GYNECOLOGIC

ONCOLOGY

39, l-15 (I!?%)

REVIEW Tumor Markers in Gynecologic Oncology B. DAUNTER University

of Queensland

and Department

of Obstetrics

and Gynaecology,

Royal

Brisbane

Hospital,

Herston

Q, 4006,

Australia

Received January 2, 1990

marker is detectable, the earlier a possible diagnosis can be made. However, the appearance of a nonspecific tumor marker, be it produced early or later in tumor development, does not preclude its use in the management of the cancer patient. The tumor marker that fills this role is the beta subunit of human chorionic gonadotropin (fi-HCG), produced by tumors composed of syncytiotrophoblast (choriocarcinoma). Similarly, to some extent, a-fetoprotein (AFP) (germ cell tumours), estrogens (granulosa cell tumours), and androgens (Sertoli-Leydig cell tumors) are useful nonspecific tumor markers. These markers, as well as the morphological criteria of the Pap smear, are well established in gynecological oncology and have recently been reviewed [1,2]. This review is a distillation of the development of tumour markers from the point of view of gynecologic oncology. The emphasis has been placed on glycoprotein markers that can be identified in serum and/or in terms of cellular morphology. It has been established that the carbohydrate moieties of the glycoproteins are the epitopes identified by monoclonal antibodies (Mabs), and that due to aberrations in glycosylation, these epitopes can vary between tumors. Thus, the new potential use of markers such as the carcinoembryonic antigen (CEA) is examined. The use of the relatively new marker CA125 is considered in relationship to other markers for improving diagnosis, management, and detection of recurrence. Potential new markers are also considered from the point of view of oncogenetic changes, oncogenes, and oncoproteins in relation to tumor growth factors.

INTRODUCTION There are a plethora of tumor markers in general, and for gynecologic oncology in particular. This has largely been brought about by the at times hasty introduction of tumor markers into a clinical setting when they are more suited to the research laboratory and has lead to their empirical clinical assessment, with the result that “new ones for old” has been the clinical request to the experimental oncologist. The situation is now changing, and “old” tumor markers are being reexamined in the light of new scientific information that considers the complexity of tumor progression, and hence the direction of tumor marker development. This review is therefore an attempt to distill the essence of the conception, birth, growth, and future direction of tumor markers in gynecologic oncology. Following the introduction, the review proceeds with an explanation and consideration of aberrant glycosylation, changes in the carbohydrate moieties of glycoproteins and glycolipids, by tumor cells. This results in antigenic, and sometimes immunogenic changes. These changes in glycosylation can be detected by polyvalent and monoclonal antibodies and lectins. Lectins expressed by normal and cancer cells, endogenous lectins, offer the potential for exploitation as tumor markers and for the selective delivery of cytotoxic drugs. Oncogenetic changes are also explained and considered along with oncogenes and the use of DNA and RNA probes and the polymerase chain reaction. The use of monoclonal antibodies to oncoproteins is examined, as is the association between oncogene products and growth factors. A tumor marker may be considered as any biological aberration that indicates the presence of a tumor. The more specific the marker is for the tumor type, the more useful it is as a marker, especially if it is related to tumor growth and development. In addition, the earlier the

TUMOR

MARKER

DEVELOPMENT

Glycoproteins and Glycolipids

The importance of glycoproteins and glycolipids in cancer may be considered to have first been observed 1

OO!+O-8258190 $1 SO

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

B. DAUNTER

in 1951, when a woman with stomach cancer was admitted to Charlottesville Hospital, Charlottesville, Virginia. The blood group of the woman was 0, universal donor, but her serum carried antibodies to all other erythrocytes except her own, and no compatible donors could be found. To assess the risk of blood transfusion, she was given 25 ml of group 0 blood; thereafter, the concentrations of antibodies against the transfused erythrocytes dramatically increased. Limited surgery was performed to remove part of the tumor and stomach, and the remaining tumor tissue underwent spontaneous regression. The woman lived to the age of 88 years, with no further signs of cancer. A number of years later, the reason for the spontaneous regression of the tumor was established. In this particular case, the woman was the first person recorded who did not display the P and P, antigens of the P blood group, as do other individuals. It has now been established that such a situation occurs in 1 of 100,000 people. What had happened was that the tumor tissue expressed the P and P, antigens, and the woman developed an immune response against these antigens. This immune response was then enhanced by the trial blood transfusion [3]. Glycoproteins and glycolipids carry blood group specificity in terms of their carbohydrate sequence [4]. The glycolipids represent blood group antigens on erythrocytes, whereas glycoproteins may represent the same antigens on other tissue [5]. A large number of tumorassociated antigens belong to a family of carbohydrate structures that includes the blood group antigens [6]. In fact, studies with Mabs have demonstrated that the majority of Mabs are directed to carbohydrate epitopes, many of which are derived from carbohydrate blood group determinants [6-S]. It appears that such tumor carbohydrate-associated antigens (epitopes) are the result of aberrant glycosylation [9]. In addition, some of the carbohydrate antigens also represent differentiation antigens [IO-l21 and receptors [4] that may be reexpressed (activation of derepressed genes) or modified (and this also applies to blood group antigens) to give and/or neo-glynew structures, i.e., neo-glycoproteins colipids [ 131. These new carbohydrate structures can express new epitopes that do not cross-react with each other [14]. In addition, certain carbohydrate sequences of neo-glycoproteins and neo-glycolipids, especially Asnlinked oligosaccharides, are markers of metastatic potential [15], and may also influence cellular permeability to cytotoxic drugs [ 161. The aberrant glycosylation of glycoproteins and glycolipids in cancer cells can be determined by the isolation of these compounds, followed by carbohydrate sequence analysis relative to that found in the normal tissue. However, this method is not practical in terms of the definition of a tumor marker for clinical use. Nevertheless,

Mabs of known carbohydrate epitope specificity can be used in flow cytofluorometry with the Mab conjugated, for example, to FITC. Similarly, cytological and histological examination can be carried out based on these standard immunohistochemical techniques. In addition, some of these antigens (glycoproteins/glycolipids) may be released into the serum, and may prove to be of value, at least in monitoring tumor development and therapy, if not in diagnosis. Lectins Another method of determining aberrant glycosylation is by the use of lectins. Lectins are glycoproteins or proteins, originally found in plants and invertebrates, that combine only with carbohydrates [16]. Most lectins react with the terminal nonreducing sugars of the carbohydrate chains of glycoproteins and glycolipids [17], but some also react with sugars within the carbohydrate chains. They may be specific for a particular sugar or sequence of sugars, and can also be specific for linkage and whether or not the sugars are in an (Y or /3 configuration and/or have substituted groups [16,17]. The lectins are no less specific than antibodies for their antigens and can be inhibited from combining with a glycoprotein or glycolipid by “free” monosaccharide (equivalent to a hapten’s inhibiting immune complex formation) [ 16,171. Therefore, the methods employed for the use of Mabs in cytology and histology can also be used with lectins. This can be achieved by directly labeling the lectin, or using antibodies directed against the lectin, which are conjugated to an enzyme or fluorescent marker. Many of the lectins react with plasma membrane glycoproteins and glycolipids, but with histological sections of tissue, intracellular reactivity may be evident. Also, lectins may react with both normal and tumor tissue in a differential manner [18], and this had been used to identify normal eosinophilic leukocytes in tissue sections [19]. Lectin binding for several tumor types is presented in Table 1 [20-321. In addition, lectins can be used to examine extracts of tumor tissue, using various electrophoretic methods, for example, in breast cancer [31]. In addition to lectins for identifying aberrant glycosylation, Mabs to carbohydrate epitopes have also been employed [7,21,33,34]. Endogenous

Lectins

Endogenous mammalian lectins can be subdivided into membrane-bound and soluble lectins. Most of the soluble lectins bind /3-galactosides and require a reducing agent to maintain their carbohydrate binding activity [35]. In contrast, membrane-bound lectins show a wider range of carbohydrate binding specificity and do not require a reducing agent [35]. The first membrane-bound lectin to

TUMOR

TABLE 1 Lectin Reactivity: Primary Tumors Lectin

Carbohydrate specificity

WGA

(pz( l-4)-D-GlcNAc),

Con A

a-o-Man+o-Glc

SBA MPA PNA RCA, UEA, LFA BPA DBA PHA WFA LCA

o-GalNAc a-o-GalNAc( 1+3)-D-Gal) /?-D-Gal( h3)-o-GalNAc p-o-Gal( t23)-o-Gal a-L-FUC

Sialic acid GalNAc wGalNAc NANA-Gal( &3)-D-GlcNAc cY-GalNAc @D-Man

Primary tumor type” S.Sem, C.Sem, CaCx, CaB, Rb S.Sem, C.Sem, CaCx, CaE, CaB, Rb CSem C.Sem, Rb ECT, CaCx, CaB CaCx ECT, CaCx, CaCol CaCx Rb ECT Rb Rb Rb

” CaB, carcinoma; S.Sem, spermatocytic seminoma; C.Sem, classic seminoma; ECT, embryonal carcinoma of the testis; CaCx, squamous cell carcinoma of the cervix; CaCol, carcinoma of the colon (rectal or sigmoid); CaE, endometrial carcinoma; Rb, retinoblastoma. See Refs. (20-32).

be identified was the specific asialoglycoprotein (glycoprotein from which sialic acid has been removed) receptor (galactose) on mammalian hepatocytes [36,37]. It is a well-characterized endocytic receptor-ligand system in which the receptor is returned to the cell surface and the ligand degraded in lysosomes [38,39]. A number of endogenous lectins appear to be expressed during embryogenesis and are involved in differentiation [40-431. These endogenous lectins may then be reexpressed (activation of derepressed genes) during carcinogenesis [43] and/or accompany neo-glycoprotein expression [44,45]. Thus, in some cases, tumor endogenous lectin expression is specific to the tumor relative to its normal tissue counterpart [46], and in other cases, there is differential expression of the endogenous lectin in favor of the tumor, relative to normal tissue. The endogenous tumor lectins so far identified are specific for monosaccharides, disaccharides, and some oligiosaccharides, and these are presented in Table 2 [42,43,46-531. Tumor endogenous lectins have the potential to specifically target antineoplastic drugs by conjugating the appropriate carbohydrates to the drug [541, and also to be used as tumor markers. In the mode of tumor markers, the endogenous lectins may be identified by flow cytofluorometry with the appropriate glycosylated bovine serum albumin (GBSA) conjugated, for example, to FITC. The GBSA may also be used in cytological and histological investigations. The GBSA would bind to the endogenous lectin and this could then be visualized with anti-BSA antibodies linked to one of the many detection systems available, such as peroxidase or streptavi-

