/>rim ifth") ctnd Pr4.H-ht 'fl uf (;e-,1ft,,,nf{11an· ./)tu,nfvt.: r~ i.:,.Ji@d b.v,
`!,lc,-ck R:ti;havan. Htiw:uxl I. Sdi.,r. S\ev¢n A. L,ib<;I, lllW Patti H.
`l.,u,ge. LiJ>pmc01h~avcn Ptt!>li,ti.,r,. Philillle'lj)hia, ,,. J ~lil.
`
`CHAPTER 78
`
`Renal Cell Carcinoma
`
`Tumor Markers
`
`fames M. Kozlowski
`
`!n 1996. cardnomas <if the renal patenthyma wlll accotrnt for
`,wer 30;(!(,0 new ca:ncercases and he re:.'JX!nsible for more th,111
`l 8.000 cancer death~. 1 (:)vert. evidence of metttstatk di:,;ecuse is
`-demonstra;ble in over 30% of patj;mn; with renaf cell carcinoma
`fRCC} at !he time of initial presentation.1 furthermore, meta,
`;tc;itic disease develops metachronously in ab<Jut 50% of patients
`who have 1.mdergone a •·curative·· radical nephrecto1ny.1·· 4 In
`genecaL th.e outkruk for both groups of patients is tfis.maL A
`LO% 1-yea;r survi.vut r.tte is :rntkipated for patie-Ot~ presenting:
`with ;;yochro11ous metastases tavernge survival is 4 months).
`Patients with a delayed onset ot' systemic refapse ean unfrdpate
`,1 median survival of appmx:imatefy I l months.5·lt
`Ratlicai nephrectomy and nephmn-sparing surgery play a
`preeminent role in. the mana~ement l>f putatively org11n-con(cid:173)
`tined disease, In fidd.itfon. ,ig:gresslve surgical therapy has peen
`,L~SOC.iil!ed with 5-year survival rates of J5% to 5()% in patients
`with RCC who presem with or whcnlevef()p apparently solitary
`metastatic lei.ions.s Withre~-µect to 1he latterphenomentm, stud(cid:173)
`ies by Skinner and associates;1 Tolia and Whitmore/ O'Dea
`and assqciates,10 an!'.! Middleton 1 1. have ~itetl an jneidet'.lce of
`JR%, .'.U%., 25%, and L6%, respectively.
`Unfortunately, the majority: of these patients harbor suoclin(cid:173)
`ical, m:i;Jtifocal~ micrometastutic dioouse and ultimate!>• sue·
`eumb to the uncontrolled prolife.ration of RCC dei;pite :iggres,.
`lijve systemic therapy, fvr example. partial and complete
`resp1mse rates of 17% (lOd 4%. respectively. have been assqci(cid:173)
`ated wit!t the use of h1gh-do11e interleukin-'.~ (IL,2}, Moi,t
`impressive is that these responses are generally durnble, and
`4.l}o/,,. o:.f such patients maintain the achieved .respon!re for at
`leax.t Z 3/tml'S. 11 Once again, most patients .dll nt>t be!'1~fi1 frmn
`thel';e l<1xic therapies, and mo11t responders ultimately imtnifeM
`evidence oJ &reuse pmgres-sion. The nature of RCC is ex(cid:173)
`tremely variable and un:predidable. fndeed, 20% to 10% of
`such patients. exhibit the phenome1mn tif dL..ea.se iitabi liza(cid:173)
`lioo. "·R This finding implies that some primary tumors and
`estabUl\hed metast;ttic foci may fail I() exhibit evidence of
`su~lllii11.,J gr .. iwth tir n1e1w,ta1ic p,,1e111iaf fof !lla11y yeqfs. Ob-
`
`vfr.us!y. the perceived benefits of ally systemrc therapy fqr
`RCC m.ui.1 be viewed within this context.
`Th.e availability of sensitive and specific markers C>f RCC
`tumor activity would be advantageou1, t'c>r several. reasons. The
`presence of secretory or degradation products measurable in the
`serum or urine would possibly faciiit.ite the detec.tion af low(cid:173)
`stage, organ-confine<t RCC il'l ix)t!J the ~eneral population and
`in those cohortll lit high risk for developing RCC .(patients With
`v9n Hippel-Liodl.W disease and (hose receiving hemodialysis).
