Page 001
`
`West-Ward Exhibit 1098
`Kondo 2001
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 001
`
`

`

`Int. J. Cancer: 91, 219–224 (2001)
`© 2001 Wiley-Liss, Inc.
`PTEN/MMAC1/TEP1 MUTATIONS IN HUMAN PRIMARY RENAL-CELL
`CARCINOMAS AND RENAL CARCINOMA CELL LINES
`Kei-ichi KONDO1, Masahiro YAO1*, Kazuki KOBAYASHI1, Shinsuke OTA1, Minoru YOSHIDA1, Shigeki KANEKO1, Masaya BABA1,
`Naoki SAKAI1, Takeshi KISHIDA1, Satoshi KAWAKAMI1, Hiroji UEMURA1, Yoji NAGASHIMA2, Yukio NAKATANI3 and Masahiko HOSAKA1
`1Department of Urology, Yokohama City University School of Medicine, Yokohama, Japan
`22nd Department of Pathology, Yokohama City University School of Medicine, Yokohama, Japan
`3Pathology Section, Yokohama City University Hospital, Yokohama, Japan
`
`Publication of the International Union Against Cancer
`
`Extensive allelotyping studies have implicated several tu-
`mor-suppressor loci on chromosomes 3p, 5q, 6q, 8p, 9pq,
`10q, 11q, 14q, 17p, 18q and 19p in human kidney tumorigen-
`esis. The PTEN (also called MMAC1 and TEP1) gene, a candi-
`date tumor suppressor located at chromosome 10q23.3, is
`mutated in a variety of sporadic malignancies as well as in
`patients with Cowden disease. To investigate the potential
`role of the PTEN gene in renal tumorigenesis, we searched
`for abnormalities of the gene in 68 primary renal-cell carci-
`nomas (RCCs) as well as in 17 renal carcinoma– derived cell
`lines, using DNA-SSCP, sequencing and microsatellite anal-
`ysis. Five of 68 (7.5%) primary RCCs exhibited intragenic
`mutations (3 missense, 1 deletion and 1 splice-site), and 1 of
`17 (5.9%) cell lines had an insertion mutation. Loss of het-
`erozygosity of the PTEN gene occurred in 25% of primary
`RCCs, including the 3 cases with intragenic mutation and the
`1 PTEN-mutated cell line. Clinical and histopathological ex-
`aminations revealed that 4 of the 5 primary tumors with
`PTEN mutation were high-grade, advanced clear-cell RCCs
`with distant metastases or renal vein tumor invasions, result-
`ing in poor prognostic courses. The other was a low-stage
`papillary/chromophilic RCC. Our data suggest that PTEN
`mutation is observed in a subset of RCCs and that, especially
`in clear-cell RCCs, it occurs as a late-stage event and may
`contribute to the invasive and/or metastatic tumor pheno-
`type.
`© 2001 Wiley-Liss, Inc.
