activation. PTEN mutations have been
etected in endometrial hyperplasias with and without
263Ortega et al Target Therapies in Gynecologic Cancers and Melanoma
typia (19% and 21%, respectively), both of them currentlyegarded as precursor lesions of EEC.13 Moreover, identicalTEN mutations have been detected in hyperplasias coex-
sting with EEC, which suggests that PTEN mutations arearly events in the development of EEC. There are contro-ersial data regarding the prognostic significance of PTENutations in EC, but there are some results that suggest
ssociation with favorable prognostic factors. In agreementith Knudson’s two-hit proposal, LOH at 10q23 frequently
oexists with somatic PTEN mutations. The coexistence ofoth alterations leads to activation of the PI3K/AKT path-ay, which plays a key role in the regulation of cellularomeostasis. Activated AKT modulates the expression of sev-ral genes involved in suppression of apoptosis and cell cyclerogression. Moreover, mutations in PIK3CA have been de-cribed in EC and may contribute to the alteration of theI3K/AKT signaling pathway in endometrial carcinoma.7,14
TEN has been shown to play several roles in tumor sup-ression and promotion of apoptosis. The importance of theI3K/PTEN/AKT survival pathway in EC raises the possi-ility that PI3K inhibitors, such as wortmannin and deriv-tives, may be used as potential anticancer agents. In fact, aecrease of AKT phosphorylation and increased apoptosisre seen in mutated PTEN human EC cells in the presencef PI3K inhibitor.15
Of particular interest among AKT targets is the down-tream effector mammalian target of rapamycin (mTOR).
Table 1 Clinical and pathological features of types I andII, endometrial carcinoma
he TOR family of proteins has pleiotropic functions andarticipates in the regulation of the initiation of mRNAranscription and protein translation in response to intracel-ular concentrations of amino acids and other essential nu-rients in the organization of the actin cytoskeleton, mem-rane trafficking, protein degradation, PKC signaling, andibosome biogenesis. mTOR regulates essential transduc-ion pathways and is involved in coupling growth stimuli toell cycle progression. AKT activates mTOR via directhosphorylation of TCS2 and by the inhibition of AMPPK,hereby activating Rheb and mTOR–Raptor activity. Onctivation, mTOR–Raptor activates S6K and inhibitsEBP1 to accelerate mRNA translation. However, theTOR pathway can also be activated by other mechanisms,
ncluding activation of tyrosine kinase receptors (epidermalrowth factor receptors EGFR1-4, PDGFR, KIT, IGFR) andas. Moreover, loss of function of p53 may also result inctivation of mTOR. mTOR inhibitors (rapamycin andapamycin derivatives) have been recently developed as
igure 1 Microscopical appearance of an endometrioid adeno-arcinoma of the endometrium.
igure 2 Microscopical appearance of a nonendometrioid (se-
ous) carcinoma of the endometrium.
pithmm(imdamn
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264 Seminars in Diagnostic Pathology, Vol 25, No 4, November 2008
otential anticancer agents. Tumors associated with PTENnactivation, like EC, are particularly susceptible to theherapeutic effects of mTOR inhibitors. Several mTOR in-ibitors are available for clinical trials: the prototype rapa-ycin and three rapamycin derivatives, CCI-779 (temsiroli-us), RAD001 (everolimus), and AP23573. Both CCI-779
temsirolimus) and RAD001 (everolimus) are currently be-ng tested in two phase II trials, in recurrent EC.16 PTEN�/�
ice is a good model to test sensitivity of EC to anticancerrugs, because they develop complex atypical hyperplasiand endometrial carcinoma. Pharmacological inhibition ofTOR by CCI-779 in PTEN�/� mice has shown reduced
eoplastic proliferation, tumor size, and S6K activity.17
Tyrosine kinase receptors are also good targets for anti-ancer therapies. The epidermal growth factor family and itsrowth factors are known to play critical roles in cell growthnd differentiation. The epidermal growth factor family isomprised of EGFR (ErbB1), HER-2/neu (ErbB2), HER-3ErbB3), and HER-4 (ErbB4). EGFR and HER-2/neu haveeen shown to be highly expressed in normal endometriumnd overexpressed in endometrial cancer, where it has beenssociated with a poor prognosis.18,19 Increased expressionf EGF-related protein and EGFR may contribute to arug-resistant phenotype. EGFR has intrinsic tyrosine ki-ase activity which is activated on ligand binding. Down-tream serine/threonine kinase cascades are then induced,eading to cell proliferation and the inhibition of apoptosis.yrosine kinase inhibitors prevent the phosphorylation ofAP kinase Erk1/2, MAP kinase MEK 1/2, CDK 1, AKT,AF 1, and Rb-1. Inhibition of EGFR with monoclonalntibodies leads to growth arrest, and a similar and poten-ially synergistic effect is anticipated with inhibition ofGFR tyrosine kinase activity. Potential agents are IressaZD1839), Herceptin (trastuzumab), and LapatinibGW572016). The signaling pathways of EGFR, under theffect of EGF and ZD1839, were evaluated in EC cell lines;he results showed that both EEC and NEEC have theapacity to respond to EGFR inhibition, but the response ofEEC may be limited by the constitutive activation of other
ignaling pathways. ZD1839 has been studied as a singlegent in a phase II trial (GOG 229C) of women withdvanced EC. Preliminary data in 29 patients have shown 1omplete response and stable disease in several patientsfter 6 months. GW572016 (Lapatinib) was used as a singlegent in a similar cohort of patients. Moreover, OSI-774howed partial responses in another phase II study in 23ecurrent or metastatic EC patients, with no previous che-otherapy and no more than 1 cycle of prior hormonal
herapy.The EGFR pathway may be induced by steroid hor-
ones, which is the principal growth-promoting mechanismn the endometrium. Estrogen and progesterone are the mostmportant steroid hormones that modulate endometrial cellroliferation and differentiation, respectively, and their re-eptors (ER and PR) are expressed in about 80% of EEC,
lthough the proportion may decrease with a loss of differ- i
ntiation. NEEC are not responsive to hormones and mayot express ER and PR, therefore they present a constitutivectivation of EGFR. Medroxiprogestererone acetate haseen studied in EC patients. Two large Gynecologic Oncol-gy Group (GOG) trials evaluating oral progestins in theseatients showed an overall response rate of 15% to 25%ith median progression-free survival of less than 4 months
nd overall survival of less than 11 months. Fulvestrantfaslodex), a novel antiestrogen classified as an ER antag-nist without known agonist activity in the endometrium, iseing evaluated in a phase II study in patients with recurrentr metastatic EC.
