Pathology International 1998; 48: 184-1 90

Original Article Expression of splice variants of CD44 in thyroid neoplasms derived from follicular cells

Jing Gu,’ Tsutomu Daa,’ Kenji Kashima,’ Shigeo Yokoyama,’ lwao Nakayama‘ and Shiro Noguchi’ ’First Department of Pathology. Oita Medim1 Univetsity, Oita and 2Noguchi Thyroid Clinic and Hospital Foundation, ~ P U . Japan

Isofom expression of CD44 In follicular carcinoma (FC) of the thyroid w1u analyzed by Immunohlstochemlcal Staining and compared to the Isoforma In folllcular adenoma (FA) and papillary carcinoma (PC) of the thyroid. Variant lsoforms of CD44 (CD44v) were detected In these neoplastic cdis but not In non-neoplastic cells. CD44v6 was expressed In PC with nodal metastaals and also in FC at slgnlflcantiy hlgher trsqwn~les than those In PC without metastasis and FA. The fmquency of expression of CD44v3 was slgnHl- can* hlgher in PC with nodal metastasls than In PC with- out mobstaals. The reverse tmnscription-polymerase chain mactlon (RT-PCR) followed by Southern blotting analysis revealed the presence of a transcript for a varlant of CD44 that contained varlant exon 6 in FA, FC and PC. DNA sequencing ofthe products of RT-PCR yielded three species of cDNA for CD44v. One of the cDNA corresponded to a tranocrlpt that contained varlant exon 6. These results suggest that immunohlstochemlcal staining and RT-PCR with southem Mottlng analysis for CD44v6 might be a usetul diagnostk too! for the differentiation of FC from FA and that the expression of CD44v3 and C W v 6 might be important for the development of nodal metastasis in cases of Pc.

Key words: base sequencing, CD44 splice variants, immunohistochemistry, RT-PCR, thyroid neoplasm

Cell adhesion molecules affect the development, morpho- logical characteristics, and functions of cells as well as the progression of malignant neoplasms.‘2 CD44 has been proposed to function as a lymph node homing receptor on circulating lympho~ytes.3.~ It is an integral membrane glyco- protein that functions as a receptor for hyaluronate, which is a major glycosaminoglycan of the extracellular ma t r i~ .~ -~ The gene for CD44 has at least 19 exons! The standard form, CD44s, or CD44H (hematopoietic), found mainly in the cells

Correspondence: lwao Nakayama. MD, PhD, First Department of Pathology, Oita Medical University, Hasama-machi, Oita 879-55, Japan. Email: <NAKAYAMAOoita-med.ac.jp>

Received 14 July 1997. Accepted for publication 30 September 1997.

of mesodermal origin, is encoded by a sequence composed of exons 1 through 5, and 15 through 19. Variant isoforms (CD44v) designated as CD44v2-CD44v10, arise as a result of the alternative splicing of combinations of exons 6 through 14 onto the CD44s framew~rk.~.’

There appears to be a positive correlation between the expression of CD44v and the progression and metastasis of tumors in the rat.s1a Such results stimulated studies of the expression of CD44v in a variety of human tumor^.''-'^ Wielenga et a/. demonstrated that the expression of CD44 that contained variant exon 6 (exon v6; CD44v6) was related to the progression of colon cancer.” In uterine cervical carcinoma, it was shown that tumors that express exon v6 are associated with an increase in the frequency of metastatic dispersal to the lymph nodes and that the expression of exon v6 is correlated with an enhanced likelihood of vascular invasion.’2 Similarly, in human breast cancer, a positive correlation has been found between the expression of CD44v6 and lymphatic metastasi~.’~ Thus, overexpression of CD44v, and in particular of CD44v6, might play a causative role in the progression and metastasis of certain human malignant tumors.

In an immunohistochemical study, Figge et a/. demonstrated the preferential expression of CD44H and CD44v6 in papillary carcinoma (PC) of the thyroid, as well as a relationship between the expression of CD44v6 and lymphatic metastasis of such tumor^.'^ By the reverse transcription-polymerase chain reaction (RT-PCR), Ermak et al. demonstrated the synthesis of mRNA for CD44v6 in various thyroid neoplasms derived from the follicular cells.’6 Furthermore, they described the splice variants in PC in detail and even identified novel splice variants.” However, the expression of CD44v remains to be characterized in follicular carcinoma (FC). The histological diagnostic criteria for differentiation of FC from follicular adenoma (FA) of the thyroid remain somewhat ambiguous.’* Therefore, in the present study, the transcription and expression of CD44v were investigated, and FC was compared with PC and FA by molecular biological and immunohistochemical analysis.