`Key words: PTEN/MMAC1/TEP1 gene; tumor suppressor; mutation;
`carcinoma; renal cell; cell line
`
`Renal cell carcinoma (RCC) is the most common malignant
`neoplasm in the adult kidney. Cytogenetic and molecular genetic
`studies have demonstrated that several cancer-associated genes,
`including oncogenes and tumor-suppressor genes, are involved in
`the development and progression of human neoplasms.1 Regarding
`the molecular pathogenesis of RCC, chromosome 3p loss has been
`observed in most clear-cell RCCs, and the von Hippel-Lindau
`(VHL) disease tumor-suppressor gene, located at 3p25, has been
`shown to be mutated and inactivated frequently in this tumor
`subtype.2,3
`In addition to the VHL gene, extensive allelotyping studies have
`implicated several other tumor-suppressor loci on chromosomes
`5q, 6q, 8p, 9pq, 10q, 11q, 14q, 17p, 18q and 19p in human kidney
`tumorigenesis.4 – 6 A putative tumor-suppressor gene, PTEN (phos-
`phatase and tensin homologue deleted on chromosome 10) also
`called MMAC1 (mutated in multiple advanced cancers-1) and
`TEP1 (TGF-␤–regulated and epithelial cell–enriched phospha-
`tase),
`located at chromosome 10q23.3, has been identified.7–9
`Somatic mutation of the PTEN gene has been found in a variety of
`sporadic tumors,
`including gliomas, melanomas and prostate,
`breast and endometrial carcinomas.7,8,10 –13 Moreover, germ-line
`mutation of the gene was identified in Cowden/Bannayan-Riley-
`Ruvalcaba syndrome, an autosomal-dominantly inherited multiple
`hamartoma syndrome with a high risk of breast and thyroid car-
`cinomas.14,15 The PTEN gene product is a dual-specificity phos-
`phatase with sequence homology to the cytoskeletal protein tensin;
`it suppresses the growth, apoptosis and migration of cells through
`negatively regulating the phosphatidylinositol 3-kinase/Akt signal-
`ing pathway.16 –19
`
`Although somatic PTEN mutation in renal carcinomas has been
`reported,8,20,21 the detailed mutational status of the gene in spo-
`radic RCC has not been well characterized. To further investigate
`the potential role of PTEN in human renal tumorigenesis, we
`searched for abnormalities of the gene in primary sporadic RCCs
`as well as in RCC-derived cell lines.
`
`MATERIAL AND METHODS
`
`Tissue samples
`Sixty-eight primary sporadic RCCs and matched normal kidney
`tissues were obtained from patients during surgery at Yokohama
`City University Hospital and its affiliated hospitals. All specimens
`were frozen rapidly with liquid nitrogen and stored at –80°C until
`nucleic acid extraction. Tumors were confirmed to be sporadic
`according to medical records. Tumor stage was determined ac-
`cording to the TNM classification. A system of 3 nuclear grades
`(G1,
`low-grade; G2,
`intermediate-grade; G3, high-grade) was
`used, according to the General Rule for Clinical and Pathological
`Studies on Renal Cell Carcinoma.22
`Cell lines
`Human renal carcinoma–derived cell lines included SGE-RC,
`VH-Renal, VMRC-RCW, VMRC-PCZ, SN12C, KC12, MTS-RC,
`KWA-RC, OUR10, OUR20, YCR-1, A498, CAKI1, SMKT-R2,
`SMKT-R3, ACHN and NSK-RC. Cell lines were maintained in
`Ham’s F12 or DMEM with 10% FBS.
`DNA-SSCP and sequencing analysis
`High m.w. genomic DNA was prepared by a proteinase K/phe-
`nol chloroform extraction method.23 We used PCR primer sets for
`DNA-SSCP analysis for all 9 exons and exon–intron boundaries of
`the PTEN gene, as described previously.8 The PCR conditions in
`each primer set were essentially as described previously8 with
`[␣-35S]dATP added to the reaction. Following 40 cycles of PCR
`amplification, products were denatured, electrophoresed on MDE
`gel (FMC Bioproducts, Rockland, ME), dried and exposed to
`X-ray films at room temperature overnight. When aberrant SSCP
`patterns were detected, PCR-SSCP was repeated with both tumor
`and normal corresponding kidney samples, to confirm the results.
`PCR products were purified using the Wizard PCR-Prep kit (Pro-
`mega, Madison, WI) and directly sequenced, or sequencing was
`performed after the product was cloned into the pCR2.1 vector,
`using a TA cloning kit (Invitrogen, La Jolla, CA). Sequencing was
`
`Grant sponsor: Ministry of Education, Science, Sports and Culture
`(Japan); Grant number: 10671488.
`
`*Correspondence to: Department of Urology, Yokohama City Univer-
`sity School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004,
`Japan. Fax: ⫹81-45-786-5775. E-mail: masayao@med.yokohama-cu.ac.jp
`
`Received 17 March 2000; Revised 13 September 2000; Accepted 18
`September 2000
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 002
`
`

`

`220
`
`KONDO ET AL.