Apoptosis is a key process in the regulation of cellularomeostasis. Deregulation of apoptosis plays an importantole in development and progression of cancer. There areany evidences suggesting that alteration of apoptosis is
mportant in development and progression of EC. Several ofhe molecular abnormalities that have been detected in ECay be associated with apoptosis deregulation. EEC show a
igh frequency of mutations in PTEN, which lead to constitu-ively active Akt, which in turn suppresses apoptosis triggeredy various stimuli. Moreover, the recent evidence that NF-�Bctivation is frequent in endometrial carcinoma20 may explainhe presence of apoptosis resistance by activation of targetenes such as FLIP and Bcl-XL. p53 alterations, which areharacteristic of NEEC, may also occur in EEC, particularlyn those neoplasms showing overlapping features betweenypes I and II tumors; and they may have an impact inpoptosis at several different levels. Also, members of thecl-2 family of genes are abnormal in endometrial carci-oma. For example, BAX is a target gene for mutations inEC with microsatellite instability and may have a role in
esistance to apoptosis in these tumors.21 Proteasome inhib-tors are currently used as chemotherapeutic drugs becausef their ability to trigger cell growth arrest or apoptosis oneveral tumors. In many different types of tumor cells,ortezomib and other proteasome inhibitors cause celleath by blocking NF-�B activity. However, in EC, protea-ome inhibitors induce cell death, but, instead of blockingF-�B, they increase its transcriptional activity. Protea-
ome inhibitors induce phosphorylation of IKK�/�, phos-horylation and degradation of IkB�, and phosphorylationf p65 NF-�B subunit on serine 536. Proteasome inhibitor-nduced cell death was accompanied by activation ofaspases and apoptotic nuclear morphology22,23 (Figure 3).
key molecule involved in resistance to TRAIL-inducedpoptosis is protein kinase CK2, by modulating FLIP.24,25
esults from our group show that pharmacological inhibi-ion of CK2 by DRB, Apigenin, or Emodin causes sensiti-ation of EC cell lines to TRAIL-induced apoptosis.
Sorafenib (BAY 43-9006, Nexavar) is a potent, orallydministered receptor tyrosine kinase inhibitor with antipro-iferative and antiangiogenic activities. Sorafenib was orig-nally described as an inhibitor of B- and c-RAF kinase, butlso has activity against several receptor tyrosine kinases,
265Ortega et al Target Therapies in Gynecologic Cancers and Melanoma
VEGFR2), platelet-derived growth factor receptorPDGFR), FLT3, Ret, and c-Kit. The antitumor activity oforafenib may be attributed to inhibition of tumor angio-enesis (VEGFR and PDGFR) and direct effects on tumorell proliferation/survival (Raf kinase signaling-dependentnd signaling-independent mechanisms). A recent multi-enter phase II trial in patients with advanced/recurrent ECas shown that Sorafenib may result in clinical benefit for ainority of EC patients. Preliminary results from our group
ave shown that pharmacological inhibition of B-RAF byorafenib sensitized EC cells to TRAIL-induced apoptosis,y down-regulating FLIP (unpublished results).