3

MARKERS

din/biotin systems. To date, lactose, galactose, and glucose and complex endogenous lectin receptors have been identified on an ovarian cell line (Daunter, unpublished data). DETECTION BY POLYVALENT AND MONOCLONAL ANTIBODIES The search for antigenic/immunogenic tumor markers started with the production of xenogenic polyvalent antisera to extracts of ovarian cancer tissue, and a number of putative antigens were identified, for example, the OCAA and OCAA-1 antigens [55], the OVD-l-OVD-6 series of antigens [561, the perchloric acid-soluble antigen [57], a thermostable glycoprotein antigen [58], a fucoserich glycoprotein [591, an embryonic protein [60], an unspecified antigen [61], the carcinoembryonic antigen (CEA) [62], and the NB/70K glycoprotein [63]. The CEA and the NB/70K antigen, perhaps more than any other putative ovarian cancer antigen, demonstrated an initial potential for use as serological and histological markers for ovarian cancer. However, it has been established that CEA has low specificity and sensitivity, whereas NB/ 70K has a high sensitivity, but low specificity [63]. To improve this situation, Mabs to some putative antigens such as CEA and NB/70K have been produced, but this has not changed the situation to any great extent. CEA is a plasma membrane glycoprotein initially discovered in colonic carcinomas, and the systemic levels of CEA were thought to be specific for this cancer [64,65]. Elevated serum levels of CEA have also been found in patients with carcinoma of the ovary, in particular, mutinous adenocarcinoma [66], and carcinomas of the cervix, uterus, and breast [67]. However, CEA levels are not usually elevated in more than 50% of patients with localized gynecologic malignancies, and are less frequently elevated in the early stages of tumor development [68,691. Elevation of CEA levels does reflect the tumor volume to some degree, but is less precise with large tumor masses [68,69]. In addition, CEA levels have been useful in the follow-up of ovarian cancer patients to monitor the response of the tumor to chemotherapy [68,69]. However, elevated levels of CEA are also found in nongynecologic malignancies [66,70,71], nonmalignant disease states [72,73], and healthy individuals [74-781. This has resulted in an unacceptable level of false-positive and false-negative results [67]. The falsepositive results are probably due to the fact that there are a number of cross-reactive CEA-like antigens, for example, the normal cross-reactive antigen (NCA), NCA-2, cross-reactive normal glycoprotein (NGP), (Yeacid glycoprotein, CEA-associated protein, colonic CEA-2, carcinoma antigen III, and tumor-assisted antigen [79-811. This demonstrates that CEA is a complex

4

B. DAUNTER

TABLE 2 Human Endogenous Tumour Lectin Carbohydrate Specificity” Lactose Tumor type Yolk sac Embryonic carcinoma Seminoma Colon carcinoma Teratocarcinoma Lung undifferentiated Small cell Cell line Melanoma C32 Leukemia K562 Hela

Galactose

Glucose

X

Fucose X

Melibiose

Mannan

Fucan

X

X

Man.b-PO,

X X

X X

X X

X

X

X X

X X X

X

X

” See Refs. [4l-43,46-531.

molecule in terms of its epitopes. This complexity has been revealed by the mapping of CEA Mab defined epitopes. The Mabs NCRC-23, C228 and 11.285.14 reacted specifically with CEA with little, if any, reaction with NCA, whereas C24, C161, and Cl98 rzaected with NCA and CEA [82]. Thus, CEA measurements may yet prove to be of greater importance with the increased specificity of Mabs NCRC-23, C228, and 11.285.14 [82]. At present, however, CEA levels can still be useful if measured simultaneously with CA- 125 and CA-19-9 (see below) to differentiate between ovarian and colorectal carcinoma [83,84]. This, together with the determination of NB/ZIOK, with one of the four Mabs available [85], to detect early-stage low-grade epithelial ovarian cancers [85], may be useful. A number of Mabs have been produced that define antigens found in most epithelial malignancies including ovarian cancer [86-881. For example, NCRC-11 is a Mab that was originally prepared against human primary breast carcinoma cells [89] and also reacts with a variety of normal tissue [90]. Similarly, B72.3 Mab has been shown to react with primary breast [91] and colon carcinomas and various histological types of ovarian cancer to different degrees [92,93], but the antigen was either not expressed or expressed in only trace amounts in normal tissue [91,92]. The Mab HMFG2 produced against a component of human milk fat globule membrane reacts strongly with lactating breast tissue and various epithelial neoplasms including breast, colonic, and ovarian cancer [94]. This antigen, however, is not generally released into the systemic circulation unless there is a malignant change, and may therefore have the potential to screen for cancer per se. The Mab OC-125, prepared against an ovarian cancer cell line, is one Mab that has considerable clinical potential. It reacts with 70-90% of nonmucinous ovarian

tumors [95-1011. This sensitivity is, however, variable and depends on the patient’s age, the type of tumor, and the stage. Its sensitivity has been reported to be 50% in premenopausal women, and 84% in postmenopausal women [102]. Similarly, the specificity of CA-125, detected by the Mab OC-125, is also variable. This is because CA-125 is also present on a variety of normal tissue, including coelomic epithelium [103,104] and the epithelium of the female reproductive tract, and can be detected in 22% of patients with nongynecological cancers and benign gynecological neoplasms [103-1071. Thus, this Mab also lacks specificity and produces a number of false-positive results [log]. The specificity of CA-125 can, however, be improved, if the cutoff point is raised from the commonly used value of 35 U/ml to 194 U/ml. This will still allow the detection of 80% of nonmucinous tumours but will exclude 95% of women with benign masses [lOl, 1091. A compromise value of 65 U/ml has been suggested to allow for acceptable sensitivity and specificity [ 110,l 1 11. However, the variables previously discussed need to be taken into account. In addition, CA-125 is expressed at higher levels in undifferentiated relative to differentiated nonmucinous tumors of the ovary, and there is little if any correlation between grade, stage, and ploidy [ 1121. Serum CA-125 is a useful marker in monitoring the progress of nonmucinous ovarian tumors, and is invaluable in the management and follow-up of patients. In follow-up, it reflects the response to therapy (surgery, chemotherapy, radiotherapy) in 80% of cases [96,110, 113,114]. The half-life of CA-125 may also be of prognostic value in that patients with a CA-125 half-life longer than 20 days had a significant increase in tumor progression [115]. However, although CA-125 is useful in the detection of recurrence [97,110,112,116,117], it does not detect tumor volumes less than 1 cm [ 1141. Never-

TUMOR

theless, the presence of CA-125 in the patient’s serum after therapy, does indicate residual tumor or metastasis [118]. Although CA-125 can be detected in early stages of ovarian cancer, it is not suitable as a screening marker in an asymptomatic population [ 1171, because normal serum levels of CA-125 do not exclude the presence of other diseases [96,119]; also, CA-125 is elevated in pelvic inflammatory disease [llO], and it has been used to aid in the evaluation of pelvic masses [102]. In addition, serum levels of CA-125 measured simultaneously with tissue polypeptide antigen (TPA), immunosuppressive acidic protein (IAP), carcinoembryonic antigen (CEA), ferritin [98], and to some extent CA-19-9 [106,120,121], but not lipid-associated sialic acid protein (LASA-P) [ 110,122], were more useful for early diagnosis, differential diagnosis, and early detection of recurrence and remission than CA-125 alone. These conclusions are supported by similar studies [ 123,124]. The use of multiple serum markers has also previously been demonstrated [125]. A monoclonal antibody (OV-TL3) raised against ovarian cancer cells was reported in 1986 to be more specific than OC-125 (Mab), and reacts with serous, mutinous, endometrioid, and clear cell cancers [108]. There was, however, a weak and irregular reactivity with benign cysts and epithelial cells of the female genital tract, including endocervical cells [108]. Similarly, more recent reports of a Mab against CA-153 indicates that this too may be another useful marker for ovarian cancer, although it has a low specificity since it is elevated in patients with carcinoma of the cervix, endometrium, and vulva [126,127]. Nevertheless, CA-15-3 values were correlated with the clinical course of the disease in 87% of ovarian cancer patients [ 126,127]. Two Mabs, MA54 and MA61, derived from immunization with a lung adenocarcinoma cell line also detect their corresponding antigens in serum from patients with both serous and mutinous ovarian cancer, endometrioid cancer, and mesonephroid cancer at 67, 64,40, and 78% respectively [128]. The advantage of these particular Mabs is that they detect mutinous ovarian cancer more frequently relative to OC-125 [128]. Other Mabs are proving useful in the diagnosis of malignancies, although the antigens cannot be detected systematically. These include the OMA, OMB, and OMC Mabs that detect predominantly cytoplasmic antigens of mutinous ovarian tumors [129]. Serum levels of squamous cell carcinoma antigen (TA4) appear to be more specific for cervical cancer. TA-4 has been reported to be elevated in 35% of stage IB patients, and increasing to 91% in stage IV, with serum levels returning to normal values during therapy in cases of complete remission [123]. Pretreatment levels of TA4 were elevated in 55% of patients compared to CA-125

MARKERS

5

(15%) and CEA (17%) [123]. There also exists the Ki67 Mab, which identifies a nuclear antigen in cells at all stages of the cell cycle except G,,. This Mab may be useful in assessing cell proliferation. In the case of cervical carcinoma, Ki67 has demonstrated considerable variation in tumor growth rates [130]. Examples of some Mabs that have been and/or are being assessed for monitoring gynaecologic malignancies are presented in Table 3 [131]. ONCOGENETIC