`
`performed using a Thermo Sequenase radiolabeled terminator cy-
`cle sequence kit (Amersham, Arlington Heights, IL) according to
`the manufacturer’s protocol. Following the sequence reaction,
`these products were electrophoresed on 6% polyacrylamide gels.
`Gels were dried and exposed to radiographic films for 1 day.
`RNA extraction, RT-PCR and cDNA sequencing
`Total RNA was isolated from primary RCCs and corresponding
`normal tissues by a guanidine thiocyanate extraction method.24
`Total RNA was treated with DNase I to avoid contamination of the
`DNA from PTEN pseudogene.25 First-strand cDNA was then
`synthesized using a reverse transcription system (Promega) ac-
`cording to the manufacturer’s protocol. For RT-PCR amplification
`of the PTEN fragment from exons 1 to 4, PCR primers (forward
`5⬘-ATG ACA GCC ATC ATC AAA GAG-3⬘, reverse 5⬘-AGG
`ATA TTG TGC AAC TCT GCA-3⬘) were used with the following
`conditions: denaturation at 95°C for 10 min; then 30 sec at 95°C,
`30 sec at 55°C and 30 sec at 72°C for 40 cycles; then 5 min at
`72°C. Following amplification, PCR products were evaluated on
`1.5% ethidium bromide–stained agarose gels, purified using the
`QIA quick Gel Extraction (Qiagen, Chatsworth, CA), cloned into
`the pCR2.1 vector and sequenced using the same protocol de-
`scribed above.
`LOH detection by PCR-microsatellite analysis
`Two microsatellite markers, D10S215 and D10S541, both
`flanking the PTEN gene, were used for determining the allelic
`imbalance of the gene.26 Another polymorphic dinucleotide-repeat
`marker, PTENCA, which contains the 5⬘ end of the gene, was also
`used for loss of heterozygosity (LOH) analysis.27 PCR conditions
`were essentially as described previously with [␣-32P]dATP added
`to the reaction.26,27 Following amplification for 20 cycles, products
`were electrophoresed through denaturing 6% polyacrylamide gels,
`dried and exposed to X-ray films at –80°C overnight. Each allelic
`intensity was measured with a Hewlett-Packard (Palo Alto, CA)
`Scan Jet digital scanner with NIH image software (version 1.55)
`for determining LOH status.
`
`RESULTS
`We screened 68 primary sporadic RCCs and matched normal
`kidney samples as well as 17 renal carcinoma–derived cell lines
`for PTEN mutations throughout all exons and exon–intron bound-
`aries using DNA-SSCP and sequencing analysis. Sixty-eight pri-
`mary RCCs consisted of 54 clear-cell and 7 papillary/chromophilic
`carcinomas, 6 sarcomatoid variants and 1 chromophobic carci-
`noma.
`
`Five primary RCCs, tumors 27, 68, 86, 263 and 333, and 1 cell
`line, OUR20, showed aberrant mobility shifts on DNA-SSCP
`analysis for exons 2, 5, 3, 3, 8 and 8, respectively (Fig. 1).
`Sequencing studies revealed that tumor 27 had a T-to-C transition
`at nucleotide (nt.) 158, and it made an amino acid change (valine
`to histidine) at codon 53. In tumor 68, a C-to-G transversion was
`found at nt. 303, and it made an amino acid change (isoleucine to
`methionine) at codon 101. In tumor 263, a T-to-C transition was
`found at nt. 202, and it made an amino acid change (tyrosine to
`histidine) at codon 68. In tumor 333, a 1 bpdeletion at nt. 968 (968
`delA) was identified. In OUR20, a 1 bpinsertion at nt. 1005 (1005
`insA) was found (Fig. 2). The presence of a single mutant allele in
`both SSCP and direct sequencing of PCR products in OUR20
`indicated that the wild-type PTEN allele is lost in this cell line.