Histone acetylation is one of the mechanisms involved inhe epigenetic control of gene expression. Altered histonecetylation in cancer cells may be responsible for abnormalxpression of oncogenes and tumor suppressor genes. His-one deacetylase inhibitors (HDACI) are promising antican-er drugs. In cancer cells, HDACI cause cell-cycle arrest,poptosis, and differentiation. HDACI cause derepressionf genes whose reactivation would promote an antiprolif-rative effect. Examples of genes upregulated by HDACIre p21, TRAIL-R2, p19ARF, Bmf, and Rap1. Paradoxi-ally, HDACI cause downregulation of important genesuch as thymidylate synthetase, Bcr-Abl, and c-Myc. Inter-stingly, microarray studies have shown that HDACI treat-ent leads to changes in RNA levels of a surprisingly small
umber of genes (around 2–5% of the genome). Sometudies have suggested that HDACI may be more effectiven tumors expressing mutant p53. Preclinical studies usingDACI in endometrial carcinoma cell lines have provided
nteresting results.26,27 For example, HDACI have beenhown to induce differentiation of endometrial carcinomaell lines, which resemble normal endometrial epithelial
igure 3 The proteasome inhibitors MG-132 and bortezomibndometrioid carcinoma of the endometrium. Tumor cells were leftr bortezomib for 24 hours. Pictures show Hoechst 33258 and cle
s
ells in the absence of ovarian steroid hormones. Moreover,ome studies have shown that HDACI had important growthnhibitory effect on EC cell lines, by decreasing the propor-ion of cells in S phase and increasing the proportion of cellsn the G0-G1 and or G2-M phases of the cell cycle. Thereas also upregulation of p21, p27, and E-cadherin, andownregulation of Bcl-2 and cyclin-D1 and -D2. Therowth-suppressor effects seem to be irrespective of the p53ene status.
varian carcinoma
olecular features of ovarian carcinoma
There are different types of ovarian carcinoma, each ofhem showing different molecular features (Table 3).28,29
erous tumors account for 30% to 40% of all ovarianumors. A dualistic model of serous ovarian carcinogenesisas recently been proposed. According to this proposal, theatural history and molecular features of low-grade serousarcinoma are different from those of high-grade ovarianerous carcinomas (Figure 4). Low-grade serous carcinomas
igure 4 Microscopical appearance of a high-grade, ovarian
apoptotic cell death in a tumor cell explant obtained from aned (UN) or treated with 0.5 �mol L�1 or 25 nmol L�1 of MG-132aspase-3 staining.
induceuntret
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266 Seminars in Diagnostic Pathology, Vol 25, No 4, November 2008
ould develop from preexisting serous borderline tumors,hrough oncogenic activation of K-RAS and B-RAF. Inontrast, high-grade serous carcinomas would develop deovo from the epithelial cells of the ovarian surface. Theain molecular feature of the “high-grade” pathway is
lteration of the p53 tumor suppressor gene. However, LOHn 13q and 17q, involving the loci of BRCA-2 andRCA-1, as well as BRCA-1 promoter hypermethylation,re also frequent in sporadic high-grade serous carcino-as.30,31
Endometrioid and clear cell carcinomas account for 10%o 20% and 6% of ovarian carcinomas, respectively. Theyre morphologically similar to their uterine counterparts,nd they frequently arise from ovarian endometriosis. Theolecular features of ovarian endometrioid carcinomas are
lso similar to those of endometrioid endometrial carcino-as. They are also characterized by the presence of micro-
atellite instability and mutations in PTEN, k-RAS,IK3CA, and CTNNB1 gene.32 However, the frequency of
hese alterations is quite different, with increased prevalencef mutations in the CTNNB1 gene. The molecular featuresf ovarian clear cell carcinoma are under evaluation. In-reased expression of hepatocyte nuclear factor 1�HNF1�) and mutations in TGF�RII (exon 7) have beeneported. In this regard, HNF1� has been proposed as aossible molecular target for therapy in ovarian clear cellarcinoma.33
Ovarian mucinous carcinomas are rare, and the vastajority of ovarian carcinomas with mucinous differentia-
ion are, indeed, metastatic tumors from carcinomas fromhe large bowel, appendix, or pancreas. K-RAS appears toe the most typical molecular feature of ovarian mucinousumors (either of the benign, borderline, or malignantype).34
Moreover, the molecular features of transitional cell car-inomas and undifferentiated carcinomas are poorly under-tood.
argeted therapies in ovarian carcinoma
Numerous potential therapeutic targets have been pro-
Table 4 Targeted therapies proposed for ovarian carcinoma
Imatinib (Gleevec) TKI c-ABTrastuzumab (Herceptin) mAb HERPertuzumab (Omnitarg) mAb HERCetuximab (Erbitux) mAb EGFLapatinib (Tykerb) ITK EGF
HER
osed based on prognostic studies, but large randomized r
rials assessing relevant agents are now only in the planningtages (Table 4). Drugs targeting some of the most commonlterations observed in ovarian cancer are those affectingEGF or its receptors (since VEGF production is common
n ovarian cancer and is the basis for development of ascitis)nd EGFR.