Expression of CD44 in thyroid neoplasms 185

MATERIALS AND METHODS

lmmunohistochemistry

All neoplasms in this series were reviewed histologically to confirm the diagnosis according to the classification of the World Health Organization.18 Neoplasms with the following characteristics were used in the study. In conventional PC, branching papillae were visible, which had a fibrovascular core covered with a single layer of neoplastic cells, and specific nuclear features such as a ground glass-like appearance and a nuclear groove. Follicular carcinoma was associated with widespread or focal invasion to adjacent thyroid tissue and/or vascular invasion; while FA had a well- defined fibrous capsule with no evidence of capsular or vascular invasion and nodal metastasis.

Thyroid neoplasms (105 cases) included: 27 cases of FA; 21 cases of FC of the widely invasive type (FCWI); 19 cases of FC of the minimally invasive type (FCMI); 19 cases of PC without metastasis to the lymph node (PCw/oM); and 19 cases of PC with metastasis to the lymph node (PCw/M). In addition, 25 cases of metastatic PC to the lymph node (MPCL) were studied. The specimens were obtained from the Noguchi Thyroid Clinic and Hospital Foundation (Beppu, Japan) between 1980 and 1994.

All samples were fixed with 10% formalin for 24h and embedded in paraffin. The tissue blocks were cut at a thickness of 4pm and mounted on silanized slides. Endogenous peroxidase activity was blocked by incubation with 3% H202 for 20min. The sections were autoclaved in 0.01 mol/L citrate buffer (pH6.0) at 121°C for 15min. Normal goat serum, diluted 1 : 10 in phosphate-buffered saline (PBS pH7.4) was used to block the non-specific binding of second antibodies. The sections were then incubated with monoclonal antibodies (mAb) against CD44H, CD44v3 and CD44v6 at dilutions of 1 :lOOo, 1:500, 1:1000 in PBS. respectively, for 16h at 4°C. These mAb were directed against different variant epitopes of the CD44 molecule and were purchased from R & D System Europe Ltd (Abingdon, UK). Sections were then incubated with the Envision Labeled Polymer Reagent (DAKO Japan, Kyoto, Japan), namely, a peroxidase-labeled polymer that has been conjugated with goat antibodies against mouse immunoglobulin (lg; second antibodies). The immunoreaction was detected by incubation with 0.02% 3,3'-diaminobenzidine tetrahydrochloride plus 0.005% H202 for 5 min, and nuclei were counterstained with hematoxylin. Negative controls were prepared with normal mouse lg instead of the primary mAb.

Positive and negative samples were identified according to the criteria described by Figge eta/." In brief, a sample with strongly membrane-stained cells that represented more than 10% of the target population was classified as positive while Others were classified as negative.

Comparison of the relative numbers of positive and negative cases was made with a cross table and significance was examined by the Chi-squared test. Differences with a probability value of less than 0.05 were regarded as significant.

RT-PCR and Southern blotting analysis

For 15 cases of FA, five of FC, four of PCw/oM and eight of PCw/M, RT-PCR was performed. The tissues were frozen in dry ice and acetone immediately after surgical removal and stored at -80°C prior to analysis.

Messenger RNA was extracted with a mRNA isolation kit (Stratagene, La Jolla, CA, USA) according to the manu- facturer's instructions. Using a First-strand cDNA synthesis kit (Pharmacia, Uppsala, Sweden) to yield first-strand cDNA, 5 pg of mRNA were reverse transcribed. The first-strand cDNA was amplified by Taq DNA polymerase (Perkin-Elmer, Foster City, CA, USA) for 30 cycles at 94°C for 1.5 min, 62°C for 0.5 min, and 72°C for 1.5 min. The primers P1 (CCGCTAT- GTCCAGAAAGGAG) and P2 (GGGTGGAATGTGTCTTG- GTC) were homologous to conserved exons 5 and 15 of the published sequence of the human gene for CD44, respec- tively.6 Therefore, the length of the expected amplified fragment was 180 base pairs (b.p.) when tumor cells expressed the standard form of CD44. Larger products of amplification were anticipated from samples that expressed variant isoforms of CD44 which contained one or more variant exons.B'9~m A volume of 10 pL of the products of PCR were subjected to electrophoresis on a 1.5% agarose gel. After denaturation of the products of RT-PCR in a solution of 0.5 mol/L NaOH and 1.5 mol/L NaCI, samples were trans- ferred onto a Hybond N' nylon membrane (Amersham, Amersham, Buckinghamshire, UK) with subsequent cross- linking by ultraviolet irradiation. The samples on the mem- brane were then allowed to hybridize with a digoxigenin (DIG)-labeled probe for CD44v6 in a solution of l.Omol/L NaCI, 50mmoVL Tris-HCI (pH7.0), 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 50pg/mL salmon sperm DNA for 16 h at 64OC. The sequence of the probe for CD44v6 was TGGCATGAGGGATATCGCCAAACACCCAGA." Hybridization was detected with a DIG detection kit (Boehringer-Mannheim, Mannheim, Germany) and the color reaction allowed to develop for 3 h.