`In tumor 86, a G-to-C transversion 5 bp from the splice donor
`site of intron 3 was found, and we suspected that it made a splice
`variant. It is typical for nucleotide changes involving splice-junc-
`tion sequences to produce a variety of splice variants together with
`a normally spliced message.28 To look for splice aberrations in this
`tumor, we reverse-transcribed mRNAs, amplified the cDNA and
`sequenced the fragments. RT-PCR and sequencing analysis re-
`vealed that tumor 86 exhibited at least 2 aberrant-sized transcripts;
`one was an abundant longer message with abnormal 52 bp se-
`quences between exons 3 and 4, and the other was a shorter, faint
`message due to the absence of all of exon 3 (Fig. 3). In addition to
`these aberrant messages, we detected a normally spliced PTEN
`transcript by RT-PCR. Unfortunately, we could not determine
`whether this normal message was derived from tumor cells with a
`splice-site mutation since we analyzed mRNAs from fresh tumor
`specimens usually containing some amount of non-cancerous cells.
`Histopathological and clinical data revealed that 4 of the 5
`primary tumors displaying PTEN mutation were high-grade, ad-
`vanced clear-cell RCCs with distant metastasis (cases 68 and 86)
`or renal vein tumor invasion (cases 263 and 333) at the time of
`nephrectomy. All 4 patients subsequently died of multiple meta-
`static lesions within 3, 3, 48 and 22 months, respectively (Table I).
`In contrast, the fifth PTEN-mutated tumor (case 27) was a papil-
`lary/chromophilic RCC with T1bN0M0G1 staging and good prog-
`nosis (Table I).
`Exons 7 and 8 of the PTEN gene contain an A(6) repeat, and it
`has been shown that these mononucleotide repeats are targets for
`mutations in replication error-positive colorectal carcinomas.29
`However, we did not find any change in these 2 A(6) repeats in our
`tumors or cell lines.
`During mutational analysis of the PTEN gene, we identified 2
`sequence polymorphisms, both of which had been reported by
`
`FIGURE 1 – DNA-SSCP analysis of the PTEN gene. Primary RCCs 27, 68, 86, 263 and 333 and the OUR20 cell line showed aberrant mobility
`shifts in exons 2, 5, 3, 3, 8 and 8, respectively. Aberrant bands (arrows) were observed only in renal carcinoma (C) or the OUR20 cell line (CL)
`but not in normal corresponding or control kidney samples (N).
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 003
`
`

`

`PTEN MUTATION IN HUMAN RENAL CARCINOMA
`
`221
`
`FIGURE 2 – Sequence analysis of the PTEN gene, demonstrating that tumor 27 had a T-to-C transition at nt. 158, tumor 68 had a C-to-G
`transversion at nt. 303, tumor 86 had a G-to-C transversion 5 bp from the splice donor site of intron 3 [nt. position 209(⫹5)], tumor 263 had
`a T-to-C transition at nt. 202, tumor 333 had an A deletion at nt. 968 and renal-carcinoma cell line OUR20 showed an A insertion at nt. 1005.
`N, corresponding normal kidney; C, renal-carcinoma sample.
`
`others.27,30 One was a C/G variant 9 bp from the splice donor site
`of the 5⬘ side of exon 1 with allelic frequencies of 0.97 (C) and
`0.03 (G). The other was a T/G variant 32 bp from the splice donor
`site of intron 8 with allelic frequencies of 0.43 (T) and 0.57 (G) in
`our panel of Japanese patients.
`We next examined primary RCCs for LOH status at the PTEN
`locus using 3 highly polymorphic dinucleotide-repeat markers,
`D10S215, D10S541 and PTENCA. LOH at the PTEN locus was
`found in 14 (25%) of 57 informative cases. Three of the 5 primary
`RCCs with intragenic mutation (tumors 86, 263 and 333) also
`showed LOH at this locus (Fig. 4), which means that the PTEN
`gene was completely inactivated in a “2-hit” manner in these
`tumors. We did not detect any aberrant patterns exhibiting micro-
`satellite instability (MSI) for these 3 dinucleotide-repeat markers
`in the 68 fresh tumors analyzed.