ntiangiogenic agents
A solid tumor depends on angiogenesis to grow in sizend to disseminate. VEGF is one of the main factors relatedo vascular proliferation. Overexpression of VEGF has beenbserved in 53% to 97% of ovarian cancers. Althoughverexpression correlates with poor survival in other ma-ignancies, including colorectal, gastric, breast, and prostateancer, its prognostic value in ovarian cancer is still con-roversial. Therefore, antiangiogenic agents represent aromising anticancer strategy. Bevacizumab, a humanizedurine monoclonal antibody against VEGF, blocks all iso-
orms of VEGF. Several phase II trials have investigated thelinical benefit of bevacizumab, either alone or in combi-ation with commonly used chemotherapy regimens in per-istent or recurrent epithelial ovarian cancer patients. TheOG 170D study, using bevacizumab as monotherapy in 62atients with persistent or recurrent ovarian cancer, showedcomplete responses and 8 partial responses with 54.8% of
he patients experiencing stable disease. This drug is actu-lly being tested in phase III first-line trials, either as main-enance therapy or in combination with chemotherapyGOG 218 and ICON 7) in ovarian cancer patients.35
GFR family
The EGFR superfamily is made up of four distinct buttructurally similar tyrosine kinase receptors: epidermalrowth factor receptor (EGFR/HER1/ErbB1), HER2ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Activation ofreceptor occurs by dimerization between two identical or
wo different receptors. This results in activation of theyrosine kinase domain with subsequent activation of inter-
chanism Route Phase
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267Ortega et al Target Therapies in Gynecologic Cancers and Melanoma
hatidylinositol 3-kinase (PI3K)–AKT pathway and theAPK pathway. These mechanisms are involved in differ-
nt cellular processes, and their disregulation is associatedith malignant transformation, being responsible for driv-
ng proliferation, invasion, and survival of cancer cells.GFR overexpression has been found in 12% to 82% ofvarian cancers, whereas aberrant HER2 expression waseported in 5% to 66% of ovarian carcinomas. Some studiesave shown that overexpression of EGFR is an independentrognosis factor for poor outcome. Understanding the rolef the EGFR family in cancer has led to the development ofeveral strategies, including development of inhibitors ofeceptor tyrosine kinase domain (TKI), and monoclonalntibodies (mAbs) against the extracellular domain.36 Bothypes of agents result in downregulation of the MAPK, andI3K/AKT pathways. The TKI gefitinib (ZD 1839, Iressa),
n a phase II study combined with paclitaxel and carboplatins second-line therapy in 40 patients platinum-resistant orensitive ovarian cancer, showed overall responses rates of5% and 71% in the resistant and sensitive groups, respec-ively. A number of phase II trails have evaluated thefficacy of gefitinib as a single agent in ovarian cancer, withinimal activity in patients without activating mutations inGFR. Two independent research groups have identified
hat the majority of patients responding to gefitinib in otherolid tumors had specific activating mutations within theyrosine kinase domain, whereas patients who had wild-typeeceptors did not have response. Both of these groups con-luded that specific mutations in the EGFR gene led toncreased growth factor signaling through this pathway andonferred susceptibility to the TKI gefitinib, as well asrlotinib, but do not seem to predict response to EGFRntibodies. Another TKI, erlotinib (OSI-774, Tarceva), in ahase II clinical trial in platinum-resistant ovarian cancer,eported 42% of stable disease in 34 patients, and 2 (5.8%)ad partial responses. A phase III trial of docetaxel andarboplatin with or without erlotinib in ovarian cancer iseing done by Scottish Randomized Trial in Ovarian Can-er. Cetuximab (Erbitux), an anti-EGFR antibody, hashown, in preliminary data from a phase II trial in combi-ation with paclitaxel and carboplatin, that it is well toler-ted in 31 previously untreated patients with advancedvarian cancer.
The HER-2 (c-erbB2) proto-oncogene encodes a trans-embrane receptor, which is overexpressed in several types
f human carcinomas and is an attractive therapeutic target.Abs to HER-2 can inhibit the proliferation of tumor cells
hat overexpress this gene. A humanized monoclonal anti-ody, Herceptin (trastuzumab), has beneficial therapeuticffects in primary breast carcinomas patients with HER-2mplification, particularly when combined with chemo-herapeutic drugs. However, only 25% of primary ovarianarcinomas show HER-2 overexpression by immuno-istochemistry. Moreover, unlike breast cancer, it isontroversial to what extent HER-2 amplification and pro-
ein overexpression correlates with prognosis in ovarian o
ancer. It has, however, been reported that HER-2 expres-ion is more frequent in ovarian carcinomas relapsing afterhemotherapy, suggesting that cells expressing this proteinave a selective growth advantage over HER-2-negativeells. This finding suggests that patients with ovarian canceray benefit from treatment with anti-HER-2 monoclonal
ntibodies plus chemotherapeutic drugs, both at an ad-anced stage (when the majority of tumor cells expressER-2) and at an earlier time, to eliminate the potentiallyore malignant HER-2-positive tumor cells. A phase IIOG trial showed, among 95 patients with persistent or
ecurrent ovarian cancer with HER2 overexpression, anverall response rate of 7.3% with 1 complete response and
partial responses.37 Interestingly, no correlation wasound between HER2 expression with clinical outcome.lso, there was no correlation between gene amplification
nd HER2 overexpression, suggesting that the biologic rolef HER2 in ovarian cancer may differ from that previouslyemonstrated in breast cancer. A different HER-2-targetedonoclonal antibody is pertuzumab. In contrast to trastu-
umab, pertuzumab sterically blocks HER-2 heterodimer-zation with other HER receptors. In other words, pertu-umab blocks ligand-activated signaling from HER-2/GFR and HER-2/HER-3 heterodimers. The pertuzumabinding site of extracellular domain II does not overlap withhe epitope on HER-2, which is the one recognized byrastuzumab. In a phase II trial, 130 patients with previouslyreated ovarian cancer received pertuzumab intravenouslylus gemcitabine or placebo. The adjusted hazard ratio forFS was 0.67 in favor of pertuzumab, with a median pro-ression-free survival (PFS) of 3.0 months versus 2.6onths. More recently, Gordon and coworkers reported the
esults of a phase II trial using pertuzumab as a single agentn 123 patients with ovarian cancer.38 PhosphorylatedER2 expression was studied, suggesting that pertuzumabay be active in ovarian cancer and that HER2 activation
tatus, in the absence of HER 2 overexpression, may beotentially predictive for identifying tumors responsive toertuzumab.