Cloning and sequencing of the products of RT-PCR

For cloning and sequencing, the products of RT-PGR obtained from one case each of PCw/M and FC were ligated to a cloning vector, pCR2.1 (lnvitrogen, San Diego, CA. USA), with an Original TA Cloning Kit (Invitrogen). Individual

colonies were then screened by the size of the insertion using digestion with EcoRI. Products of RT-PCR of bigger than 180 b.p. in length that had been ligated to the vector were seq-.

RESULTS

lmmunohlstochernlcal stalnlng

In almost all cases, the products of the immunoreaction specific for CD44H were observed on the plasma membrane of the non-neoplastic follicular cells that surrounded the neoplasms and on the plasma membrane of the neoplastic cells in FA, FC, and PC, but not in the cytoplasm and nucleus (Fig. la-c). The non-neoplastic thyroid tissue reacted less strongly in cases of FA than the neoplastic tissue (Fig. la). The neoplastic cells of FCWl were intensely immunostained in both intra- and extracapsular regions (Fig. 1 b), although the intensity of immunostaining varied considerably from one area of the neoplastic tissue to another, even within a single tumor. The localization and intensity of the products of a CD44H-specific positive immunoreaction in PC were almost identical to those in FC (Fig. Ic).

The products of a positive immunoreaction specific for CD44v3 and CD44v6 were detected on the neoplastic cells of FA, FC, and PC but not on the non-neoplastic follicular cells that surrounded these neoplasms. The relative levels of cells that expressed CD44v diered considerably from one type of tumor to another. The products of positive immunoreactions specific for CD44v3 and CD44v6 in the thyroid neoplasms were similarly localized to those for CD44H (Fig. 169.

The relative levels of positive cells calculated after

TsMe 1 Results of immunostaining for splice variants of CD44

No. Tumor cases

cD44v3 Positive cases (%) P

CD44v6

cases (%) P Positive

FA 27

FCWl 21

FCMl 19 PCWM 19

PCwIoM 19 MPCL 25

9

6 3 :=7°.g7 (16) 0.31

14 (74) ] 0.02 6 (32) 13 (52)

FA, follicular adenoma; FCWI, follicular carcinoma of the widely invasive type: FCMI, follicular carcinoma of the minimally invasive type; PCw/M, papillary carcinoma with metastasis to the lymph node; PCw/oM, papillary carcinoma without metastasis to the lymph node; MPCL, metastatic papillaty carcinoma to the lymph node; C W v 3 , monoclonal antibody against an epitope defined by exon v3 of the CD44 molecule; C W v 6 , a monodonal antibody against an epitope defined by exon v6 of the C M molecule. Pvaiues are the resuks of x' tests.

immunostaining for CD44v3 and CD44v6 in FA, FC, and PC are summarized in Table 1. Statistical analysis indicated that FCWl and FCMl were associated with significantly higher relative levels of CD44vG-positive cases (90 and 84%, respectively) than was FA (44%; P=0.002 and 0.013, respectively). The relative levels for CD44v3 did not, however, differ among FCWI, FCMl (29 and ley', respectively), and FA (33%; P= 0.97 and 0.31, respectively). In contrast, the expression of CD44v3 and CD44v6 was detected at significantly higher frequencies (74 and loo%, respectively) in PCw/M than in PCw/oM (Table 1). Metastatic PC to the lymph node also gave higher frequencies for the expression of CD44v3 and CD44v6 (52 and 88%, respectively) than PCw/oM, but the difference was not statistically significant.