CHANGES

Tumors are composed of diploid and aneuploid cells. The diploid cell component of a tumor is made up from a portion of normal diploid cells and diploid tumor cells. Although not all patients with a diploid tumor have a good prognosis, aneuploidy is certainly a poor prognosis [132,133]. Aneuploidy is a reflection of increased DNA synthesis, and the chromosomes may have abnormal shapes and be damaged. Such gross changes are readily identified by cytogenetic examination of tumor cells, using the chromosome spread technique with trypsin/ Giemsa staining of chromosomes [134]. More subtle changes can also be identified from stained chromosomes. It was by such a method that a shortened chromosome 22 was identified in CML and named the Philadelphia chromosome, in honor of the city of its discovery [135]. The staining of chromosomes results in a banding pattern, and the bands of normal and malignant cells can be compared. In this way, high-resolution chromosomal banding has revealed that the Philadelphia chromosome was not just a deletion of chromosome 22, but the result of reciprocal and unequal exchange of chromatin between chromosomes 9 and 22 [136]. Similarly, other lymphoid malignancies have been defined in this manner [137]. Although these techniques have been applied to other malignancies and precancerous conditions, such as carcinoma of the cervix [138,139], their promised potential has not been completely attained. This is because chromosomal bands represent gross chromosomal changes, and such gross changes do not always accompany malignancy. In addition, chromosomal banding techniques are more diagnostic with regard to the tumor than prognostic with regard to the patient, relative to other methods. Apart from karyotyping, previously mentioned, which can be prognostic, there is also the doubling time (DT) of the tumor mass. However, this requires sequential measurements of the tumor mass prior to therapy and is therefore to some degree unethical. Those few studies that have been conducted on DT have been equivocal [140]. The potential DT of a tumor can, however, be determined in vitro by the tritiated thymidine labeling index (LI), using cytological autoradiography, or by scin-

6

B. DAUNTER

TABLE 3 Examplesof Monoclonal Antibodies and Their CorrespondingTumor-AssociatedAntigens in GynecologicMalignancies Monoclonal

antibody

OC-lZS(IgG,) OC-133(IgG,) OMl(IgM) MOvl(W,) MW(W) 3C2(IgM) 4WWd ID,UgG,) DU-Pan-2 (IgM) F 36/22(&G,) 4F,/7A,oUgG) OV-TL3(IgG,) DWW, ) B72.3 (IgG,) 2W2FAWJ MF 116 (IgG,,) CEA 1I-HS(IgG,) 19-9(IgG,) 3.14.A3(IgG,) H17-E2 (IgG,)

Antigen characteristics

Gynecologic cancer”

“CA-125” >200-kDa GPb 80-kDa GP “SGA” 360-kDa GP High-MW mucin High-MW mucin/glycolipid

CX, ov ov ov ov ov ov ov ov EN, ov EN, ov CX, ov EN, cx, ov EN, ov

-

High-MW mucin High-MW mucin >700-kDa GP “gp 48” 48-kDa GP

300- to 400-kDa GP “TAG-72” >lOOO-kDa mucin 60-kDa GP 105kDa GP “CEA” I80-kDa GP “CA-19-9” sialylated lacto-N-fucopentakose “HMFG-2” >400-kDa GP “PLAP” 67-kDa GP

Source. Adapted from Ref. [125]. ” CX, cervix; EN, endometrium; FT, fallopian tube; MT, mixed miillerian ’ GP = glycoprotein.

tillation counting. Thus, the uptake of tritiated thymidine is proportional to the potential DT. The actual DT (in viva) is always much greater than the potential DT (in vitro). Nevertheless, there is an acceptable correlation between tritiated thymidine uptake in vitro and actual DT in vivo [140]. With the development of rapid flow cytometry, DT analysis has given way to the more precise analysis of ploidy and cell cycle as prognostic indicators [141]. Cell flow cytometry (CFC) involves staining the DNA of cells with, for example, ethidium bromide and mithramycin. Characteristics of normal diploid cells can then be compared with those of cancer cells; it is thus possible to determine cell cycle events, as well as cell ploidy. Benign ovarian and cervical neoplasms are diploid, with an increase in the proportion of S-phase cells; in diploid and aneuploid malignant tumors, the percentage of S-phase cells is also increased. With respect to FIG0 staging (1 to IV) of ovarian cancers, all stage III and IV cancers appear to be aneuploid, although they do have variable populations of diploid cells, whereas 60% of stage I and II cancers may actually be diploid. There is also a correlation between DNA ploidy abnormalities and grade of differentiation in ovarian cancer. In the case of cervical cancer, there does not appear to be such a close relationship between staging and flow cytometry parameters. In contrast, malignancy grading reflects tumor proliferation (S phase). Flow cytometric and morphometric

II

EN, FT. Mt, OV

OV OV EN, MT, OV, VA OV (cell lines) ov OV

tumor; OV, ovary.

parameters, for both ovarian and cervical cancer have been analyzed by multivariate statistics, to improve the prognostic index. The success of flow cytometry is further evidenced by the development of methods to examine the nuclei of cells from fixed and/or paraffinembedded tissues. Further work is in progress to bring flow cytometric techniques into on-line clinical practice [ 142-1521. It should be noted that confusion can occur when reference is made to cell ploidy based on CFC studies, which are a measure of DNA content and not the number of chromosomes. Thus, DNA diploid range, etc., should be used, because diploid means normal chromosomes, and chromosomes of cancer cells are obviously not normal. In addition, it is possible to have a DNA content that is not reflected in terms of chromosome number.

ONCOGENES In 1911, Peyton Rous found that he could induce sarcoma in chickens using a cell-free filtrate from a similar tumor [153]. The active component of this filtrate was shown to be a virus, which is now referred to as Rous sarcoma virus (RSV). Then, in 1970, Martin [154] demonstrated that the ability of RSV to induce sarcoma in chickens was due to a single gene product. This gene became known as the src gene, an abbreviation for sar-

.

TUMOR

coma; hence the term oncogene came into existence. Similarly, other viral oncogenes are named after the tumors with which they are found to be associated. For example, the gene associated with chicken myelocytomatosis is myc; myb is derived from chicken myeloblastosis, and rus from rat sarcoma. Two major rus gene families are named after the discoverers Harvey (Ha or H-ras) and Kirsten (ki or K-ras) [155]. It should be noted that oncogenic viruses are rare and are usually defective forms of their normal counterparts because they contain additional coding sequences derived from normal cellular genes [156,1571. The concept of oncogenes was further developed by Stehelin er al. [158] in 1976, who discovered that the src gene or v-src, i.e. viral oncogene, had homologs in normal cells. These homologs of v-src are found in yeast, the fruit fly Drosophila, and various species of vertebrate cells, and they differ from each other approximately according to phylogenetic distance [153,154,158]. This conservation of the v-src homologs, and this applies to all other homologues of viral oncogenes, is that they are normal genes. These genes are essential to the function of normal cells in the regulation of growth and differentiation [159]. Thus, because these normal genes can become oncogenic, they are referred to as proto-oncogenes, and are designated by the prefix “c” (for cellular) to the viral counterpart [155], for example c-src. It should, however, be noted that not all cellular oncogenes (proto-oncogenes) have viral homologs. The proto-oncogenes become oncogenes by (i) mutation, (ii) amplification, and/or (iii) activation and/or amplification by translocation/rearrangement. Thus, in general terms, we may consider that inappropriate activation of protooncogenes results in deregulation of cell growth and differentiation. In terms of differentiation, this is exemplified in retinoblastoma, from which the term unri-oncogene has arisen [160]. Basically, anti-oncogenes are involved in differentiation and produce cancer in a recessive mode, that is, by loss or inactivation of both alleles. In contrast, oncogenes produce cancer in the heterozygous state (one active allele) and continued activation appears to be required to maintain carcinogenesis. It should not be concluded that only one active oncogene or a deletion of both anti-oncogene alleles is required to initiate carcinogenesis, because carcinogenesis is a multistep process. For example, retinoblastoma involves both an anti-oncogene [160] and an N-myc oncogene which becomes amplified [161]. In essence, carcinogenesis is a cascade effect, and only rate-limiting steps can at present be identified [ 1621. With this proviso in mind, Table 4 summarizes some of the proto-oncogenes associated with 15-20% [163] of neoplastic growths, when the proto-oncogenes become oncogenes.

7

MARKERS TABLE4

Proto-oncogenes Proto-oncogene (one)

Normal chromosome and region

c-myc

9(W)

N-myc

2(p23-24) 6(q22-25) 2O(q12-13) 15(q25-26)

c-myb c-srcc-fes c-abl c-erbB c-fms

I

wq34

Leukemia Leukemia

Yq34)

3(p25) 4

c-raf-2 c-mos

WV

c-Ha-ras-1

ll(Pl5)

c-H-ras-2

X

cKi-ras-1 c-Ki-ras-2

6(p23-ql2) 2(p1205-pter) l(pl31-221) (or p21-ten) l(q12-qter) 17(pll-q21) 22(q123-131) 2 l(p32) 12

N-ras

1

B-lym- 1 int-1

Lymphoma

wl22)

c-raf-l

c-ski c-erb*c-sis c-fos

Oncogene tumor association

Carcinoma Leukemia Carcinoma/sarcoma Leukemia/carcinoma

Lymphoma

ONCOPROTEINS The products encoded by viral oncogenes and protooncogenes are obviously proteins. What is known about these proteins in terms of their function has largely been based on investigations of the src gene. The protein encoded by the src gene has a molecular weight of 60kDa and is referred to as 60psrc (p = protein). This type of nomenclature applies to all “oncoproteins.” These proteins can be classified with respect to their function, as follows: (i) cytoplasmic protein kinases (serine, threonine, and tyrosine phosphorylation in proteins); (ii) growth factors; (iii) growth factor receptors, composed of an external ligand binding domain, a transmembrane portion, a protein-tyrosine kinase domain, and a regulatory domain; (iv) truncated growth factor receptors, i.e., some receptors are composed of subunits and not all the subunits may be produced and/or various domains may be inactive; (v) intracellular signal transducers, e.g., binding GTP, and acting as coupling factors in systems relaying signals such as from hormones; (vi) autokinase (threonine self-phosphorylation) using GTP instead of ATP; and (vii) nuclear acting proteins, possibly bind DNA [159]. In terms of anti-oncogene proteins, only one to date has been isolated. This is the protein encoded by the Rb (retinoblastoma) anti-oncogene 15OpRb [163]. It appears