Lapatinib is an orally active small molecule that revers-bly inhibits EGFR and HER2 tyrosine kinases. Varioushase I trials have been conducted in patients with solidumors including ovarian cancer, with preliminary evidencef antitumor activity in heavily pretreated patients. How-ver, no phase II studies have been reported to date inynecological malignancies. The potential advantages ofapatinib over other tyrosine kinase inhibitors are that itnhibits two receptors and thus may inhibit multiple receptorimers and their corresponding downstream signaling path-ays.Imatinib mesylate (STI-571, Gleevec), an adenosine
riphosphate-binding selective inhibitor of BCL/ABL, KIT,nd PDGFR, has been evaluated in some phase II trials. Inormal ovarian surface epithelium, c-ABL was expressedniversally. PDGFR-� was expressed in the majority (93%)
f samples of normal ovarian epithelium, whereas the c-KIT
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268 Seminars in Diagnostic Pathology, Vol 25, No 4, November 2008
rotein was undetectable in normal ovarian surface epithe-ium. Overall, c-ABL was expressed in 71% of serousarcinomas [more frequently in the low-grade serous carci-omas (81%) compared with the high-grade serous carci-omas (65%)]. PDGFR-� expression was observed in 81%f serous carcinomas overall and was observed more fre-uently in higher-grade tumors. c-KIT immunohistochemi-al staining was absent in low-grade tumors but was presentn 26% of high-grade serous carcinomas. In conclusion, theajority of ovarian serous carcinomas express one or more
f the kinases targeted by the tyrosine kinase inhibitor,matinib mesylate, suggesting the potential usefulness ofhis drug in the treatment of ovarian carcinoma. However, in
phase II clinical trial, including patients with recurrentvarian cancer whose tumors overexpressed at least one ofhe known molecular targets of imatinib, there were noesponses during a median follow-up of 6.6 months.
strogen receptor signaling pathway
Estrogen growth factors, including EGF, insulin, insulin-ike growth factor-1, and TGF-�, activate estrogen receptor,hich can activate the PI3K/AKT cell survival pathway and
an also activate the EGF family of receptors.
argeting the apoptosis pathway
G3139 (Oblimersen) is an antisense phosphorothioateligodeoxynucleotide that suppresses BCL2 expression.CL2 family proteins play a central role in controlling theitochondria pathway. This family includes proteins that
uppress apoptosis, such as BCL2. Overproduction of BCL2rotein prevents programmed cell death, enhances meta-tatic potential, and promotes resistance to chemotherapy.igh expression of BCL2 proto-oncogene is found in var-
ous solid tumors, including ovarian cancer. Moreover, mo-ecular targeting of BCL2 and Bcl-XL proteins by syntheticCL2 homology 3 domain peptide has shown to enhance
he efficacy of chemotherapy.39
argeting signal transduction
Aberrant or overactive signal transduction has beenound to enhance proliferation, invasiveness, metastasis,nd angiogenesis, and to confer poor response to conven-ional cytotoxic agents. One of these pathways is the RAS/AF/MAPK. Constitutive activation of the RAS/RAF/APK pathway components has been seen in primary
umor cell. In this way, RAS gene mutations have beenbserved in 18% of ovarian cancers, especially in mucinousumors, but also in low-grade serous carcinomas. BAY3-9006 (sorafenib) is currently in evaluation in severalhase II assays for the treatment of recurrent or refractoryvarian cancer alone or with paclitaxel, carboplatin, and
emcitabine. An in vitro and clinical/pharmacological phase
study has recently tested the combination of irinotecan andorafenib.40
elanoma
olecular features of melanoma
Development of malignant melanoma is associated withllelic deletions (LOH) at several chromosomes, includingp, 6q, 9p, 10q, 11q, and 17q. Moreover, there are some locihat correspond to familial susceptibility genes (1p36, 9p21,nd 12q14) which contain tumor suppressor genes that aremportant in melanoma development. There are evidencesuggesting that transition from normal melanocyte to mela-ocytic dysplastic nevus involves the loss of genes at 1p, 9p,nd 10q. Moreover, the progression from melanocytic dys-lastic nevus to the radial growth phase of malignant mel-noma involves further losses on 9p, 10q, and 6q, and thevolution to the vertical growth phase of malignant mela-oma involves 1p, 11q, and 17q.41
Genes controlling cell cycle machinery are very impor-ant in melanoma tumor development and progression. TheDKN2A gene (p16) is located on chromosome 9p21,ithin a region that is frequently deleted in sporadic mela-omas. Moreover, CDKN2A germline mutations have beenetected in many melanoma kindreds. CDKN2A is a pivotalell cycle regulator that binds to CDK4 and CDK6 andnhibits their ability to phosphorylate pRB. Inactivation ofDKN2A increases cell division by altering control of the
1-S checkpoint. CDK4 gene, located on chromosome2q14, shows recurrent focal amplification, and germlineutations in CDK4 have been detected in melanoma kin-
reds. Moreover, CCND1, a binding partner of CDK4, isrequently amplified in melanomas.42,43