`In nddition, such easily assay able markets could pen:nit the early
`detection •of local recurrenc..e ot sysremic .refapse .and could prg,(cid:173)
`vide a means of prompt asi;es~ment of the anti:turnor ucrivily of
`udministere(,J systemic therapies. RCC-assoctated monoclonal
`antibodies (MABs) could be used to detei;t antigen~ shed or
`secreted into blood. urine. or renal cystic tluid, 11 Radloimmilno:(cid:173)
`scjntigmphy ui;.ing RCC~rcactive MA.Bs could pos.:;ibly facili(cid:173)
`tate the loculization of metastatic ttiseare and the evaluat1cm
`of complex cystic renal masses. 1~ .Finally. MABs capable of
`largeting RCC (pel io vivo coulr! provide the delivery vehicle
`for potentially effective therapy by Hnkag:eto chemotlier;.1pet1tic
`agents. radiomiclides, or toxins. u
`N(,highly sensitive and :;pecific markers of RCC tumoractiv·
`ity Cifpable of fulfilling all the prevrously. enumeruttd fiinctions
`are avaHabte. Certain biologic markers have been evaluated
`with re1>pect to their abl!ity lo dt;tect RCC tumor activity and
`have proved unreliable because of unacceptably low sensitivity
`and specificity profiles. These markers include tenirt, i 5 erythro·
`po!etin,15 lipid-associated sla.tic acid, 1" ca.rdnoembryoniG. l).Jlti(cid:173)
`gen.17 fibrintigen, ui haptogJobin/1 and C-reactive ptotein, 10
`More recent investigatlorts have defined :uilverse a.rnty of bio(cid:173)
`fogic marker!! that hold much greater promille with respect tt)'.
`.their itb,lity 10 detect RCC-rel:tted tuluor activity and include
`the folkiwing: (h serum ferritin (SF): (2} nephrocalcin (NCJ:
`en y-enofase: {4l ~ldofase; (5) S- EOOUi:, protein; t6bnterleukin·
`t1!· (IL,fl> ,ind other cytukines; (7) l:lask fil:lt'Obla.st growth factor
`{bFGFl anti other angiogenk peptides: (!f) para:thymid~related
`protein WRP): and t9) RC'C-reactive MABJ;. The remainder of
`ihis chaph:r pt·ovJdes a briel' t:wervlew of e,1ch {Jf these area1,.
`
`813
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`
`SERUM FERRI1'1N
`
`Ferritin is an intracellular iron-storage protein whose main
`function is to sequester iron in a nontoxic fom1 and to release
`it to iron pools when required for metabolic activity. '.'I.'.''.' Apof(cid:173)
`erritin is the protein component of ferritin and is composed of
`a variable number of polypeptide subunits responsible for the
`different isomeric fonns of ferritin."-' The basic isoferritins are
`predominantly located in the liver and spleen and are found in
`greatest concentration in those cells involved in iron storage
`(macrophages and hepatocytes).:, 4 Acidic isoferritins have been
`found in most mammalian cells, with substantial concentrations
`in the nonnal heart, pancreas. placenta, and kidney. 1 1.::>-1 With
`respect to the kidney. immunocytochemical studies have dem(cid:173)
`onstrated that acidic isoferritin is confined to the proximal tubu(cid:173)
`lar cells of the maturing fetal nephron and the adult kidney. 21
`Although the SF concentration generally reflects the magni(cid:173)
`tude of available iron stores in the body, in the following condi(cid:173)
`tions, for example. SF is disproportionately elevated: ( I J various
`infections and inflammatory disorders: (2) anemia of chronic
`disease; (3) hepatic disease or damage; and (4) certain neo(cid:173)
`plastic conditions such as Hodgkin's disease, acute lymphatic
`leukemia, hepatoma. and carcinomas of the breast stomach.
`pancreas, and kidney. 2 u-1 That the acidic isoferritins are prefer(cid:173)
`entially expressed in these malignant conditions helps to explain
`their alternative designation as the oncofetal ferritins. 24
`In 1987, Flemmin~g used polyclonal antiferritin antisera to
`perfonn an immunocytochemical localization of ferritin in 18
`nephroblastomas, 4 renal sarcomas, 26 RCCs, and 8 renal onco(cid:173)
`cytomas.2' Ferritin was located within the neoplastic cells of
`54% of RCCs and 63% of renal oncocytomas. Ferritin was not
`seen in the other tumor types. Although stromal macrophages
`also contain ferritin. these studies tend to support the contention
`that the "ferritinemia" of cancer patients is directly related to
`the production and release of ferritin by tumor cells. 2"
`Esen and associates analyzed SF levels in 32 patients with
`RCC using a ferritin radioimmunoassay. Serum samples were
`evaluated before and after nephrectomy in this cohort. The mean
`SF concentration was significantly higher in patients with RCC
`than in age- and sex-matched controls (259.10 ng/mL versus
`61.30 ng/mL, P = .001).21 In addition. the SF levels rose pro(cid:173)
`portionately and significantly with advancing tumor stage. In(cid:173)
`deed, SF was elevated in 33%, 80%, 100%,, and 93% of patients
`with stages I. II, Ill, and IV. respectively. In this study, the
`mean preoperative SF level for patients with stage 1-11 disease
`was 132.27 ng/mL, and only this cohort exhibited a statistically
`significant diminution in this parameter after nephrectomy
`(mean = 77.39 ng/mLJ. In contrast, patients with stage Ill-IV
`disease had a mean preoperative SF level of 361.42 ng/mL.