8

B. DAUNTER

that the presence of the Rb gene 15OpRb controls or is involved in controlling cell replication. This is demonstrable by the fact that Rb is expressed in cells other than retinal cells and can be inactivated by the oncogenic adenovirus protein EIA, which leads to cell replication [163]. DNA AND RNA PROBES

It follows that if proto-oncogenes and their conversion to oncogenes can be identified, then it is possible to differentiate between a normal cell and a cancer cell. In essence, we are searching for gene activation and/or amplification (oncogenes) and/or loss or inactivation of genes (anti-oncogenes). It is possible, in some cases, to detect gene amplification cytogenetically, but this applies more to cells in vitro, as such changes in vivo are rarely detected [164]. The ease of detection of gene amplification in vitro is apparently due to culture conditions which can select for gene amplification [165]. The cytogenetic method for detecting gene amplification employs the chromosome spread technique with trypsin/ Giemsa staining of chromosomes [166]. Amplification is identified by the presence of double minute chromosomes and homogeneously staining chromosomal regions [167]. The more general method of detecting gene amplification is by the sensitive method of DNA hybridization. This involves the use of a single-stranded DNA probe, which in terms of oncogenes is the viral oncogene DNA or some sequence of it that has been cloned (cDNA) and radioactively labeled, usually with 32P. The DNA from the cells of interest is extracted and treated with bacterial restriction endonucleases to produce DNA fragments which are fractionated by agarose gel electrophoresis, and the fragments are denatured to give single-stranded DNA. The [32P]cDNA probe is then used to locate the complementary strand of DNA in one or more of the fragments, after the DNA fragments have been transferred to a nitrocellulose filter paper. The detection of binding of the [32P]cDNA probe is by autoradiography (photographic film placed over the nitrocellulose filter paper after interaction with the [32P]cDNA probe). Therefore, amplification of a particular oncogene can be determined by reference to DNA from normal tissue [168-1791. Using similar methods, mRNA can also be analyzed with [32P]cDNA probe [169,180-1851. DNA probes can also be used to detect translocations and rearrangements. This again requires the use of bacterial restriction endonucleases which recognize a specific sequence of four to six nucleotides in DNA. These enzymes can therefore be used to determine the position of a gene in relationship to the recognition sequence of the restriction endonuclease. For example, in normal

cells, the hexanucleotide sequence GAATTC occurs at the two ends of the c-myc gene. Therefore a restriction endonuclease (EcoRI) which recognizes this sequence will produce fragments of DNA containing the whole gene. If a translocation or rearrangement has occurred, with breaks within the gene, the restriction fragments with parts of the gene attached are likely to have different molecular weights. Thus, the [32P]cDNA probe will be able to identify these fragments if the probe recognizes all three exons of the gene [153,180,381]. Such a translocation break of the c-myc gene has been reported for Burkitt’s lymphoma [ 1861. POLYMERASE

CHAIN

REACTION

The sensitivity of DNA probes can be increased by using the polymerase chain reaction (PCR) technique. This involves amplification of the DNA sequence of interest by a primer-directed enzymatic amplification by Taq polymerase [ 1871. For example, for HPV DNA, the specificity of the PCR is based on two oligonucleotide primers that flank the HPV DNA to be amplified, which hybridize to opposite stands. Cycles of DNA heat denaturation, annealing of primers to their complementary sequences, and primer extension with Taq polymerase are repeated a number of times. Thus, the extended sequence of one primer serves as a new template for another primer, and each cycle doubles the amount of target DNA. Thus, by use of such a method, one molecule of HPV DNA has been detected in 100,000 cells [188]. In one study [ 1881, HPV-11 and HPV-16 were detected by the PCR technique in all cervical smears from 38 women, HPV-16 in 95% and both HPV-16 and HPV-11 in 58%). Importantly, 7 of 10 other women who had no cytological abnormality were found to be infected with either HPV-11 or HPV-16. Thus the presennce of HPV DNA in women with normal or abnormal cervical epithelium is much higher than has previously been reported. To date, however, there is no unequivocal evidence to prove or disprove that HPV is or is not a causative factor in cervical cancer [ 1881. MONOCLONAL

ANTIBODIES

TO ONCOPROTEINS

Since the DNA sequences of a number of oncogenes are known, it has been possible to synthesize peptides or obtain recombinant fusion proteins for Mab production [189]. The advantage of using Mabs to oncoproteins lies in their versatility, in that they can be more easily applied to cytological and histochemical analysis and flow cytometry. Examples of oncoproteins to which Mabs have been produced include p62C.“‘“’ [ 1891, p2 l’-‘“” [190], and ~40, a protein identified by Mab MycL9E1, raised against a synthetic peptide of ~62~~~” [ 1531. These

TUMOR

Mabs have been used to define differential rus gene expression in benign and malignant colonic neoplasia [191] and c-myc expression in yolk sac tumors [I921 and lung cancer [ 1931. The Mab to c-myc, Mycl-9E1, has also been used to detect the breakdown product of ~62~~~~’ and p40 in the urine of cancer patients and pregnant women [194]. In addition, natural antibodies against ~53 have been detected in the sera of women with breast cancer [195] and against a ~84, p62c-mY’-associated antigen in the sera of several cancer patients [196]. In addition to serum, cytological, and histological analyses, anti-oncoprotein Mabs can also be used in cytometric studies. In this way, oncogene expression on a cell-to-cell basis can be correlated with the cell cycle and ploidy. These methods can be applied both to fresh tissue and to nuclei extracted from paraffin-embedded tissues [197,198], if the oncoprotein is nuclear associated. For example, p62C+“y’ is nuclear associated [199], and has been shown to be significantly elevated in both teratoma and seminoma; and in teratoma with increasing differentiation, there is a significant increase in ~62’.“” [200]. Similarly, ~62~+“?~ has been found to be elevated in nuclei from ovarian diploid carcinoma, and borderline low-potential malignancy exhibited ~62~+“~ nuclear levels between those of nuclei from normal ovarian and ovarian cancer cells [201]. Application of these methods to cervical cancer has, however, not been so informative; the level of ~62~~~~~ was lower in carcinomas than in normal tissue, and aneuploidy, with a well-defined G, peak was not apparent [ 1551. Nevertheless, the level of p60C-“““ progressively decreased from CIN I to CIN III [155]. GROWTH

FACTORS

A number of tissue growth factors have been identified, for example, platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factors (TGF,, TGF,). TGFs are produced by tumors and an autocrine self-stimulating role has been proposed. On the basis of this proposal, it can be hypothesized that there should be a link between growth factors and oncogenes. That this may be possible is suggested by the sequence homology between and function of the v-erbB oncogene product and the cytoplasmic and membrane domain of the EGF receptor (EGFR), and also by the binding of TGF to EGFR [202,203]. EGFR has shown promise as a marker in breast cancer [204] in that tumors that are EGFR positive do not generally express estrogen or progesterone receptors, and therefore are not suitable for hormonal therapy [205]. In addition, EGFR status is associated with early recurrence and death, regardless of the histological subtype

9

MARKERS

of the breast cancer [203]. In contrast, in ovarian carcinoma, EGFR expression is associated with a good response to chemotherapy and increased survival time relative to patients with EGFR-negative carcinomas [206]. Ovarian cancer appears to be the exception, since tumor development is generally associated with growth factor expression [207,208]. Similarly, the study of cell lines from various tumors demonstrates the role of growth factors in tumor development [209-2121. In conclusion, the more we understand about the molecular biology of tumors, not only oncogenes, but also aberrations in cellular metabolism, the closer we will come to using tumor markers to their full potential in diagnosis, prognosis, and therapeutic management of the cancer patient. ACKNOWLEDGMENT I thank Professor E. V. Mackay for the many discussions in the preparation of this manuscript.

REFERENCES Mackay, E. V., Khoo, S. K., and Daunter, B. Tumour markers, in Gynecologic oncology (M Coppleson, Ed.), Churchill Livingstone, London, 2nd ed., pp. 270-282 (1987). 2. Shield, P. W., Daunter, B., and Wright, R. G. The Pap smear revisited, Aust. N.Z. J. Obstet. Gynaecol. 27, 269-283 (1987). 3. Hakomori, S. Glycosphingolipids. Sci. Amer. 254, 32-41 (1986). 4. Feizi, T., and Childs, R. A. Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour associated antigens and components of receptor systems. Trends Biochem. Sci., Jan., pp. 24-29 (1985). 5. Feizi, T. Carbohydrate antigens in human cancer, Cancer Surv. 4(l), 245-269 (1985). 6. Feize, T. Monoclonal antibodies reveal saccharide structures as differentiation and tumour associated antigens of haemotopoietic and epithelial cells of man and mouse, in Peptides ofthe biological jluids (M. Peters, Ed.), Pergamon Press, Oxford, UK, Vol. 31(C.5), pp. 993-996 (1984). 7. Leoni, F., Miotti, S., Canevari, S., Sonnino, S., Ripamonti, M., and Colnaghi, M. I. Carbohydrate epitope defined by an antitumour monoclonal antibody detected on glycoproteins and glycohpids by immunoblotting, Hybridoma 5(4), 289-2% (1986). 8. Yogeeswaran, G. Cell surface glycolipids and glycoproteins in malignant transformation, Adv. Cancer Res. 38, 289-250 (1983). 9. Mann, P. L., Lopez-Colberg, I., and O’Kelley, R. Cell surface oligosaccharide modulation during differentiation. I. Modulation of lectin binding, Mech. Ageing Dev. 38, 207-217 (1987). 10. Mann, P. L., Swartz, C. M., and O’Kelley, R. Cell surface oligosaccharide modulation during differentiation. II. Membrane mobility of oligiosaccharide lectin conjugates, Mech. Ageing Dev. 38, 219-230 (1987). 11. Reading, C. L., Hickey, C. M., and Yong, W. Analysis of cell surface glycoprotein changes related to hematopoietic differentiation, J. Cell. Biochem. 37, 21-36 (1988). 12. Rademacher, T. W., Parekh, R. B., and Dwek, R. A. Glycobiology. Annu. Rev. Biochem. 57, 785-838 (1988). 13. Hakomori, S. Aberrant glycosylation in cancer cell membranes I.