`
`DISCUSSION
`In the current study, we examined the mutational status of the
`PTEN1/MMAC1/TEP1 gene in primary RCCs as well as in renal-
`carcinoma cell lines and found somatic PTEN mutations in 5 of 68
`(7.5%) primary tumor specimens and 1 of 17 (5.9%) cell lines. The
`PTEN gene was completely inactivated in 3 of the 5 primary
`tumors and in 1 cell line by “2-hit” mechanisms.31 Histopatholog-
`
`ical and clinical data revealed that 4 of the 5 primary tumors with
`PTEN mutation were high-grade, advanced clear-cell RCCs with
`poor prognosis.
`In sporadic gliomas, PTEN mutation was observed exclu-
`sively in high-grade, high-stage tumors.11 Loss of PTEN ex-
`pression was associated with tumor progression and poor prog-
`nosis in these malignancies.32 Moreover, functional analysis
`demonstrated that wild-type PTEN can inhibit cell spreading
`and motility by suppressing both the focal adhesion kinase and
`the mitogen-activated protein kinase pathways, while mutated
`PTEN that had lost these functions enhanced the spreading and
`migration of tumor cells.16,33 Our data suggested that PTEN
`inactivation also occurs as a late-stage event in the tumorigen-
`esis of clear-cell RCCs and may contribute to the invasive
`and/or metastatic tumor phenotype.
`In contrast, the fifth PTEN-mutated tumor (case 27) was a
`relatively early-stage,
`low-grade papillary/chromophilic carci-
`noma. The patient is alive without tumor recurrence more than 11
`years after surgery. This subtype of RCC is morphologically and
`genetically distinct from clear-cell RCCs.34 –36 Indeed, we have
`previously analyzed tumor samples in the present series for so-
`matic VHL mutations. Three clear-cell RCCs with PTEN mutations
`(tumors 86, 263 and 333) also showed somatic VHL mutations,
`while tumor 27 had no VHL mutation3 (data not shown). These
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 004
`
`

`

`222
`
`KONDO ET AL.
`
`FIGURE 3 – Results of RT-PCR analysis of PTEN transcripts and schematic illustration of aberrant splicing identified in primary RCC. RT-PCR
`of PTEN exons 1–4 in renal tumor sample 86 (No. 86) showed 2 aberrant-sized transcripts together with a normal-sized transcript identical to
`that in a normal corresponding kidney sample (Control). Sequencing analysis revealed that the longer transcript contained 52 bp aberrant
`insertions between exons 3 and 4, the shorter band lost all of exon 3 and a normal-sized transcript had normally spliced cDNA sequences. Marker,
`100 bp DNA ladder (GIBCO-BRL, Gaithersburg, MD) was used as a size marker.
`
`Mutation
`
`Exon/intron
`
`Consequence
`
`LOH1
`
`Histopathology
`
`TNM2
`
`Grade3
`
`Outcome (months)4
`
`TABLE I – SUMMARY OF PTEN MUTATIONS IN PRIMARY RENAL-CELL CARCINOMAS AND RENAL-CARCINOMA CELL LINES
`Renal
`vein
`invasion
`(⫺)
`
`Tumor/
`cell line
`
`27
`
`Exon 2
`
`NI
`
`T1bN0M0
`
`G1
`
`Alive, NED (133)
`
`158T3C
`Val53 Gly
`Papillary/
`chromophilic
`303C3G
`Ile101 Met
`Exon 5
`68
`Dead of disease (3)
`G3
`(⫺)
`T3aN2M1
`Clear/compact
`No
`209 (⫹5)G3C
`Dead of disease (3)
`G3
`(⫺)
`T3aN0M1
`Clear/compact
`Yes
`Splice mutation
`Intron 3
`86
`202T3C
`Tyr68 His
`Dead of disease (48)
`G3
`(⫹)
`T3bN0M0
`Clear/compact
`Yes
`Exon 3
`263
`Dead of disease (22)
`G3
`(⫹)
`T3bN0M0
`Clear/compact
`Yes
`Frameshift
`Exon 8
`968 delA
`333
`—
`—
`—
`—
`—
`Yes
`Frameshift
`Exon 8
`OUR20 1005 insA
`1NI, not informative.–2TNM classification.–3Nuclear grading system: G1, low grade; G2, intermediate grade; G3, high grade.–4Survival
`months were calculated from the date of surgery. NED, no evidence of disease.