`These patient~ did not demonstrate a significant diminution in
`this marker after nephrectomy. Concomitant immunohisto(cid:173)
`chemical studies demonstrated a marked increase in intracellu(cid:173)
`lar ferritin content in the RCC tissues when compared with
`normal parenchyma. In the latter. only proximal tubular cells
`demonstrated ferritin content.
`Ferritin appears to constitute more than an inert marker of
`RCC tumor activity. For example. iron is required for the prolif(cid:173)
`eration of nonnal and neoplastic cells. Moreover. proteins in(cid:173)
`volved with iron metabolism (ferritin and transfcrrin) may fa(cid:173)
`vorably modulate those iron supplies available to malignant
`
`cells and may enhance their growth potential. 2' Further, a
`tumor-derived acidic ferritin appears to possess a distinct imnrn(cid:173)
`nosuppressive effect, particularly with respect to inhibiting the
`activation of T lymphocytesY Indeed. this same phenomenon
`has been associated with the acidic ferritins derived from the
`placenta, and investigators have suggested that the high serum
`levels of placental ferritin during pregnancy may be responsible
`for suppression of the immune system of the mother, thus insur(cid:173)
`ing" immune tolerance" of the maturing fetus. :, 2 In this respect,
`acidic or oncofetal ferritin may facilitate both neoplastic pro(cid:173)
`gression and embryonic development through its inhibition of
`the host immune system.22
`
`NEPHROCALCIN
`
`NC is a glycoprotein derived from the cells of the proximal
`tubule and thick ascending limb. 26 NC is present in mammalian
`urine, and its major function is that of a urinary calcium oxalate
`monohydrate crystal growth inhibitorY Patients with a history
`of calcium oxalate stones generally have decreased urinary NC
`activity, which is otherwise detectable at stable levels in healthy
`controls."x The specific elution pattern of urinary NC in healthy
`adults has been thoroughly described and demonstrates the pres(cid:173)
`ence of approximately 5 isomers whose generation is contingent
`on differences in the phosphorylation of the NC molecule. 29
`Nakagawa and associates used polyclonal antibodies specific
`to NC and measured urinary and serum NC levels by an en(cid:173)
`zyme-linked immunosorbent assay (ELISA) in 19 patients with
`newly diagnosed (localized) RCC and 21 controls.26 The mean
`level of urinary NC in the patients with RCC was 0.241 ±
`0.34 I µg NC/mg creatinine. In contrast. the average urinary NC
`level in the controls was 0.022 ± 0.012 µg NC/mg creatinine (P
`= .0112). Only 4 of 19 patients with RCC had urinary NC
`levels within the control range. No obvious correlation was seen
`between the urinary NC levels demonstrated and such factors
`as tumor stage, tumor size. tumor histotype, or tumor grade.
`The serum and urinary NC concentrations were closely corre(cid:173)
`lated. In previous studies. this group had demonstrated elevated
`urinary NC levels in patients with RCC with documented meta(cid:173)
`static disease. 27 This latter observation suggests that NC maybe
`a useful tumor marker even when RCC tissue is not contact
`with the urothelial svstem. Furthermore, the NC isolated from
`RCC cells demonstl:ates a distinctly abnormal elution pattern
`characterized by the following: ( I J increased amounts of isomer
`1; (2) low phosphate content: and (3) larger amounts ofcarhohy(cid:173)
`drate residues. 211•27
`NC. unlike y-enolase and aldolase, appears to be kidney(cid:173)
`specific. 2r, Another distinction between NC and these other
`markers (discussed later) is that NC has been localized to RCC
`of all grades and histologic types. 26 These observations, coupled
`with the apparent close correlation between urinary and serum
`levels, suggest that NC maybe a unique and important marker
`of RCC tumor activity.