10

B. DAUNTER as focused on glycolipids. Overview and perspectives, Cancer 45, 2405-2414 (1985). Dennis, J. W., and Laferte, S. Tumour cell surface carbohydrate and metastatic phenotype, Cancer Metastas. Rev. 5, 195-204 (1987). Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants, Biochim. Biophys. Acta 455, 152-162 (1976). Sharon, N. Lectins, Sci. Amer. 236(6), 108-119 (1977). Damjanov, I. Biology of disease: Lectin cytochemistry and histochemistry, Lab. Invest. 57(l), 5-20 (1987). Hsu, Su-M., and Ree, H. J. Histochemical studies on lectin binding in reactive lymphoid tissues, J. Histochem. Cytochem. 31(4), 538-546 (1983). Lee, M. C., Turcinov, D., and Damjanov, 1. Lectins as markers for eosinophilic leukocytes, Histochemistry 86, 269 (1987). Lee, M. C., Talerman, A. Ooterhuis, J. W., and Damjanor, I. Lectin histochemistry of classic and spermatocytic seminoma, Arch. Pathol. Lab. Med. 109, 938-942 (1985). Teshima, S., Hirohashi, S., Shimosato, Y., Kishi, K., Ino, Y., and Matsumoto, K. Histochemically demonstrable changes in cell surface carbohydrates of human germ cell tumours, Lab. Invest. 50(3), 271-277 (1984). Teshima, S., Hirohashi, S., Shimosato, Y., Kishi, K., et u/. Histochemical demonstrable changes in cell surface carbohydrates of human germ cell tumours, Lab. Invest. 50(3), 271-277 (1984). Dansey, R., Murray, J., Ninin, D., and Bezwoda, W. R. Lectin binding in human breast cancer: Clinical and pathologic correlations with fluorescein-conjugated peanut, wheat germ and concanavalin A binding, Oncology 45(4), 300-302 (1988). Rhodes, J. M., Black, R. R., and Savage, A. Glycoprotein abfjnormalities in colonic carcinoma, adenoma and hyperplastic polyps shown by lectin peroxidase histochemistry, J. C&n. Pathol. 39, 1331-1334 (1986). Matsushita, Y., Yonezawa, S., Nakamura, T., Shimuzi, S., Ozawa, M., and Miramatsu, T. Carcinoma-specific Ulex europaeus agglutinin 1 binding glycoproteins of human colorectal carcinoma and its relation to carcinoembryonic antigen, J. Natl. Res.

14.

15.

16. 11. 18.

19. 20.

21.

22.

23.

24.

25.

Cancer

Inst.

75(2),

219-226

(1985).

26. Louis, C. J., Wyllie, R. G., Sheung, T. C., and Tamara, S. Lectin binding of human epidermal tumours and related conditions, Amer. J. Clin. Pathol. 75(5), 642-647 (1980). 27. Irimura, T., Ota, D. M., and Cleary, K. R. ullex europeus agglutinin I-reactive high molecular weight glycoproteins of adenocarcinoma of distal colon and rectum and their possible relationship with metastatic potential, Carbohyd. Res. 115, 209-220 (1987). 28. Bychkov, V., and Tota, P. D. Lectin binding to normal dysplastic and neoplastic cervical epithelium. Amer. .Z. C/in. Pathol. 85(5), 542-547 (1986). 29. Davina Hans, J. M., Stadhouders, A. M., VanHaelst, U. J. G. M., Lamers, G. E. M., et al. Gynecol. Oncol. 22, 212223 (1985). 30. Bychkov, V., and Toto, P. D. Wheat germ and peanut agglutinin binding to normal, dysplastic and neoplastic cervical epithelium, Gynecol. Obstet. Invest. 21, 158-163 (1986). 31. Ng, R. C. Y., Roberts, A. N., Wilson, R. G., Latner, A. L., and Turner, G. A. Analysis of protein extracts of human breast cancers: Changes in glycoprotein content linked to the malignant phenotype, Brit. J. Cancer 55, 249-254 (1987). 32. Bialasiewicz, A. A., and Salowsky, A. Binding patterns of lectins

to human retinoblastoma Prevent.

10, 63-70

cells, Prelim.

Findings

Cancer

Detect.

(1987).

33. Feizi, T. Monoclonal antibodies reveal saccharide structures as differentiation and tumour associated antigens of haemotopoietic and epithelial cells of man and mouse, in Protides of the Biological Fluids (M. Peters, Ed.), Chap. 5, pp. 993-996 (1984). 34. Schrump, D. S., Furukawa, K., Yamaguchi, H., and Lloyd, K. 0. Recognition of galactosylgloboside by monoclonal antibodies derived from patients with primary lung cancer, Proc. Nat/. Acad. Sci. USA 85, 4441-4445 (1988). 35. Barondes, S. H. Soluble lectins: A new class of extracellular proteins, Science 223, 1259-1264 (1984). 36. Ashwell, G., and Harford, J. Carbohydrate specific receptors of the liver, Annu. Rev. Biochem. 51, 531-544 (1982). 37. Harford, J., and Ashwell, G. The hepatic receptor for asialoglycoproteins, in The glycoconjugates (M. Horowitz, Ed.), Academic Press, New York, Vol. IV, pp. 27-55 (1982). 38. Bridges, K., Harford, J., Ahswell, G., and Klausner, R. D. Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes, Proc. Natl. Acad. Sci. USA 79, 350354 (1982). 39. Weigel, P. Evidence that the basic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature, Biochem. Biophys.

Res.

Commun.

101, 1418-1425

(1981).

40. Barondes, S. H. Vertebrate lectins: Properties and functions, in The Iectins: medicine,

Properties,

Academic

functions

and upplications

in biology

and

Press, New York, Chap. 7, pp. 437-466

(1986). 41. Grabel, L. B., Singer, M. S., Martain, G. R., and Rosen, S. D. Isolation of a teratocarcinoma stem cell lectin implicated in intercellular adhesion, FEBS 183(2), 228-231 (1985). 42. Grabel, L. B. Isolation of a putative cell adhesion mediating lectin from teratocarcinoma stem cells and its possible role in differentiation, Cell DifJ 15, 121-124 (1984). 43. Whitney, P., Maxwell, S., Ryan, U., and Massaro, D. Synthesis and binding of lactose-specific lectin by isolated lung cells, Amer. J. Physiol.

l&WC, 258-264

(1985).

44. Feize, T. Carbohydrate antigens in human cancer, Cancer Surv. 4(l), 245-269 (1985). 45. Yogeeswaran, G. Cell surface glycolipids and glycoproteins in malignant transformation, Adv. Cancer Res. 38, 289-350 (1983). 46. Gabius Hans, J., Engelbart, R., and Cramer, F. Expression of endogenous lectins in human small cell carcinoma and undifferentiated carcinoma of the lung, Carbohyd. Res. 164, 33-41 (1987). 47. Monsigny, M., Kieda. C.. and Roche. A. C. Membrane glycoproteins, glycolipids and membrane lectins as recognition signals in normal and malignant cells, Cell Biol. 47, 95-1 IO (1983). 48. Gabius, H. J., Engelhart, R., and Cramer, F. Endogenous tumour lectins: Overview and perspectives, Anticancer Res. 6,573-577 (1986). 49. Gabius. H. J.. Engelhart, R., Casper. J., Scimoll. H. J.. Nagel, G. A., and Cramer, F. Comparison of endogenous lectins in human enbryonic carcinoma and yolk sac carcinoma Tumour Biol. 6, 471-482 (1985). 50. Gabius, H. J., Engelhart, R., Casper, J., Reile, D., Schumacher, S., and Schmoll, H. J. Cell surface lectins of transplantable human teratocarcinoma ceils: Purification of a new mannan-specific endogenous lectin, Tumour Biol. 6, 145-156 (1985). 51. Grabel, L. B., Singer, M. S., Martin, G. R., and Rosen, S. D. Teratocarcinoma stem cell adhesion: The role of divalent cations and a cell surface lectin, J. CeN Biol. 96, 1532-1537 (1985).