Several genes have been shown to be abnormally ex-ressed in melanoma (Table 5); these include B-RAF, N-AS, PTEN, c-KIT, CCND1, and CDKN2A. However, the
Table 5 Molecular features of malignant melanoma
CDKN2A/p16 Deleted in somatic tumors; germlinemutations in familial tumors
CDK4 Amplified in somatic tumors; germlinemutations in familial tumors
CCND1 Amplified in sporadic tumorsB-RAF mutation Melanomas without chronic sun-
induced change (together with PTENloss)
N-RAS mutation Melanomas without chronic sun-induced change
PTEN loss Melanomas without chronic sun-induced change (together withB-RAF mutation)
c-KIT mutation Mutated in acral and mucosalmelanomas
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269Ortega et al Target Therapies in Gynecologic Cancers and Melanoma
requency of abnormalities in these genes seems to beelated to the natural history of the tumor. Melanomas onkin without chronic sun-induced damage, which representhe most common type of melanoma, had frequent muta-ions in B-RAF, together with losses of chromosome 10PTEN), or mutations in N-RAS alone. Interestingly, muta-ions in B-RAF and N-RAS are mutually exclusive events.s mentioned below, these alterations lead to activation of
he RAS/B-RAF/MAPK pathway, and could be responsiveo therapeutic interventions targeting such signaling path-ay. On the other side, other types of melanoma do notsually have mutations in B-RAF or N-RAS, but havencreased numbers of copies of CCND1 or CDK4. Further-ore, losses at the CDKN2A locus (p16) are also frequent,
nd they occur more commonly in mucosal and acral mel-noma, which also exhibit mutations in c-Kit.43,44
argeted therapies in melanoma
Malignant melanoma is extremely resistant to chemo-herapy. Empirical use of chemotherapy and/or immuno-herapy in the treatment of patients with melanoma hashown that the majority of melanomas are resistant to mostgents.45 For that reason, it is very important to look for newtrategies (Table 6). There are several molecules that maye good targets for therapy.46 It is now appreciated thatelanomas have defects that include loss of regulatory
unctions or the gain of proliferative or antiapoptotic func-ions that are no longer under normal regulatory control.here are numerous signaling pathways that play a signif-
cant role in melanoma tumor development and progression.ome of them are activated as a result of activating muta-
ions (B-RAF, N-RAS, PTEN), whereas others show in-reased expression of some of the key members (Bcl-2,F-kb, CDK2, cyclin D1). Many agents, such as mAb,
ntisense oligonucleotids, and TKI, are under investigationn melanoma. Unfortunately, most of these are phase I/IItudies, and the real efficacy of agents oriented to theolecular target can only be assessed with well-designed
Table 6 Targeted therapies proposed for malignant melanoma
The mitogen-activated protein kinase (MAPK) signalingascade, which normally regulates cell growth, survival, androliferation, appears to be activated in melanoma.47 Two ofhe most important members of the pathway are frequentlybnormal in melanoma; mutations in N-RAS or B-RAF areeen in approximately 90% of cases. RAS is an importantpstream regulator of the MAPK and PI3K pathways and washe first oncogene to be identified in melanoma. In normalells, RAS proteins are activated by cell-surface receptor bind-ng of growth factors. The RAS proteins then activate RAFs,hich phosphorylate and activate MEK, leading to subsequent
ctivation of the extracellular signal-regulated kinase, ERKs,nvolved in cell-cycle progression and proliferation. B-RAFlso contributes to neoangiogenesis by stimulating autocrineEGF secretion. In normal cells, B-RAF is downstream fromRAS, but in melanoma can be constitutively activated by autation in codon V600 (Figure 5). This mutation has been
dentified in 66% of malignant melanomas, and this mutationepresents more than 80% of all B-RAF mutations. Because-RAF mutations can increase chemotherapy resistance, RAF
nhibitors such as sorafenib are being evaluated.48–50 Althoughorafenib has demonstrated limited activity as a single agent,
igure 5 Phenogram showing the typical V600 mutation in
Target Phase
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270 Seminars in Diagnostic Pathology, Vol 25, No 4, November 2008
hase II data from patients with advanced melanoma haveuggested that this drug in combination with chemotherapycarboplatin, paclitaxel, temozolomide, and dacarbazine) isuch more active.51 Phase III trials of sorafenib in melanoma
re under development, and additional B-RAF inhibitors areurrently in preclinical development. In vitro data have shownhat melanomas with B-RAF mutations may be selectivelynhibited by two MEK inhibitors, AZD6244 and PD0325901,hich are currently being tested in clinical trials. Interestingly,
nthrax toxin inactivates both MEK1 and MEK2, providinghe basis for study in the treatment of melanoma.52 However,t should be noted that one potential problem of MEK inhibi-ion is that MEK is also activated by tumor progression locus
(TPL2), a protein kinase that regulates innate immunity, sohronic MEK inhibition might have negative effects on innatemmunity.