`
`y-ENOLASE
`
`Enolascs are gfycolvtic enzymes that catalyze the react.ion
`pathway bctwcc0n - 2-phosphoglyceratc and phosphoenolpyr(cid:173)
`uvate. m These enzymes are dimers composed of a, fl, and y.
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`,ubunits. Five distinct isozymes have been recognized ( aa. /3/3.
`yy, a/3. and cry), with each dimer having an approximate molec(cid:173)
`ular weight of !00.000.31 The a subunit appears to be ubiqui(cid:173)
`rnusly distributed. whereas the /3 subunit is preferentially ex(cid:173)
`pressed in striated muscle and heart. 31 With respect to y(cid:173)
`~nolase. high levels have been found in neuronal and neuroen(cid:173)
`docrine cells. For this reason. y-enolase is alternatively desig(cid:173)
`nated neuron-speci fie enolase. 12 ··34
`Haimoto and associates used immunohistochemical tech(cid:173)
`niques to detennine the localization of enolase isozymes in nor(cid:173)
`nial renal tubules. 10 In that study, the aa dimer was found
`preferentially in the proximal tubules, whereas the yyform was
`located in the loops of Henle. Finally, the medullary collecting
`ducts and macula densa exhibited the aa, yy, or ay isozymes.
`The reason for the preferential expression of y-enolase in the
`loops of Henle may be that this isozyme is more stable in the
`presence of chloride ions that undergo active transepithelial
`rransport in that segment of the nephron.1<us
`Haimoto and associates detected evidence of y-enolase
`apression in all 36 RCCs subjected to immunohistochemical
`,malysis. 36 Using a coJToborative immunoassay. these same in(cid:173)
`vestigators demonstrated that the levels of y-enolase in the nor(cid:173)
`mal cortex were 16.8 ± 3.7 ng/mg protein. In contrast, the
`levels expressed in RCC were approximately 55-fold higher
`(928 ± 554 ng/mg protein) than those documented in normal
`cortical tissue. In this same study, serum y-enolase levels were
`elevated in 49% (20 of 4 l) of patients with RCC. The sera of
`I 00 healthy adults were analyzed for y-enolase activity, and a
`mean value of 3. l ± 0.9 ng/mL was documented. Similarly,
`in the 41 patients with RCC, the mean serum y-enolase concen(cid:173)
`tration was 9.1 ± 11.0 ng/mL. The values in excess of 6 ng/mL
`were considered elevated and were consequently documented in
`20 of 4 l patients:10
`The preceding observations were expanded in the study con(cid:173)
`ducted by Kusama and associates.n In that study. all 13 RCCs
`tested demonstrated strong immunohistochemical reactivity for
`neuron-specific enolase. Serum y-enolase levels were also mea(cid:173)
`sured in these patients by means of radioimmunoassay, with
`IO ng/mL designated as the upper limit of normal. A mean
`value of 24.7 ± 33.2 ng/mL was detected. In all, the y-enolase
`levels were elevated in 65 % ( l l of 17) patients. Six of 7 of the
`patients with stage I disease exhibited no1mal serum levels. In
`contrast, all the patients ( IO of IO) with stage II-IV disease had
`elevated y-enolase serum levels. These results suggested that
`neuron-specific enolase activity is correlated with tumor vol(cid:173)
`ume and may be most useful as an indicator of advanced-stage
`RCC.
`The preceding observations validated the findings of Tabshi
`and associates.11 These investigators demonstrated that tissue
`y-enolase levels were 34 times higher in RCC than in the
`healthy renal cortex. Similarly, 51% (53 of 103) of patients
`with RCC had serum y-enolase levels higher than 6 ng/mL.
`The mean serum y-enolase level in RCC was 8.0 ± 5.7 ng/
`mL, in contrast to a value of 3.1 ± 0.9 ng/mL noted in healthy
`subjects. Patients with stage III-IV tumors had higher mean
`serum y-enolase levels (9.9 ± 6.8 ng/mL) those with stage I(cid:173)
`ll neoplasms (5.8 ± 3.0 ng/mL). Positive serum detection rates
`of 34% and 22'/c, were noted in patients with stage I and stage
`II disease, respectively. In contrast. positive detection rates of
`80%, 61 %, and 6 l %, were noted in patients with stage III,
`stage IV, and recurrent disease, respectively. Complete surgical
`
`RE's.\L CELL G\RC!i\:0\1,\
`
`/
`
`815
`
`excision was associated with a significant reduction in serum
`y-enolase levels. Finally. 7 of 8 patients whose values normal(cid:173)
`ized after nephrectomy had increased values at the time of sy,(cid:173)
`temic relapse. No apparent relationship was noted between
`serum y-enolase levels and the site of metastatic disease. Al(cid:173)
`though patients with granular cell histotypes demonstrated
`higher serum y-enola,e levels than those with clear cell tumors,
`these results were not statistically significant.