TUMOR 52. Gabius, H. J., Engelhart, R., Cramer, F., Batge, R., and Nagel, G. A. Pattern of endogenous lectins in human epithelial tumours, Cancer Res. 45, 253-257 (1985). 53. Raz, A., and Lotan, R. Lectin-like activities associated with human and murine neoplastic cells, Cancer Res. 41, 3642-3647 (1981). 54. Monsigny, M., Roche, A. C., and Midoux, P. Uptake of neoglycoproteins via membrane lectins of L1210 cells evidenced by quantitative flow cytofluorometry and drug targeting, Biol. Cell 51, 187-196 (1984). 55. Bhattacharya, M., and Barlow, J. J. Tumour markers for ovarian cancer, Int. Adv. Surg. Oncol. 2, 155-176 (1979). 56. Mamura, N., Takhashi, T., Lloyd, J. O., and Lewis, J. L. Analysis of human ovarian tumour antigens using heterologous antisera: Detection of new antigenic systems, ht. J. Cancer 21, 570577 (1978). 57. Knauf, S., and Urbach, G. I. Ovarian tumour specific antigens, Amer. .I. Obstet. Gynecol. 119, 966-970 (1974). 58. Burton, R. M., Hope, N. J., and Lubbers, L. M. A thermostable antigen associated with ovarian cancer, Amer. J. Obstet. Gynecol. 125, 472-477 (1976). 59. Hamazaki, M. H., and Hotta, K. Ovarian cyst fluid specific antigens, Experientia 31, 241-243 (1975). 60. Tatarinov, Y. S., and Kalashnikov, V. V. New embryonic antigen and its presence in some adenocarcinoma, Nature (London) 265, 638-639 (1977). 61. Was, M., Searle, A., and Bagshaw, K. D. Radioimmunoassay of the normal serum glycoprotein (CXl) in monitoring ovarian malignancy, Eur. J. Cancer 17, 1267-1273 (1981). 62. Marchand, A., Fenoglio, C. M., Pascall, R., and Richart, R. M. Carcinoembryonic antigen in human ovarian neoplasms, Cancer Res. 35, 3807-3810 (1975). 63. Dembo, A. J., Chang, P. L., and Urbach, G. I. Clinical correlations of ovarian cancer antigens NB/70K: A preliminary report, Obstet. Gynecol. 65(5), 710-714 (1985). 64. Gold, P., and Freedman, S. 0. Demonstration of tumour specific antigens in human colonic carcinoma by immunological tolerance and absorption techniques, J. Exp. Med. 122, 439-462 (1965). 65. Gold, P., and Freedman, S. 0. Specific carcinoembryonic antigens of the human digestive system, J. Exp. Med. 122, 467-481 (1965). 66. Khoo, S. K., Warner, N. L., Lie, J. T., and Mackay, I. R. Carcinoembryonic antigenicity of tissue extracts: A quantitative study of malignant and benign neoplasms, cirrhotic liver normal adult and fetal organs, Znt. J. Cancer 11, 681-687 (1973). 67. Crum, G. P., and Fenoglio, C. M. The immunoperoxidase technique: A review of its application to diseases of the female genital tract, Diagn. Gynecol. Obstet. 2(3), 103-115 (1980). 68. Ludwig, H. Antigenic markers of genital carcinoma, Eur. J. Obstet. Gynaecol. Reprod. Biol. 28, 104-108 (1988). 69. Sikorska, H., Shuster, J., and Gold, P. Clinical applications of carcinoembryonic antigen, Clin. Detect. Prevent. 12, 321-355 (1988). 70. Wahren, B., Lidbrink, E., and Wallgren, A. CEA and other tumour markers in tissue and serum or plasma of patients with primary carcinoma, Cancer 42(4), 1870-1878 (1978). 71. Jullienne, A., Calmettes, C., Moukhtar, M. S., and Milhaud, G. Physicochemical characteristics of carcinoembryonic antigen extracted from medullary carcinoma of the thyroid, Oncodev. Biol. Med. 1, 137-142 (1980).

MARKERS

11

72. Galen, R. S. When is a tumour marker a laboratory test, Ann. Clin. Lab. Sci. 7, 51-56 (1977). 73. Delwiche, R., Zamcheck, N., and Marcoon, N. Carcinoembryonic antigen in pancreatitis, Cancer 31, 328-330 (1973). 74. Pustaszeri, G., and Mach, J. P. Carcinoembryonic antigen (CEA) in non-digestive cancerous and normal tissues, Immunochem 10, 197-204 (1973). 75. Egan, M. L., Pritchard, D. G., Todd, C. W., and Go, V. M. Isolation and immunochemical and chemical characterization of carcinoembryonic antigen-like substances in colon lavages of healthy individuals, Cancer Res. 37, 2638-2643 (1977). 76. Fritsche, R., and Mach, J. P. Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa, Immunochem 14, 119-127 (1977). 77. Kim, Y. D., and Hirata, A. A. CEA-like substances in human cavity fluids, Immunol. Commun. 5(7,8), 619-629 (1976). 78. Burtin, P., Sabine, M. C., and Chavanel, G. Presence of carcinoembryonic antigen in children’s colonic mucosa, Int. J. Cancer 10, 72-76 (1972). 79. Ochi, Y., Ura, Y., Hamazu, M., Fujiyama, Y. V., Kajita, Y., and Ishida, M. Immunochemical identification of an cr,-acid glycoprotein-antigenic determinant on carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA), Clin. Chem. Acta 138, 9-19 (1984). 80. Primas, F., Freeman, J. W., and Golderberg, D. M. Immunological heterogeneity of carcinoembryonic antigen: Purification from meconium of an antigen related to carcinoembryonic antigen, Cancer Res. 43, 679-685 (1983). 81. Von Kleist, S., and Burtin, P. Antigens cross-reacting with CEA, in Immunodiagnosis of cancer, Part I (R. B. Herberman, and K. R. McIntire, Eds.), Marcel Dekker Inc., New York, pp. 322342 (1979). 82. Price, M. R., Edwards, S., Jacobi, E., Pawluczyk, I. Z. A., Byers, V. S., and Baldwin, R. W. Mapping of monoclonal antibody-defined epitopes associated with carcinoembryonic antigen (CEA), Cancer Immunol. Immunother. 25, lo-15 (1987). 83. Quentmeier, A., Moller, P., Schwarz, V., Abel, U., and Schlag, P. Carcinoembryonic antigen CA-19-9 and CA-125 in normal and carcinomatous human colorectal tissue, Cancer 60,22612266 (1987). 84. Gocze, P. M., Szabo, D. G., Than, G. N., Csaba, I. F., and Krommer, K. F. Occurrence of CA-125 and CA-19-9 tumourassociated antigens in sera of patients with gynaecologic trophoblastic and colorectal tumours, Gynecol. Obstet. Invest. 25, 268-272 (1988). 85. Knauf, S. Clinical evaluation of ovarian tumour antigen NB/70K: Monoclonal antibody assays for distinguishing ovarian cancer from other gynecologic disease, Amer. J. Obstet. Gynecol. 158, 1067-1072 (1988). 86. William, G. R., DeBoer, J. M., and Nayman, J. Intestine-associated antigens in ovarian tumours. An immunohistological study, Pathology 13, 447-455 (1981). 87. Tagliabue, E., Menard, S., Della-Torre, D., Barbanti, P., Mariant-Costantini, R., and Porro, G. Generation of monoclonal antibodies reacting with human epithelial ovarian cancer, Cancer Res. 45, 379-385 (1985). 88. Pant, K. D., Samora, P. O., Rhodes, B. A., Sachatello, C. R., Hagihora, P. F., and Griffen, W. 0. Characterization of a common antigen of colorectal and mutinous ovarian tumours, cota, Tumour Biol. 5, 243-254 (1984). 89. Ellis, I. O., Robins, R. A., Elston, C. W., Blarney, R. W.,

B. DAUNTER

12

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

Ferry, B., and Baldwin, R. W. A monoclonal antibody, NCRC11 raised to human breast carcinoma. 1. Production and immunohistological characterization, Histopathology 8,501-509 (1984). Price, M. R., Edwards, S., Powell, M., and Baldwin, R. W. Epitope analysis of monoclonal antibody NCRG-11 defined antigen isolated from ovarian breast carcinomas, &it. J. Cancer 54, 393 (1986). Nuti, M., Teramoto, Y. A., Mariani-Costantini, R., Hand, P. H., Colcher, O., and Schlom, J. A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumour associated antigen in human mammary cell populations, Inr. J. Cancer 29, 539-545 (1982). Thor, A., Gorstein, F., Ohuchi, N., Szpak, C. A., Johnston, W. W., and Schlom, J. Tumour-associated glycoprotein (TAG72) in ovarian carcinomas defined by monoclonal antibody B72.3, .I. Natl. Cancer Ins?. 76(6), 995-1003 (1986). Stramignoni, D., Bowen, R., Atkinson, B. F., and Schlom, J. Differential reactivity of monoclonal antibodies with human colon adenocarcinoma and adenocarcinoma, Inr. J. Cancer 31,543-552 (1983). Arklie, J., Taylor-Papadimitriou, J., Bodmer, N. F., Egan, W. M., and Millis, R. Differentiation antigens expressed by epithelial cells in the lactating breasts are also detectable in breast cancers, Int. J. Cancer 28, 23-29 (1981). Kabawat, S. E., Bast, R. C., Welch, W. R., Knapp, R. C., and Calvin, R. B. Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumours of serous, endometrioid and clear cell types, Amer. .I. C/in. Pathol. 79, 98-104 (1983). Szymendera, S. Clinical usefulness of three monoclonal antibodydefined tumour markers CA-19.9, CA-50 and CA-125, Tumour Biol. 7, 333-342 (1986). Nouwen, E. J., Pallet, D. E., Eerdekyns, M. W., Hendrix, P., Lombardi, F., and Bolis, G. Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen and cancer antigen 125 in normal and neoplastic human lung, Cancer Res. 46, 866-876 (1986). Kabawat, S. E., Bast, R. C., Bhan, A. K., Welch, W. R., and Ishihara, M. Tissue distribution of a coelomic epithelium related antigen recognized by the monoclonal antibody OC-125, In?. J. Gynecol. Pathol. 2, 275-285 (1983). Halila, H., Lehtovirta, P., Stenman, U. H., and Seppala, M. CA125 in the follow-up of patients with ovarian cancer, Acta Obstet. Gynecol.

&and.

67, 53-58

(1988).

100. Xinguo, L., Dixa, C., Schwartz, P. E., and Zhan, Y. A study of the monoclonal antibody OC-125 to diagnose malignant ovarian turnours, Gynecol. Oncol. 32, 327-330 (1989). 101. Di-Xia, C., Schwartz, P. E., Xinguo, L., and Zhan, Y. Evaluation of CA-125 levels in differentiating malignant from benign tumours in patients with pelvic masses, Obstet. Gynecol. 72, 23-27 (1988). 102. Finklwe, N. J., Benacerraf, B., Lavin, P. T., Wojciechowski, C., and Knapp, R. C. Comparison of serum CA-125, clinical impression and ultrasound in the preoperative evaluation of ovarian masses, Obstet. Gynecol. 72(4), 659-663 (1988). 103. Nouwen, E. J., Pallet, D. E., Eerdekyns, M. W., Hedrix, P. G., Briers, T. W., and De Broe, M. E. Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen and cancer antigen 125 in normal and neoplastic human lung, Cancer Res. 46, 866-876 (1986). 104. Kabawat, S. E., Bast, R. C., Bhan, A. K., Welch, W. R., Knapp, R. C., and Colvin, R. B. Tissue distribution of a coelomic epi-

105.