RT-PCR and Southern blot analysis

For RT-PCR analysis and Southem blotting, products that hybridized with the v6 probe were detected in 10 cases of FA (67%; lanes 2,411, and 14; n = 15) and in all cases of FC (1 00%; lanes 16-20; R = 5; Fig. 2). Eleven cases of PC (92%; lanes 21 and 23-32; n = 12) yielded the bands of reaction products which hybridized with the probe, although the intensity of the products of positive hybridization reactions was relatively weak in lanes 23, 25, 29, and 30 (Fig.2). All cases of PCw/M (lanes 21, 23, 24, and 2-0; n = 8) and three cases of PCw/oM (lanes 25, 31, and 32) gave positive hybridization bands with the probe. The remaining case of PCw/oM (lane 22) was negative (Fig.2). There were no statistically significant differences among the frequencies of positive results by RT-PCR Southern blotting analysis among FA, FC, and PC. However, the products of hybridization from both PCw/M and PCw/oM were distributed between the range 320 and 1400b.p. as one or several bands, whereas the products of hybridization in two cases each of FC (lanes 19 and 20) and FA (lanes 4 and 5) corresponded to fragments between the range 320 and 1800b.p. Three other cases of FC (lanes 1 6 1 8) gave no positive bands of about 1800 b.p. and the main hybridizing band corresponded to a product of 800 b.p., which was commonly detected in PC and FA (Fig. 2).

Cloning and sequencing of the products of RT-PCR

The cloning and sequencing of the products of RT-PCR of more than 180b.p. in length revealed three types of clones for both FC and PC. These clones are represented by lanes 19 and 28 in Fig.2, respectively. The exon structures deduced from the base sequences are shown in a schem- atic diagram of CD44H and CD44v in Fig.3. Clone 1 corresponded to the epithelial form of CD44, and clone 2

Expression of CD44 in thyroid neoplasms 187

Figure 1 lmmunohistochemical staining of CD44H and CE44v. Micrographs show products of positive immunoreactions for C W H in (a) follicular adenoma (FA; right), (b) follicular carcinoma (FC) and (c) papillary carcinoma (PC); for CD446 in (d) FC and (a) PC; and for (1) CD44v3 in FC. (a) The reaction products specific for CD44H are visible on the plasma membrane of the non-neoplastic tissue (left), and immunostaining is weaker in non-neoplastic tissue than in neoplastic tissue. Products of the immunoreactions spec& for C W 6 and C W V 3 are seen only in the neoplastic tissue of FA, FC and PC with the same localization as CD44H.

188 J. Gu eta/.

included exon v3 in addition to the exons of the epithelial fom. All variant exm, with the exception of exon v2, were detected in done 3.

MSCUSSKlN

The vast majority of patients with thyroid neoplasms suffer from PC, which has an excellent prognosis, although nodal metastasis to the neck region is encountered at high

frequency at the time of operation. By contrast, FC is found at a significantly lower frequency than PC, and some cases of FC are associated with extensive metastasis to distant organs.18 Therefore, these types of thyroid carcinoma seem to differ in terms of their biological behavior as well as their histological characteristics. Furthermore, FA and FC have similar histological features, with the exception of the presence or absence of capsular or vascular invasion and nodal metastasis. Thus, it is important to clarify the differences in the expression of CD44v between FC and FA.

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 t 4 1 5

bP 1800- 1400- 800- 500-

300- 200-

100-

16 I ? 10 19 20. 21 22 23 24 25 26 27 20 29 30 31 32

800 - 500 - 300 - 200 - 100 -

Figure 2 Southern blotting analysis of products of amplification by reverse transcriptase -polymerase chain reaction (RT-PCR) for CD44v6. Upper and lower panels show products of RT-PCR for specimens obtained from follicular adenoma (lanes 1-15). follicular carcinoma (lanes 16-20) and papillary carcinoma (lanes 21-32) after Southern hybridization with the V6 probe. Papillary carcinoma with metastasis to the lymph node, lanes 21,23.24. and 26-30; papillary carcinoma without metastasis to the lymph node, lanes 22,25.31, and 32.

Expression of CD44 in thyroid neoplasms 189

In a recent study with an experimental model, it was demonstrated that the expression of CD44v6 by tumor cells allows those cells to mimic the behavior of lymphocytes and facilitates the lymphatic spread of the tumor cells." Thus, the expression of CD44 and, in particular, of CD44v6 seems to be important for the establishment of lymphatic metastasis in experimental tumors as well as in certain human tumors.