`y-Enolase is not a tumor marker specific for RCC. Nonethe(cid:173)
`less, ii is avidly expressed by most such tumors at the tissue
`level. and elevated serum levels are frequently demonstrable in
`patients with advanced-stage disease.
`
`ALDOLASE
`
`Fructose-I, 6-bisphosphate aldolase is a tetrameric enzyme
`involved in the anaerobic glycolytic pathway.38 It is composed
`of three immunologically distinct subunits (A, 8, and C).w Al(cid:173)
`dolase A and C are the predominant isozymes expressed in the
`fetal kidney.40 In contrast, aldolase B represents 94.1 % of total
`aldolase content in the normal adult kidney, with A and C iso(cid:173)
`zymes contributing 5.8% and 0. I%, respectively:1~.4 1 Aldolase
`B is localized predominantly in the proximal tubules of the
`nephron.
`Previous studies have demonstrated altered expression of al(cid:173)
`dola,e isozymes during carcinogenesis, and most have demon(cid:173)
`strated reversion to the fetal isozymes (A and C) during this
`process. 42,.·1 In concert with these observations. Zhu and associ(cid:173)
`ates analyzed RCC tissues by means of sensitive enzyme immu(cid:173)
`noassays capable of discriminating the various aldolase iso(cid:173)
`zymes and demonstrated that these tumor tissues contained
`97.6% of A isozyme. with Band C isozymes contributing 1.7%
`and 0.7%, respectively.]8 Furthermore, these investigators
`noted that the concentrations of aldolases A and C in RCC
`exceeded the concentrations documented in normal renal cortex
`tenfold and fivefold. respectively. This quantitative analysis by
`enzyme immunoassay was further correlated with immunohis(cid:173)
`tochemical studies that demonstrated the presence of aldolases
`A and C in all ten RCC specimens subjected to this analysis.
`Conversely, aldolase B was only faintly expressed in 20% of
`these specimens. 38
`These same investigators determined the serum levels of al(cid:173)
`dolase A in patients with RCC (n = 40), patients with benign
`urogenital diseases (n = 48), and in patients with prostatic,
`testicular. and urothelial cancers (n = 34).18 The average serum
`aldolase A level in nonnal controls was 198 ± 51 ng/mL. The
`upper limit of nomial for serum for aldolase A was established
`at 300 ng/mL. The mean serum aldolase A level in patients
`with RCC was 472 ± 258 ng/mL. Using these parameters,
`elevated serum aldolase A levels were detected in 75%, 6.3%,
`and 21 %. of patients with RCC, benign urologic diseases, and
`non-RCC malignant diseases. respectively. When stratified by
`tumor stage, elevated serum aldolase levels were noted in 53%
`(9 of 17) of patients with stage I disease. All patients with stage
`II (4 of 4) and stage Ill (7 of 7) disease demonstrated elevated
`levels. Finally, 83% ( 10 of 12) patients with stage IV RCC
`demonstrated elevated serum levels of this "fetal" isozyme.
`Aldolase is apparently similar to y-enolase in several re(cid:173)
`spects. First, neither marker is specific for RCC. Nonetheless,
`both markers appear to be consistently elevated in patients with
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`
`advanced-stage disease. Finally, both markers are expressed in
`low concentrations in the healthy kidney cor1ex. but both iso(cid:173)
`zymes undergo a resurgence of expression during carcinogene(cid:173)
`sis. The latter observation legitimizes their designation as "on(cid:173)
`cofetal ·' tumor markers.
`
`S-IOOAo PROTEIN
`
`The S-100 protein family consists of dimers composed of
`two subunits. a and /3. Three isofonns have been identified and
`include the following: (I) S-1 (){)a0 • aa: (2) S-1 OOa. a/3: and (J)
`S-IOOb, /3/3. 44 The S-100a0 isoform is expressed in particularly
`high concentrations in the heart, skeletal muscle, kidney. carti(cid:173)
`lage tissue, and brain. 44 The S-IOOail protein is preferentially
`localized in the proximal tubules of the nonnal adult kidney.
`The a antigen is not expressed in the epithelial cells of the
`distal and collecting tubules.