106.

107.

108.

109. 110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

thelium related antigen recognized by the monoclonal antibody OC-125, Int. J. Gynecol. Parhol. 2, 275-285 (1983). De Bruijn, H. W. A., Van Beech Calkoen-Carpay, T., Jager, S., Duk, J. M., Aadders, J. G., and Fleuren, G. J. The tumour marker CA-125 is a common constituent of normal cervical mucus, Amer. J. Obstet. Gynecol. 154(5), 1088-1091 (1986). Malkasian, G. D., Podratz, C., Stanhope, C. R., and Podratz, K. C. CA-125 in gynaecologic practice, Amer. J. Obstet. Gynecol. l%(3), 515-518 (1986). Masahashi, T., Matsuzawa, K., Ohsawa, M., Narita, O., Asai, T., and Ishihara, M. Serum CA-125 levels in patients with endometriosis: Changes in CA-125 levels during menstruation, Obstet. Gynecol. 72(3), 328-331 (1988). Poels, L. G., Peters, D., and Van Megen, Y. Monoclonal antibody against human ovarian tumour associated antigens, J. Nutl. Cancer Inst. 76(5), 781-791 (1986). Jones, 0. W., and Surawit, E. A. Meigs syndrome and elevated CA-125, Obstet. Gynecol. 73, 520-521 (1989). Altaras, M. M., Goldberg, G. L., Levin, W., Bloch, B., Darge, L., and Smith, J. A. The role of cancer antigen 125 (CA125) in the management of ovarian epithelial carcinomas, Gynecol. Oncol. 30, 26-34 (1988). Li-juan, L., Xiu-Feng, H., Wen-Shu, L., and Ai-Ju, W. V. A monoclonal antibody radioimmunoassay for an antigenic determinant CA-125 in ovarian cancer patients, Chin. Med. J. 99,721726 (1986). Friedlander, M., Leary, J., and Russel, P. An evaluation of CA125, CA-I and peanut lectin immunoreactivity in epithelial ovarian neoplasms: Correlation with histopathological features, prognostic variables and patient outcome, Pathology 20, 38-44 (1988). Alvarez, R. D., To, A., Boots, L. R., Shingleton, H. M., Hatch, K. D., Hubbard, J., Soong, S. J., and Potter, M. E. CA-125 as a serum marker for poor prognosis in ovarian malignancies, Gynecol. 0~01. 26, 284-289 (1987). Khoo, S. K., Hurst, T., Webb, M. J., Dickie, G. J., Kearsley, J. H., and Mackay, E. V. Predictive value of serial CA-125 antigen levels in ovarian cancer evaluated by second-look laparotomy, Eur. J. Cancer Clin. Oncol. 23, 765-771 (1987). Vanderburg, M. E. L., Lammes, F. B., Van Putten, W. L. J., and Stoter, G. Ovarian cancer: The prognostic value of the serum half-life of CA-125 during induction of chemotherapy, Gynecol. Oncol. 30, 307-312 (1988). Stratton, J. A. S., Rettenmaier, M. A., Phillips, H. B., Herbabutya, S., and Di Saia, P. J. Relationship of serum CA-125 and lipid-associated siahc acid tumour associated antigen levels to the disease status of patients with gynaecologic malignancies, Obstet. Gynecol. 71(l), 20-26 (1988). Zurawski, V. R., Knapp, R. C., Einhorn, N., Kenemans, P., Mortel, R., and Ohmi, K. An initial analysis of preoperative serum CA-125 levels in patients with early stage ovarian carcinoma, Gynecol. Oncol. 30, 7-14 (1988). Szymendera, S. Clinical usefulness of three monoclonal antibodydefined tumour markers CA-19.9, CA-50 and CA-125, Tumour Biol. 7, 333-342 (1986). Maughan, T. S., Fish, R. G., Shelley, M., Jasani, B., Williams, G. T., and Adams, M. Antigen CA-125 in tumour tissue and serum from patients with adenocarcinoma of the ovary, Gynecol. Oncol. 30, 342-346 (1988). Fioretti, P., Gadducci, A., Ferdeghini, M., Bartolini, T., Bianchi, R., and Facehini, V. Correlation of CA-125 and CA-19-

TUMOR

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133. 134.

135. 136.

137.

138.

9 serum levels with clinical course and second-look findings in patients with ovarian cancer, Gynecol. Oncol. 28,278-283 (1987). Gocze, M., Szabo, D. G., Than, G. N., Csaba, I. F., and Krommer, K. F. Occurrence of CA-125 and CA-19-9 tumour-associated antigens in sera of patients with gynecologic, trophoblastic and colorectal tumours, Gynecol. Obstet. Invest. 25, 268-272 (1988). Patsner, B., Mann, W. J., Vissicchio, M., and Loesch, M. Comparison of serum CA-125 and lipid-associated sialic acid (LASAP) in monitoring patients with invasive ovarian adenocarcinoma, Gynecol. Oncol. 30, 98-103 (1988). Panza, N., Pacilio, G., Campanella, L., Peluso, G., Battisha, C., and Amoriello, A. Cancer antigen 125, tissue polypeptide antigen, carcinoembryonic antigen, beta-chain human chorionic gonadotrophin as serum markers of epithelial ovarian carcinoma, Cancer 61(l), 76-83 (1988). Lahousen, M., Stattner, H., Hellmuth, P., Urdl, W., and Parstner, P. The predictive value of a combination of tumour markers in monitoring patients with ovarian cancer, Cancer 60(9), 22282232 (1987). Satjadi, S., Daunter, B., Mackay, E. V., Magon, H., and Khoo, S. K. A multiparametric approach to tumour markers detectable in serum in patients with carcinoma of the ovary or uterine cervix, Gynecol. Oncol. 10, 113-124 (1980). Scambia, G., Benedetti, P., Baiocchi, G., Perrone, L., Greggi, S., and Di-Roberto, P. CA-15-3 as a tumour marker in gynaecological malignancies, Gynecol. Oncol. 30, 265-273 (1988). Scambia, G., Benedetti, P., Baiocchi, G., Perrone, L., Greggi, S., and Di-Roberto, P. CA-15-3 serum levels in ovarian cancer, Oncology 45, 263-267 (1988). Nozawa, H., Yajima, M., Kajima, K., Iizuka, R., Mochizaki, H., and Sugawara, T. Tumour associated mucin-type glycoprotein (CA-54/Gl) defined by two monoclonal antibodies (MA-54 and MA-61) in ovarian cancer, Cancer Res. 49, 493-498 (1989). Sakakibara, K., Ueda, R., Ohta, M., Nakashima, N., Tomoda, Y., and Takahashi, T. Three novel mouse monoclonal antibodies OM-A, OM-B and OM-C reactive with mutinous type ovarian tumours, Cancer Res. 48, 4639-4645 (1988). Brown, D. C., Cole, D., Gatter, K. C., and Mason, D. Y. Carcinoma of the cervix uteri: An assessment of tumour proliferation using monoclonal antibody Ki67, Brit. .Z. Cancer 57, 178-181 (1988). Smith, L. H., and Teng, N. N. H. Clinical application of monoclonal antibodies in gynaecologic oncology, Cancer 60(8), 20682074 (1987). Wolman, S. R., and Horland, A. A. Genetics of tumour cells, in Cancer: A comprehensive trentise (F. F. Becker, Ed.), Vol. 3, Plenum, New York, pp. 155-198 (1975). Watson, J. V. Oncogenes, cancer and analytical cytology, Cytometry 7, 400-410 (1986). Cowell, J. K. Double minutes and homogeneously staining regions: Gene amplification in mammalian cells, Annu. Rev. Genet. 16, 21-52 (1982). Nowell, P. C., and Hungerford, D. A. A minute chromosome in human chronic granuloytic leukemia, Science 132, 1497 (1960). Rowley, J. D. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining, Nature (London) 243,290-293 (1973). Haluska, F. G., Tsujimoto, Y., and Croce, C. M. Oncogene activation by chromosome translocation in human malignancy, Annu. Rev. Genet. 21, 321-345 (1987). Murty, V. V. V. S., Mitra, A. B., Singh, I. P., and Luthra, U. K.

MARKERS

139.

140.

141. 142.

143.

144.

145.

146.

147. 148.

149.

150.

151.

152.

153. 154.

155. 156. 157.