`
`mutational data suggested that clear-cell and papillary/chro-
`mophilic RCCs have different tumorigenic pathways and that
`PTEN may play distinct roles in these 2 subtypes of RCC.
`PTEN was frequently mutated in endometrial carcinomas with
`MSI, suggesting that this gene might be one of the mutational
`targets for a mismatch repair defect.13,37 MSI has been observed in
`only a small fraction of sporadic RCCs.6,38 Indeed, most of the
`tumor specimens in the present series were the subject of previous
`studies, including VHL mutations and MSI status, and we have
`found infrequent MSI in these RCC tumors.38 In the current
`analysis, again, we did not find any aberration of the 2 A(6) repeats
`in the PTEN coding, which have been shown to be targets in
`replication error (RER)-positive colorectal cancers,29 nor did we
`find MSI in 3 dinucleotide-repeat markers in our 68 RCC samples.
`The MSI/RER⫹ phenotype, therefore, appears to be rare, and there
`is no observable correlation between PTEN mutation and MSI in
`sporadic RCCs.
`Somatic PTEN mutation was originally found in 1 of 6 primary
`RCCs by Steck et al..8 Then, Sakurada et al.39 and Cairns et al.40
`reported no PTEN mutations out of 24 and 15 RCC samples
`analyzed, respectively. Alimov et al.20 found somatic PTEN inac-
`
`tivation in 3 of 54 (5.6%) primary RCCs and 1 of 9 (11%) RCC
`cell lines. In this study, we identified somatic PTEN mutation in
`primary RCCs and renal-carcinoma cell lines at a relatively low
`incidence (7.5% in primary tumor samples and 5.9% in RCC cell
`lines). Based on these findings, we conclude that PTEN alteration
`is likely to be involved in a subset of RCCs; nevertheless, our data
`suggest that inactivation of this tumor-suppressor gene occurred as
`a late-stage event and may be associated with invasive and meta-
`static tumor phenotypes in some clear-cell RCCs. Further detailed
`analysis including mutational detection and functional study will
`be needed to evaluate the importance of the PTEN gene in human
`kidney tumorigenesis.
`
`ACKNOWLEDGEMENTS
`We thank Drs. I. Kondo and T. Miura of the Kanagawa Cancer
`Center Hospital, M. Moriyama of the Yokohama City Municipal
`Hospital, H. Fukuoka of the Yokohama Minami Kyousai Hospital
`and K. Kitami of the Kokusai Shinzen Hospital for providing
`kidney tissue samples. We also thank Ms. R. Shimizu, Ms. Y.
`Nakamura and Ms. M. Ishii for excellent technical assistance.
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 005
`
`

`

`PTEN MUTATION IN HUMAN RENAL CARCINOMA
`
`223
`
`FIGURE 4 – Dinucleotide repeat analysis of uncultured tumor DNA. LOH analysis for selected cases and markers is shown. Arrows, lost alleles;
`N, corresponding normal kidney DNA; C, renal-carcinoma DNA.
`
`3.
`
`6.
`
`REFERENCES
`cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer
`1. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer.
`Res 1998;58:23312334.
`Cell 1996;87:159–170.
`19. Tamura M, Gu J, Takino T, Yamada KM. Tumor suppressor PTEN
`2. Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, et al.
`Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat
`inhibition of cell invasion, migration, and growth: differential in-
`volvement of focal adhesion kinase and p130Cas. Cancer Res 1999;
`Genet 1994;7:85–90.
`59:442–449.
`Shuin T, Kondo K, Torigoe S, Kishida T, Kubota Y, Hosaka M, et al.
`20. Alimov A, Li C, Gizatullin R, Fredricksson V, Sundelin B, Klein G,
`Frequent somatic mutations and loss of heterozygosity of the von
`et al. Somatic mutation and homozygous deletion of PTEN/MMAC1
`Hippel-Lindau tumor suppressor gene in primary human renal cell
`gene of 10q23 in renal cell carcinoma. Anticancer Res 1999;19:3841–
`carcinomas. Cancer Res 1994;54:2852–2855.