`Using a sandwich enzyme immunoassay system ti.)r S-1 OOai,
`protein,45 Kato44 and Kimura45 and their associates demonstrated
`that the average content of S-100a0 in RCC was fourfold higher
`than that expressed in notmal kidney tissue (650 ng/mg protein
`versus 160 ng/mg protein, respectively), When serum S-IOOa0
`levels were measured in healthy adults (n = 40), all patients ex(cid:173)
`hibited levels less than 0.3 ng/mL. Indeed. 78<¼, of healthy con(cid:173)
`trols exhibited serum levels less than 0.2 ng/mL, the lowest
`limit of detectability for their assay. 44 Elevated levels of S(cid:173)
`J 00a0 ( >0.5 ng/mL) were detected in 53% ( 17 of 32) of patients
`with RCC at the time of diagnosis. Serial determinations were
`also made in 13 patients at various times during their clinical
`course, and the S-IOOai) levels diminished after nephrectomy,
`but they increased at the time of clinically documented relapse.
`Unfortunately, a detailed subset analysis of these patients (i.e ..
`tumor stage. tumor grade, and histologic type) was not reported.
`The ultimate utility of S-100a0 as a tumor marker for RCC
`will depend on the outcome of larger and more rigorously de(cid:173)
`signed clinical trials. Elevations of this marker are not unique
`to RCC, but have also been detected in patients with lung cancer
`(30% ), breast cancer (20% ), and chronic nephritis ( 15% ).44 In
`chronic nephritis, obstruction of the proximal tubules as a com(cid:173)
`ponent of the inflammatory process probably accounts for the
`increased serum level of S-100a0 protein.
`
`INTERLEUKIN-6 AND OTHER CYTOKINES
`
`IL-6 is a pleiotropic cytokine whose synonyms include inter(cid:173)
`feron beta1 • hepatocyte-stimulating factor. and B-cell stimula(cid:173)
`tory factor-2. 46 The gene for human IL-6 is located on the short
`arm of chromosome 7 p J 5-p21. consists of five exons and four
`introns. and is homologous with the gene for granulocyte col(cid:173)
`ony-stimulating factor (G-CSF).46 The IL-6 gene transcribes a
`1.3-kb mRNA that is subsequently translated into a 212-amino
`acid (26-kd) precursor protein. The final 184-amino acid protein
`is obtained after removal of a 28-amino acid signal peptide.
`Before secretion. IL-6 is glycosylated. and this posttranslational
`modification apparently may play an important role in the phar(cid:173)
`macokinetics of JL-6. as well as in tissue-specific functions. 41'
`Disturbances of homeostasis. including infection. tissue in(cid:173)
`jury. immunologic disorders. and neoplastic growth. are the
`major catalysts of IL-6 production. Endothelial cells. fibro-
`
`blasts. and mo11ocytes and macrophages constilllle !he major
`sources of IL-6 during elicitation of the acute-phase response.46
`Both interleukin- I (IL- I J and tumor necrosis factor-alpha
`(TNF-a) stimulate the synthesis of IL-6. whereas steroid hor(cid:173)
`mones tend to be inhibitory. The interaction of IL-6 with its
`numerous target cells is contingent on the expression of the
`IL-6-receptor by those target cells. The IL-6-receptor cDNA
`transcribes a protein of 468 amino acids that contains a signal
`peptide ( 19 amino acids) and a domain resembling that of the
`immunoglobulin superfamily (90 amino acids). 46 The molecu(cid:173)
`lar weight of the IL-6 receptor protein is approximately 80 kd.