In an immunohistochemical study using antibodies against CD44H and CD44v6, Figge et al. suggested that the de- regulated expression of CD44v6 on the plasma membrane of PC cells contributed to the ability of these cells to metastasize to the regional lymph node^.'^ In the present study, the expression of CD44H was detected in both non- neoplastic and neoplastic thyroid tissue in almost all cases. However, the expression of CD44v and, in particular, that of CD44v6 was limited to the plasma membrane of neoplastic tissue. The frequency of expression of CD44v6 was significantly higher in both FCWl and FCMl than in FA (P= 0.002 and 0.01 3, respectively). Furthermore, the detection of both CD44v3 and CD44v6 was significantly more frequent in PCw/M than in PCw/oM. These results suggest that the expression of CD44v and that of CD44v6 in particular, might be important for the diagnostic differentiation of FC from FA and for the development of nodal metastases from PC of the thyroid. Compared with previously reported re~ults,'~ the present study revealed a much higher frequency of

onno no. 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 Variant exon 110. v2 v3 v4 v5 VB v7 VB Vgv10

P1 P2

Standard form

E p it tm I i a I fwm

clone 1

clone2

Gbn3

expression of CD44v6 in FC, although an elevated frequency of expression of CD44v6 in PC was found in both this and the earlier study.15 The discrepancy between frequencies of expression of CD44v6 in FC might reflect the subtypes of FC or the sensitivities of the reactions of the mAb with CD44v6.

Reverse transcription-PCR was performed using RNA from several specimens of PC, FC, and FA to confirm the immunostaining results of CD44v6. Using semiquantitative RT-PCR to examine PC, nodular goiter, FA, and thyfoiditis, Ermak eta/. found that PC was associated with significant overexpression of a CD44 transcript of between 750 and 1000 b.p., with an especially prominent Wontaining isofom of 750 b.p., which was also Seen in the majority of cases of goiter and FA but not in the cases of thyroiditis.16 They concluded from the data that CD44, including CD44v6, plays an important role in the pathogenesis of various thyroid diseases.

To date, there have been no reports describing the deregulation of alternative splicing of transcripts for CD44 in FC. Reverse transcriptase-PCR and DNA sequencing technology might provide relevant information for the diagnostic discrimination of FC from FA, and an explanation for the fundamental differences between FC and PC by revealing the details of alternative splicing.

The analysis by RT-PCR and Southern blotting in the present study revealed the presence of the CD44v6 transcript in both PC and FC at a frequency of 92 and loo%, respectively, while in FAthe mRNAthat included exon v6 was found at a somewhat lower frequency (67%). The products of RT-PCR that were hybridized with the v6 probe had different pattern lengths for FC (range, 320-1800b.p.) and for PC (range, 32O-14OO b.p). The most prominent v6containing cDNAwas 800 b.p. in length for the majority of neoplasms. In the present study, the CD44v6 transcription of Basedow's disease was tested instead of the normal thyroid surrounding the neoplasm. No transcription of the CD44v6 was detected in the thyroid of Basedow's disease (data not shown). Therefore, it appears that deregulation of alternative splicing of the transcript for CD44 is an important factor in the neoplastic growth of thyroid follicular cells, and that molecular biological events of different types might oaxr in FC and PC with variations in the length of the mRNA for CD44v6. Furthermore, synthesis of the mRNA for CD44v that yields the 8oDb.p. product of RT-PCR. which contains exon v6, miaht be one of the key stws in the neo~lastic ~rol i ferat ion of

clone 3. containing att variant exons with the exception of exon @. that OCCUIS between the transcription of mRNA to the

190 J.Guetal.

expression of the synthesized protein. The aforementioned discrepancy might reflect the differences in these steps for the neoplastic cells tested in the present study. Woodman et a/. have described that comparative studies on the tran- scription and translati of CD44 gene products in tumor cells have revealed some striking and unexpected discrepancies in the proportions of the prevalent mRNA and protein isofoms expressed by this

Cloning and sequencing of the products of RT-PCR generated three types of cDNA for C W v from both PC and FC: the epithelial form, which is expressed by most epithelii organs and two novel species containing additional variant exons in addition to those of the CD44 epithelial form; that is, containing both exon v3 and v6 (done 3) or exon v3 but not axon v6 (clone 2), in addition to the exons of the epithelial form. Thus, the results of RT-PCR with Southern blotting analysis were confirmed by the DNA sequencing of the RT-PCR products. It is likely that CD44v3 and/or CD44v6, which were detected immunohistcehemicalty in PC and FC, are expressed as a resutt of the translation of the cloned sequences. and their expression probabty induces malignant behavior, such as invasion and/or metastasis, as a result of an effect on the intercellular and/or cell-extracellular matrix adhesion.=

In conclusion, immunostaining specific for CD44v6 yielded positive results at a significantly higher frequency for FC than for FA, and RT-PCR with Southern blotting analysis confirmed the presence of a transcript for CD44 that contained exon v6 in all cases of FC. Therefore, immunohistochemical staining for CD44v6 and RT-PCR with Southem blotting analysis appear to be useful diagnostic tools for differentiating between FC and FA, and the expression of CD44v3 and C W v 6 appear to be important in the development of nodal metastasis in PC cases.