`3846.
`4. Morita R, Ishikawa J, Tsutsumi M, Hikiji K, Tsukada Y, Kamidono S,
`21. Gesk S, Siebert R, Wacker HH, Nurnberg N, Harder L, Lehman J, et
`et al. Allelotype of renal cell carcinoma. Cancer Res 1991a;51:820–
`al. Lack of deletions of the PTEN/MMAC1 and MXI1 loci in renal
`823.
`cell carcinoma by interphase cytogenetics. Cancer Genet Cytogenet
`5. Morita R, Saito S, Ishikawa J, Ogawa O, Yoshida O, Yamakawa K, et
`2000;118:87–88.
`al. Common regions of deletion on chromosomes 5q, 6q, and 10q in
`22. Japanese Urological Association, Japanese Society of Pathology and
`renal cell carcinoma. Cancer Res 1991b;51:5817–5820.
`Japan Radiological Society. General rule for clinical and pathological
`Thrash-Bingham CA, Greenberg RE, Howard S, Bruzel A, Bremer M,
`studies on renal cell carcinoma, 3rd edition. Tokyo: Kanehara, 1999.
`Goll A, et al. Comprehensive allelotyping of human renal cell carci-
`23. Sambrook J, Fritsch EF, Maniatis T, Molecular cloning: a laboratory
`nomas using microsatellite DNA probes. Proc Natl Acad Sci USA
`manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor
`1995;92:2854–2858.
`Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, et al. PTEN,
`Laboratory Press, 1989.
`24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by
`a putative protein tyrosine phosphatase gene mutated in human brain,
`acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
`breast, and prostate cancer. Science 1997;275:1943–1947.
`Biochem 1987;162:156–159.
`Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, et
`25. Teng DH, Hu R, Lin H, Davis T, Iliev D, Frye C, et al. MMAC1/PTEN
`al. Identification of a candidate tumour suppressor gene, MMAC1, at
`mutations in primary tumor specimens and tumor cell lines. Cancer
`chromosome 10q23.3 that is mutated in multiple advanced cancers.
`Res 1997;57:5221–5225.
`Nat Genet 1997;15:356–362.
`26. Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, et al. A
`Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus,
`comprehensive genetic map of the human genome based on 5,264
`is a novel protein tyrosine phosphatase regulated by transforming
`microsatellites. Nature 1996;380:152–154.
`growth factor beta. Cancer Res 1997;57:2124–2129.
`27. Wang SI, Puc J, Li J, Bruce JN, Cairns P, Sidransky D, et al. Somatic
`10. Guldberg P, thor Straten P, Birck A, Ahrenkiel V, Kirkin AF, Zeuthen
`mutations of PTEN in glioblastoma multiforme. Cancer Res 1997;57:
`J. Disruption of the MMAC1/PTEN gene by deletion or mutation is a
`4183–4186.
`frequent event in malignant melanoma. Cancer Res 1997;57:3660–
`28. Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single
`3663.
`base-pair substitutions in mRNA splice junctions of human genes:
`11. Rasheed BK, Stenzel TT, McLendon RE, Parsons R, Friedman AH,
`Friedman HS, et al. PTEN gene mutations are seen in high-grade but
`causes and consequences. Hum Genet 1992;90:41–54.
`29. Guanti G, Resta N, Simone C, Cariola F, Demma I, Fiorente P, et al.
`not in low-grade gliomas. Cancer Res 1997;57:4187–4190.
`Involvement of PTEN mutations in the genetic pathways of colorectal
`12. Cairns P, Okami K, Halachmi S, Halachmi N, Esteller M, Herman JG,
`et al. Frequent inactivation of PTEN/MMAC1 in primary prostate
`cancerogenesis. Hum Mol Genet 2000;9:283–287.
`cancer. Cancer Res 1997;57:4997–5000.
`30. Ueda K, Nishijima M, Inui H, Watatani M, Yayoi E, Okamura J, et al.