`The liver. a particularly sensitive target organ for IL-6, is
`largely responsible for the induction of acute-phase proteins by
`that organ.47 These proteins include the following: ( l) C-reac(cid:173)
`tive protein; (2) serum amyloid A; (J) haptoglobin; (4) alpha 1-
`antichymotrypsin; (5) fibrinogen; (6) alphal-antitrypsin; and (8)
`alpharmacroglobulin. 46
`IL-6 also plays an obligatory role in the tenninal differentia(cid:173)
`tion of B cells into immunoglobulin-secreting cells. 46·47 IL-6
`induces the proliferation of the pluripotential hematopoietic
`stem cells in synergy with IL-3, IL-4. granulocyte-macrophage
`colony-stimulating factor (GM-CSF). and macrophage colony
`stimulating factor (M-CSF).46 Finally, IL-6 is an endogenous
`pyrogen. along with IL- I, TNF-a. and interferon beta.46
`IL-6 is constituitively produced by many human nonhemato(cid:173)
`poietic tumor cell lines. most notably RCC.48 ···00 Gast I and asso(cid:173)
`ciates demonstrated in vitro secretion of IL-6 (>20 pg/mL/72
`hours) in a total of94% (17 of 18) of the RCC cell lines tested. 48
`These observations are not limited to RCC cell lines. Studies
`conducted by Takenawa and associates documented of IL-6
`gene expression by Northern blot analysis in over 50% (22
`of 43) of fresh primary RCC tissues. In addition. these same
`investigators used DNA-polymerase chain reaction and de(cid:173)
`tected the presence IL-6 receptor transcripts in all RCC tissues
`( 43) and cell lines (7). 49 These observations contrast with analy(cid:173)
`ses performed in normal kidney tissues that reveal that mesan(cid:173)
`gial cells. but not tubular epithelial cells, produce IL-6. 49·"0
`Some studies have suggested that IL-6 may function as an auto(cid:173)
`crine growth factor in RCC." 1 This perception remains contro(cid:173)
`versial. however.52 IL-6 has also promoted tumor-cell motility
`in vitro. 5·1 Compelling evidence also indicates that IL-6 plays
`a major role in many paraneoplastic syndromes observed in
`RCC patients including fever. erythrocytosis. leucocytosis. am(cid:173)
`yloidosis. and increased acute-phase proteins.49 Finally. some
`observations suggest that IL-6 is an etiologic factor in the he(cid:173)
`patic dysfunction syndrome (Stauffer"s syndrome). 54· " 5
`Several studies have suggested that IL-6 expression in RCC.
`at both tissue and serum levels. is an adverse prognostic vari(cid:173)
`able. In the study performed by Takenawa and associates. clini(cid:173)
`cal and pathologic data were available in 24 of 4'.l patients with
`RCC. 49 These investigators performed a subset analysis based
`on the ratio of tumor to nonnal tissue of IL-6 gene expression
`(i.e .. :s 1.0 or > 1.0). Patients exhibiting high-level expression
`of IL-6 (>I.OJ had a significantly greater incidence of both
`lymph node metastasis and substantially greater levels of serum
`C-reactive protein. These investigators did not observe any sig(cid:173)
`nificant difference in tumor size. tumor grade. or distant metas(cid:173)
`tasis.
`Tsukamoto and associates used an ELISA assay for IL-6 and
`analyzed the serum levels in 71 p.iticnts with RCC."' 7 Because
`none of the controls exhibited IL-6 serum levels higher than
`
`NOVARTIS EXHIBIT 2023
`Breckenridge v. Novartis, IPR 2017-01592
`Page 4 of 10
`
`

`

`IO pg/ml. rhis value wui,: estahlbhed as the upper limit t,f nor(cid:173)
`mal. Using this pan1i:t1cwr definiti<m, 25,1%. llf the patients wi1h
`RCC had elevatw IL-6 le.vefs. ln thi.~ study. the likelihood of
`:ll:lnorm.il IL-6 levels closely ct)rtelated with advancing tumor
`·;lage. Conttury m the ohservations of Takenawa and assoi:i(cid:173)
`:ttes."~' these investigators did not d(.)cument any ol:lviou1,. rela(cid:173)
`li9nship between nothtl sHuus ai1d a~r1om1al sermn lL-6 levels~
`i1/>wever, atmormul IL~6 expre~'.siort did roughly i;orre:Iate with
`· u,m>r g:ntde, because ratients with grade I. gra4e 2, and gra<le
`} tfoiease had abnonmil IL-6 levels in 6.3'Yo. 27;<)%. aJ:ld 46.2%
`,if ca.sei:. res:pectively. l\foreover. the 5-yeu.r disease-specific
`'\~FVIV'UI WU!, significantly greater in those p.atients )NithSta;ge
`.\4H (jisea;se who n\anifexted nonnai serum IL-6 levels. These
`findings were further vafid.t1ted by Blay and associates, who
`,lbserved th.at patients with elevated JL-6.serum levels exhibited
`;i poor response to systemic IL-2 therapy aoo generally mani•
`,ested a shotter survival interval when compared with patients
`wJth RCC who had normal or undetectable levels of thii; cyro(cid:173)
`kine.5';
`Other t.')'lokrnes have also been identified in RCC cell lines
`,lml fi!lsues. For example, Q-CSF has been identified. as a 11ec;re-
`1ory product i)l the RCC cell Jine BAI 119 and may be responsi(cid:173)
`ble. tot the striking polymorphonudear leuk-0cytosis doc.u(cid:173)
`mented jn nude mice bearing. thi11 xenograft.M.55 G-CSF
`production tn!lY contribute to .similar pa;raneoplastic. findings in
`patients with RCC Moreover. the sequence ltornology shared
`between IL-6 ood G-CS1'1 l1. important.~"
`Similarly. Ga!ltl and associates deiededJL-10 wecretion P40
`pg/mL/12 hours) iri 22% of RCC c~d lines {4 of IS) ctJltivated
`in vitro}t IL-10 is gener.ally coexptes$ed with IL-6 in hoth
`lymphoid .cells and c.ells of the macrophage lineage.57 8.0!h
`cytokines appear to contain homologou.,; non<.'Oding region se(cid:173)
`quences a~socr.ated v.,·ith transcriptional regt11ation.5M IL-IO is
`rapable of inhibiting num~ous aspect.-. of the immune system.