REFERENCES

Edelman GM. CeH adhesion and the molecular processes of morphogenesis. Annu. Rev. Biochem. 1985; 54: 135-169. Ledey J, Hyman R, Kincade PW. CD44 and its interaction with exhacellular matrix. A&. lmmunol. 1993; 54: 271-335. Jalkanen S, Reichert RA, Gallatin WM, Bargatze RF. Weissman IL, Butcher EC. Homing receptors and the control of lymphocyte migration. /muno/. Rev. 1986; 91: 39-60. Jalkanen S, Bargatze RF, de 106 Toy- J, Butcher EC. Lymphocyte recognition of high endothelium: Antibodies to distinct epitopes of an 85-9OkDa glycoprotein antigen differentially inhibe lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J. Cell Biol. 1987; 105: 983-990. Aruffo A, Stamenkovic I, Melnick M, Undethill CB, Seed 8. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990; 61: 1303-1313.

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. P m . NabAcad. SN. USA1992;89: 12160-12164. Brooks L, Niedobitek G. Agathanggelou A, Farrell PJ. The expression of variant CD44 in nasophatyngeal carcinoma is unrelated to expression of LMP-1. Am. J. Pathol. 1995; 146: 1102-111 2. Guntheit U, Hofmann M, Rudy W et a/. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell1991; 65: 13-24. Rudy W. Hofmann M, Schwartz-Albiez R et a/. The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: Each one individually suffices to confer metastatic behavior. Cancer Res. 1993; 53:

Seiter S, Arch R, Reber S eta/. Prevention of tumor metastasis formation by anti-variant CD44. J. fip- Med. 1993; 177: 443-455. Wielenga VJ, Heider KH, Offerhaus GJ et a/. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res. 1993; 53: 4754-4756. Kainz C, Kohlberger P, Sliutz G et a/. Splice variants of CD44 in human cervical cancer stage IB to IlB. Gynecol. Oncol. 1995; 57: 383-387. Kaufmann M, Heider KH, Sinn HP, von Minckwii G, Ponta H, Herrlich P. CD44 variant exon epitopes in primary breast cancer and length of survival. Lancet 1995; 345: 615-619. Li H, Hamou MF, de Tribolet N eta/. Variant CD44 adhesion molecules are expressed in human brain metastases but not in glioblastomas. Cancer Res. 1993; 53: 5345-5349. Figge J, del Rosario AD, Gerasimov G et a/. Preferential expression of the cell adhesion molecule CD44 in papillary thyroid carcinoma. fxp . Mol. Pathol. 1994; 61: 203-211. Ennak G, Gerasimov G, Troshina K et al. Deregulated alternative splicing of CD44 messenger RNA transcripts in neoplastic and non-neoplastic lesions of the human thyroid. Cancer Res. 1995; 55: 4594-4598. Ermak G, Jennings T, Robinson L, Ross JS, Figge J. Restricted patterns of CD44 variant exon expression in human papillary thyroid carcinoma. Cancer Res. 1996; 56: 1037-1 042. Hedinger C, Williams ED, Sobin LH. Histological typing of thyroid tumors. In: World Health Organization lntemational Histological Classification of Tumors. Springer-Verlag, Berlin, 1988; 3-11. Screaton GR, Bell MV, Bell JI, Jackson DG. The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor: Comparison of all 10 variable exons between mouse, human, and rat. J. Biol. Cbem. 1993; 268: 12235- 12238. Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet 1992; 340: 1053-1 058. Arch R, Wirth K, Hofmann M et a/. Participation in normal immune responses of a metastasis-inducing splice variant of CD44. Science 1992; 257: 682-685. Woodman AC, Sugiyama M, Yoshida K et a/. Analysis of anomalous CD44 gene expression in human breast, bladder, and colon cancer and correlation of observed mRNA and protein isofoms. Am. J. Pathol. 1996; 149: 1519-1530. Tanabe KK, Saya H. The CD44 adhesion molecule and metastasis. Crit Rev. Oncog. 1994; 5: 201-212.

1262-1268.