he PI3K pathway
Another signaling pathway important in melanoma is theI3K pathway. PI3K is mutated in 3% of metastasic mela-omas, and PTEN is inactivated in up to 20% of advancedelanomas. Moreover, AKT is overexpressed in 60% ofelanomas. In melanoma, the accumulation of phosphory-
ated AKT suppresses apoptosis, thereby enhancing survivalnd promoting proliferation of malignant cells.53 Phosphor-lated AKT is associated with tumor progression and de-reased survival and has been observed in 54% of nevi, in1.3% of primary melanomas, and in 71% of metastaticelanomas. In addition, overexpression of AKT has been
bserved in severely dysplastic nevi and metastatic mela-omas, but not in benign nevi, and correlates adversely withatient survival. Agents that target PI3K, AKT, and otherownstream components of this pathway such as mTOR areeing developed.54 Among these, mTOR inhibitors such asCI-779 and RAD001 are the most advanced.
argeting cell surface receptors: c-kit
The proto-oncogene c-KIT is necessary for melanocyteevelopment, differentiation, proliferation, survival, andigration. c-KIT mutations occasionally occur in some
ypes of lentiginous melanoma (mucosal, acral lentiginous,nd lentigo malignant melanoma), but are very infrequent inhe most common types of cutaneous melanoma. Manytudies have linked the loss of normal c-KIT expressionith the development of primary and metastatic melano-as.44
Some preclinical studies have provided evidences that-KIT may be a good target for therapy in melanoma. Atudy done by our group demonstrated a clear antiprolifera-ive effect of imatinib mesylate (STI-571, Gleevec) on 8 of9 (42.1%) human cutaneous melanoma-derived cell lines,upporting a possible use of STI571 as potential treatmentor a subset of patients with disseminated malignant mela-
oma55 (Figure 6). However, the mechanism of the inhibi- f
ory effect of STI571 on melanoma cells was not clear sincehere was not good correlation between response to STI571nd immunohistochemical expression of c-Kit. The clinicalfficacy of STI571 in patients with disseminated cutaneousalignant melanoma is under debate. Results from three
ifferent phase II trials have been reported. They havehown lack of clinical benefit in the vast majority of pa-ients, although a striking objective response has been ob-ained in individual cases. Moreover, the efficacy of STI571n a c-Kit-negative, cutaneous melanoma cell line of murinerigin in a mouse model has also raised the speculation thatTI571 could have some molecular targets, other than c-Kit,
n cutaneous malignant melanoma.56
Promising data about STI571 related to c-Kit expressionave recently been reported in melanoma of uveal origin. Iteems that c-Kit expression in uveal melanoma (analyzedy tissue microarrays) is present in 87% of cases, beingtrong in 69% of them. Moreover, depletion of c-Kit inveal melanoma cell lines expressing c-Kit and its growthactor (SCF) strongly inhibits melanoma cell proliferation.ome clinical effects of STI571 have been reported in twoatients with disseminated ocular melanoma. Since c-KITutations have not been detected in uveal melanomas, the
ffect of STI571 has to be explained by specific inhibitionf tyrosine kinase activity of wild type c-Kit.57
At present, STI-571 and other c-KIT inhibitors (sunitinibalate, SU011248) are currently being evaluated in patientsith metastatic mucosal and acral melanomas. It is worthentioning that these drugs also target other tyrosine kinase
eceptors. For example, imatinib mesylate also inhibitsDGFR and ABL, whereas sunitinib blocks PDGFR, RET,nd VEGFR.
argeting survival signals: NF-�B and BCL-2
Nuclear factor kappa B (NF-��) is a transcription
igure 6 c-Kit expression in 95% of cells of JG melanoma celline which responded to STI571 treatment (flow cytometry).