`including; antigen pre&.entafinn, cytokine production. matro-
`11hage activalion.
`l!lld untigen-specfflc T-ce!l. prolifera(cid:173)
`tion.41!51-w Although the ultimate importance of IL-10 with re•
`sJJect to a human RCC remains. to be elucidated, Jnvestiga1ors
`have sugges:ted that IL-6 and IL-10 repre.sent major elements
`l'lf an importarn cyiokine network operative within themicroeJT.·
`vfrumhent of some solid tumors.41!
`
`BASIC FIBROBLAST GROWTH FACTOR. AND
`OTffER ANGIOGENIC PROTEINS
`
`Hypervasculatity is a prototypic trmr demonstrable in over
`85% of RCCs.. The production of angiogenic growth fucrors by
`RCC tumor cells ts po:.tulated to be responsll;ile for this fea(cid:173)
`ture, &ti Indeed. strong evidence suggests that the angiogenesi1,
`exhibited by RCCs in vivon1ay involve the cooperntlve intera1.1-
`tfon <.)f t.hree specific angiogenic factors: (!) b1'1GF: (2) vai-1cular
`tndothelia.f growtll tac.tor (VEGF}, und (3) placenta growth fa.;(cid:173)
`tor {PiGf.'}.1" The potential impti1tunceufe,.ichofthese proteins
`is brietty stirnmariied in this sectfon.
`bFGF is a member of th:e heparin-binding growth foctor fam(cid:173)
`ily. which includes acidic fibroblast growth factor, bFGF, int-2.
`hst-.LFGF-5. h.st-2/FGF-6, und 11:erutinocytJ::growth fador.()r,,,::!
`The gene for bPGF is !(,cared on chromosQme 4. appears fo be
`in1ern:1pted hy at least :! intrans. ei-tcnds over .t{} kb, and
`
`rrunsc1·ibes two to five mRNA species that µltimately translate
`a; protein l.'Ontaining (57 amino ucid..:; with a molecular weight
`ol' l1,:iOA.<'2 ·.,... bFGF confains four cysteine residues, two of
`which are conserved ;unMg an members o( the heparinabinding
`growth factor family.6'~ bFGP ha.<; been itiemificd in virtually
`in all organs. sol.id tissues. iumors,.and cultured cells subjected
`to vigorous Charuderiz.ation.112 Simllurly. reeeptoi:s for bFGF
`have been identified in numerotfs cultured cells, focluding endo(cid:173)
`thefra1 cells, baby hamster kidney eel.ls. imd fibroblasts. includ(cid:173)
`ing those of prostatit 01igin:"u'6 Two receptors with molecular
`weight'> of 125Jlt)() and i4S,OOO have been.Identified. Members
`of the heparin-binding growth tattor family appear to ha,ve
`overlapping teceptor 11ffinity. That receptor-expression is
`downregufared by the production of endogenous. bFGF ,is sug(cid:173)
`gested by the observed in.verse relationship between the receptor
`densiw per cell and the latter's conteni ofbPO P. li7 bf'.OF mRN A
`li16ks a definitive signal sequence. For this reason, the relea,<ie
`of bPGF into the surround111g milieu does not occur through
`normal secretocy pathways. lndeed, no definite mechanism for
`.bFGF release h;i.<i been idenHfied other than as a consequence
`of cell deatft,!12
`bFGP demonstrates a diverse array of b'Jologic activity, Jn(cid:173)
`cludlng the following: ( l) induciiondurlng embryonic devetop(cid:173)
`men.t: (2) inc~eased ceH.gr-0wth.and migration; (3} induction of
`plasminogen .activator and