`13. Kong D, Suzuki A, Zou TT, Sakurada A, Kemp LW, Wakatsuki S, et
`Infrequent mutations in the PTEN/MMAC1 gene among primary
`al. PTEN1 is frequently mutated in primary endometrial carcinomas.
`breast cancers. Jpn J Cancer Res 1998;89:17–21.
`Nat Genet 1997;17:143–144.
`31. Knudson AG. Antioncogenes and human cancer 1993. Proc Natl Acad
`14. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, et al. Germline
`Sci USA 1996;90:10914–10921.
`mutations of the PTEN gene in Cowden disease, an inherited breast
`32. Sano T, Lin H, Chen X, Langford LA, Koul D, Bondy ML, et al.
`and thyroid cancer syndrome. Nat Genet 1997;16:64–67.
`Differential expression of MMAC/PTEN in glioblastoma multiforme:
`15. Marsh DJ, Dahia PL, Zheng Z, Liaw D, Parsons R, Gorlin RJ, et al.
`relationship to localization and prognosis. Cancer Res 1999;59:1820–
`Germline mutations in PTEN are present in Bannayan-Zonana syn-
`1824.
`drome. Nat Genet 1997;16:333–334.
`33. Gu J, Tamura M, Pankov R, Danen EH, Takino T, Matsumoto K, et
`16. Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM.
`al. Shc and FAK differentially regulate cell motility and directionality
`Inhibition of cell migration, spreading, and focal adhesions by tumor
`modulated by PTEN. J Cell Biol 1999;146:389–403.
`suppressor PTEN. Science 1998;280:1614–1617.
`34. Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, et al.
`17. Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C,
`Germline and somatic mutations in the tyrosine kinase domain of the
`Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell sur-
`MET proto-oncogene in papillary renal carcinomas. Nat Genet 1997;
`vival by the tumor suppressor PTEN. Cell 1998;95:29–39.
`16:68–73.
`18. Cheney IW, Johnson DE, Vaillancourt MT, Avanzini J, Morimoto A,
`Inoue K, Karashima T, Chikazawa M, Iiyama T, Yoshikawa C,
`Demers GW, et al. Suppression of tumorigenicity of glioblastoma
`Furihata M, et al. Overexpression of c-met proto-oncogene associated
`
`7.
`
`8.
`
`9.
`
`35.
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 006
`
`

`

`224
`
`KONDO ET AL.
`
`with chromophilic renal cell carcinoma with papillary growth. Vir-
`chows Arch 1998;433:511–515.
`36. Yang ZQ, Yoshida MA, Fukuda Y, Kurihara N, Nakamura Y, In-
`azawa J. Molecular cytogenetic analysis of 17 renal cancer cell lines:
`increased copy number at 5q31-33 in cell lines from nonpapillary
`carcinomas. Jpn J Cancer Res 2000;91:156–163.
`37. Tashiro H, Blazes MS, Wu R, Cho KR, Bose S, Wang SI, et al.
`Mutations in PTEN are frequent in endometrial carcinoma but rare in
`other common gynecological malignancies. Cancer Res 1997;57:3935–
`3940.
`
`38. Kobayashi K, Kondo K, Ikeda I, Kaneko S, Sakai N, Yoshida M, et
`al. Microsatellite instability occurs infrequently in sporadic renal cell
`carcinoma. Oncol Rep 1997;4:941–944.
`39. Sakurada A, Suzuki A, Sato M, Yamakawa H, Orikasa K, Uyeno S,
`et al. Infrequent genetic alterations of the PTEN/MMAC1 gene in
`Japanese patients with primary cancers of the breast, lung, pancreas,
`kidney, and ovary. Jpn J Cancer Res 1997;88:1025–1028.
`40. Cairns P, Evron E, Okami K, Halachmi N, Esteller M, Herman JG, et
`al. Point mutation and homozygous deletion of PTEN/MMAC1 in
`primary bladder cancers. Oncogene 1998;16:3215–3218.
`
`West-Ward Exhibit 1098
`Kondo 2001
`Page 007
`
`