actor that occupies a pivotal point in survival and growth
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271Ortega et al Target Therapies in Gynecologic Cancers and Melanoma
athways and can be activated through either the PI3K/kt or the MEK/ERK pathway. NF-�B mediates anti-
poptotic, proliferative, metastatic, and proangiogenicffects primarily through inducing gene expression ofroteins critical to these activities. Constitutive activa-ion of the NF-�� pathway has been identified in manyumor types, including melanoma. Proteasome inhibitors,uch as PS-341 (Bortezomib, Velcade), selectively andeversibly inhibit the 26S proteasome and prevent thereakdown of many regulatory proteins through the in-ibition of the ubiquitination-proteasome process.58 Onef these proteins whose breakdown is impaired is I�B,he protein that inactivates NF-�B. In some tumor types,nhibition of the proteasome leads to NF-�B inactivation,ut in other types of tumor (for example, endometrialarcinoma), proteasome inhibitors activate NF-�B. Inelanoma, Velcade has been shown to inhibit NFKB
ctivity and reduce cell growth in vitro. In a study doney our group,59 the effect of four structurally differentroteasome inhibitors (Bortezomib, PS-341, ALLN, MG-32 and epoximicin) was assessed on 16 human cutane-us melanoma-derived cell lines, which are original andere obtained from patients who were treated by 2 mem-ers of the clinical team. Proteasome inhibitors inhibitedhe in vitro growth of melanoma cells, and this effect wasue either to a reduction in cell proliferation rate or annduction of both caspase-dependent and caspase-inde-endent cell death (Figure 7). Moreover, release of AIFas observed in the absence of detectable caspase acti-ation. Also, caspase 2 processing was observed in Bort-zomib-induced cell death. Interestingly, Velcade syner-izes with temozolamide in human melanoma xenografts,roviding the rationale for using Velcade to overcomerug resistance.58
BCL-2, which suppresses apoptosis, is overexpressed inelanoma, suggesting a direct association with chemoresis-
ance. This idea is supported by studies showing that anti-ense suppression of BCL-2 increases melanoma cell apo-tosis and sensitivity to chemotherapy.60 Oblimersen, anntisense oligonucleotide, suppresses BCL-2 and has shownimited promise in initial melanoma clinical trials. A phaseII study has shown some benefit in progression-free sur-ival and overall response when used in combination withacarbazine, but not in overall survival. At present,
igure 7 The proteasome inhibitors MG-132 and bortezomib indr were treated with 0.5 �mol L�1 or 25 nmol L�1 of MG-132
aspase-3 staining.
blimersen is the only target agent tested in a phase III trialn melanoma.61,62
ngiogenesis
Angiogenesis has proven to be a critical step in mela-oma transformation. A number of angiogenic factors haveeen shown to be released by melanoma cells and/or hostells within the tumor microenvironment. These includeEGF, PDGF, interleukin-8, and basic fibroblastic growth
actor. Several human melanoma xenografts models showhat these factors play a role in tumor progression. Antibod-es against VEGF are effective at blocking tumor growthnd metastasis. Serum levels of VEGF in melanoma pa-ients increase with clinical stage and can be predictive oforse prognosis. However, there are no encouraging phase
I data, and because sorafenib, which is thought to workhrough an antiangiogenic mechanism, provides only a mar-inal response in melanoma, it is possible that targetingngiogenesis alone will be an ineffective treatment for mel-noma. In this way, available results from phase II trialsonfirm it. Carson and coworkers evaluated a regimen ofevacizumab alone or in combination with low-dose IFN-�,howing responses only in patients receiving the tworugs.63
SU011248, AG013736, and ZD6474 are all oral tyrosineinase inhibitors that have already shown promising resultsn other cancers, including clear cell renal carcinoma. Theyre inhibitors for the VEGFR2 receptor signaling but alsolock one or more other receptors. Targets for bothU011248 and AG13736 include VEGFR2, PDGF, and-kit, whereas ZD6474 blocks both VEGFR2 and EGFR1.he ability of these agents to inhibit multiple targets makes
hem of interest to investigate in patients with melanomalone, with chemotherapy, or with other targeted agents. Its anticipated that many such studies will be done in theoming years.46
mmune escape
Negative regulation of antitumor immunity in melanomas a focus of continuing research. Tumors may evade im-unosurveillance (“tumor escape”) through downregulation
optosis in M17 melanoma cell line. M17 cells were left untreatedezomib for 24 hours. Pictures show Hoechst 33258 and cleaved
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272 Seminars in Diagnostic Pathology, Vol 25, No 4, November 2008
f various immunoregulatory components via tumor-de-ived soluble factors, alterations in signal transduction, andmmunologic tolerance. In addition, many tumor cells pro-uce antigens that are also expressed on normal cells. Con-equently, these cells are recognized as “self” and are notargeted for immune destruction. Tumors also often secretemmunosuppressing factors as they grow, thereby prevent-ng immune activation. Many of the inhibitory processessed by melanoma cells to evade immune system are po-ential therapeutic targets. The cytotoxic T-lymphocyte an-igen 4 (CTLA4) is a promising novel therapeutic target ands a CD28-family receptor that inhibits T-cell function.ntibodies directed against this receptor block CTLA4,
riggering the immune system by reversing the “invisibility”f several cancer antigens. The ultimate effect of anti-TLA4 therapy is to initiate an immune response against
he invading tumor cells, leading to tumor cytotoxicity viaymphocyte-mediated cell death. Anti-CTLA4 therapy hashown activity and prolonged responses in metastatic mel-noma.64,65
ormones
Melanoma cells express receptors for a number ofrowth factors, including hormones. Our group has recentlyested the effects of somatostatin analogues in correlationith the expression of somatostatin receptors, and the re-
ults show that somatostatin analogues have some impact oniability of melanoma cells, suggesting that they may besed in combination with other anticancer drugs.66
cknowledgments
This work is supported by Grants FISPI060832,ISPI060577, FISPI070304, FISPI070276, SAF2002-0529-E, Marató de TV3 2005-47, 2004XT00090, andD06/0020/1034. XD holds a postdoctoral fellowship from
he Fondo de Investigaciones Sanitarias, Ministerio deanidad y Consumo (CP05/00028). DLL holds a predoc-
oral fellowship from the Fondo de Investigaciones Sanitar-as, Ministerio de Sanidad y Consumo (FI05/00191).
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