13

Heteromorphism of C bands in patients with precancerous and cancerous lesions of the cervix uteri, Oncology 44, 164-168 (1987). Murty, V. V. V. S., Mitra, A. B., Das, B. C., Marthy, N. S., and Luthra, V. K. Chromosomal phenotypes in patients with precancerous lesions of the uterine cervix progressed to cancer during follow-up, Oncology 45, 384-388 (1988). Tubiana, M., and Koscienlyn, S. Cell kinetics, growth rate and the natural history of breast cancer, Eur. J. Cancer Clin. Oncol. 24, 9-14 (1988). Murray, A. W., and Szostak, J. W. Chromosome segregation in mitosis and meiosis, Annu. Rev. Cell Biol. 1, 289-315 (1985). Hedley, D. W., Friedlander, M. L., Taylor, I. W., and Rugg, C. A. Methods for analysis of cellular DNA content of paraffinembedded pathological material using flow cytometry, J. Histothem. Cytochem. 31(11), 1333-1335 (1983). Vindelov, L. L., Christensen, I. J., and Nissen, N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis, Cytometry 3(5), 323-327 (1983). Friedlander, M. L., Taylor, I. W., Russell, P., Musgrove, E. A., Hedley, D. H., and Tattersall, M. H. Ploidy as a prognostic factor in ovarian cancer, Znt. .Z. Gynecol. Pathol. 2, 55-63 (1983). Friedlander, M. L., Headley, D. W., and Taylor, I. W. Clinical and biological significance of aneuploidy in human tumours, J. Clin. Pathol. 37, %l-974 (1984). Kalhoniemi, 0. P., Pannonen, R., Mattila, J., Lehtinen, M., and Koivula, T. Prognostic significance of DNA index, multiploidy and S-phase fraction in ovarian cancer, Cancer 6(2), 334-339 (1987). Iversen, 0. E. Prognostic value of the flow cytometric DNA index in human ovarian carcinoma, Cancer 61(5), 971-975 (1988). Wils, J., Van Geuns, H., and Baak, J. Proposal for therapeutic approach based on prognostic factors including morphometric and flow-cytometric features in stage III-IV ovarian cancer, Cancer 61(9), 1920-1925 (1988). Johnson, T. S., Adelson, M. D., Sneige, N., Williamson, K. D., Lee, A. M., and Katz, R. Cervical carcinoma DNA content, Sfraction and malignancy grading. I. Interrelationships, Gynecol. Oncol. 26, 41-56 (1987). Adelson, M. D., Johnson, T. S., Sneige, N., Williamson, K. D., Freedman, R. S., and Peters, L. J. Cervical carcinoma DNA content, S-fraction and malignancy grading. II. Comparison with clinical staging, Gynecol. Oncol. 26, 57-70 (1987). Christov, K., and Vassilev, N. Flow cytometric analysis of DNA and cell proliferation in ovarian tumours, Cancer 60(l), 121-125 (1987). Rodenburg, C. J., Comelisse, C. J., Hermans, J., and Fleuren, G. J. DNA flow cytometry and morphometry as prognostic indicators in advanced ovarian cancer: A step forward in predicting the clinical outcome, Gynecol. Oncol. 29, 176-187 (1988). Chan, S., and Sikora, K. The potential of oncogene products as tumour markers, Cancer Surv. 6(2), 185-207 (1987). Martin, G. S. Rous sarcoma virus, a function required for the maintenance of the transformed state, Nature (London) 227,10211023 (1970). Watson, J. V. Oncogenes, cancer and analytical cytology, Cytometry 7, 400-410 (1986). Baltimore, D. Oncogenes: The beginning of the molecular description of a cancer, Cell Biophys. 9(1,2), 17-31 (1986). Temin, H. M. Evolution of cancer genes as a mutation-driven process, Cancer Res. 48, 1697-1701 (1988).

14

B. DAUNTER

158. Stehelin, D., Varmus, H. E., Bishop, J. M., and Vogt, P. K. DNA related to the transforming genes of avian sarcoma viruses is present in normal avain DNA, Nature (London) 260, 170-173 (1976). 159. Hunter, T. The proteins of oncogenes, Sci. Amer. 252(2), 60-69 (1984). 160. Knudson, A. G. Hereditary cancer oncogenes and anti-oncogenes, Cancer Res. 45, 1437-1443 (1985). 161. Schwab, M., Alitalo, K., Klempnauer, K. B., Varmus, H. E., Bishop, J. M., and Gelbent, F. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour, Nature (London) 305, 245-248 (1983). 162. Klein, G., and Klein, E. Evolution of tumours and the impact of molecular biology, Nature (London) 315, 190-195 (1985). 163. Weinberg, R. A. Finding the anti-oncogene, Sci. Amer., 259(3), 34-41 (1988). 164 Alitalo, K. Amplification of cellular oncogenes in cancer cells, Med. Biol. 62, 304-317 (1984). 165. Stark, G. R., and Wahl, G. M. Gene amplification, Annu. Rev. Biochem. 53, 447-491 (1984). 166. Cowell, J. K. Double minutes and homogenously staining regions: Gene amplification in mammalian cells, Annu. Rev. Gene?. 16, 21-52 (1982). 167. Barker, P. E. Double minutes in human tumour cells, Cancer Genet. Cytogenet. 5, 81-94 (1982). 168. Collins, S., and Groundine, M. Amplification and endogenous myc-related DNA sequences in a human myeloid cell line, Nature (London) 298, 679-681 (1982). 169. Della-Favera, R. D., Wong-Staasl, F., and Gallo, R. C. Oncogene amplification in promyelocystic leukaemia cell line HL-60 and primary leukaemia cells of the same patient, Nature (London) 299, 61-63 (1982). 170. Nowell, R., Finan, J., Favera, R. D., Gallo, R. C., Ar-Rushdi, A., and Romanczu, K. G. Association of amplified oncogene cmyc with an abnormally banded chromosome 8 in a human leukaemia cell line, Nature (London) 306, 494-497 (1983). 171. Alitalo, K., Schwab, M., Lin, H. E., Varmus, H. E., and Bishop, J. M. Homogeneously staining chromosomal regions containing amplified copies of an abundantly expressed celular oncogene (cmyc) in malignant neuroendocrine cells from a human colon carcinoma, Proc. Natl. Acad. Sci. USA 81, 4534-4538 (1983). 172. Kozber, D., and Croce, C. M. Amplification of the c-myc oncogene in one of five human breast carcinoma cell lines, Cancer Res. 44, 438-441 (1984). 173. Little, C. D., Nau, M. M., Carney, D. N., Gazder, A. F., and Minna, J. D. Amplification and expression of the c-myc oncogene in human lung cancer cell line, Nature (London) 306, 194-196 (1983). 174. Schwab, M., Alitalo, K., lempnauer, K. B., Varmus, H. E., Bishop, J. M., and Gelbent, F. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour, Nature (London) 305, 245-248 (1983). 175. Alitalo, K., Winqvist, R., Liu, C. C., dela Chapelle, Schwab, M., and Bishop, J. M. Aberrant expression of an amplified c-myb oncogene in two cell lines from a colon carcinoma, Proc. Natl. Acad. Sci. USA 81, 4534-4538 (1984). 176. Konopka, J. B., Watanabe, S. M., and Wittle, 0. N. An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity, Cell 37, 1035-1042 (1984).

177. Uhich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., and Tam, A. W. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells, Nature (London) 309, 418-425 (1984). 178. Zhou, D. J., Gonzalez-Cadavid, N., Ahuja, H., Battifora, H., Moore, G. E., and Cline, M. J. A unique pattern of proto-oncogene abnormalities in ovarian adenocarcinomas, Cancer 62, 1573-1576 (1988). 179. Biunno, I., Pozzi, M. R., Pierotti, M. A., Pilotti, S., Cattoretti, G., and Della Porta, G. Structure and expression of oncogenes in surgical specimens of human breast carcinomas, Brit. J. Cancer 57, 464-468 (1988). 180. LeGrys, V. A., Leinbach, S. S., and Silverman, L. M. Clinical application of DNA probes in the diagnosis of genetic disease, CRC Crit. Rev. Clin. Lab. Sci. 24(4), 255-274 (1987). 181. Matthews, J. A., and Kricka, L. J. Analytical strategies for the use of DNA probes, Anal. B&hem. 169, l-25 (1988). 182. Rathberg, P. G., Ericman, M. D., Diehl, R. E., Roviatti, U. G., et al. Structure and expression of the oncogene c-myc in fresh tumour material from patients with hemopoietic malignancies, Mol. Cell. Biol. 4, 1096-1103 (1984). 183. Spandidos, D. A., and Kerr, I. B. Elevated expression of human rots oncogene family in premalignant and malignant tumours of the colorectum, Brit. J. Cancer. 49, 681-687 (1984). 184. Spandidos, D. A., and Agantis, N. J. Human breast tumours have elevated expression of the Harvey ras oncogene, Anticancer Res. 4, 265-272 (1984). 185. Salman, D. J., de Kernion, J. B., Verma, I. M., and Cline, M. J. Expression of cellular oncogenes in human malignancies, Science 224, 256-262 (1984). 186. Adams, J., Geronkakas, S., Webb, E., Corcoran, L. M., and Cory, S. Cellular myc gene is altered by chromosomal translocation to immunoglobulin locus in mouse plasmacytomas and is rearranged similarly in human Burkitt lymphoma, Proc. Natl. Acad. Sci. USA 80, 1982-1986 (1983). 187. Erlich, H. A., Gelfand, D. H., and Saiki, R. K. Product review: Specific DNA amplification, Nature (London) 331, 461-462 (1988). 188. Young, L. S. The epidemiology of human papilloma virus infection in relation to cervical cancer, Meanwell CA Cancer Surv. 7(3), 481-497 (1988). 189. Evan, G., Lewis, G. K., Ramsey, G., and Bishop, J. M. Isolation of monoclonal antibodies specific for human and c-myc protooncogene products, Mol. Cell. Biol. 5, 3610-3616 (1985). 190. Furth, M. E., Davis, L. J., Fleurdelys, B., and Scalnick, E. Monoclonal antibodies to the ~21, product of the transforming gene of Harvey murine sarcoma virus and cellular ras gene family, J. Virol. 43, 294-304 (1982). 191. Thor, A., Horan-Hand, P., Wunderlich, D., Caruso, A., Muraro, R., and Schlom, J. Monoclonal antibodies define differential rus gene expression in malignant and benign colonic disease, Nature (London) 311, 562-564 (1984). 192. Sikora, K., Evan, G., Steward, J., and Watson, J. V. Detection of the c-myc oncogene product in testicular cancer, Brit. J. Cancer 52, 171-176 (1985). 193. Chan, S. Y. T., Evan, G., Ritson, A., Watson, J., Wright, P., and Sikora, K. Localization of lung cancer by a radiolabelled monoclonal antibody against c-myc oncogene product, Brit. J. Cancer 54(5), 761-769 (1986). 194. Niman, H. L., Thompson, A. M. H., Yu, A., Markman, M., Wil-

TUMOR

195.

196.

197.

198.

199.

200.

201.

202.

203.

hams, J. J., and Herwig, K. R. Anti-peptide antibodies detect oncogene-related proteins in urine, Proc. Natl. Acad. Sci. USA 82, 7924-7929 (1985). Crawford, L. V., Pim, D. C., and Bulbrook, R. D. Detection of antibodies against the cellular protein ~53 in sera from patients with breast cancer, Znt. J. Cancer 30, 403-408 (1982). Ben-Maharez, K., Thierry, D., Sorokine, I., Danna-Muller, A., and Kohiyama, M. Detection of circulating antibodies against cmyc protein in cancer patients’ sera, Brit. J. Cancer 57, 529-534 (1988). Headley, D. W., Friedlander, M. I., Taylor, I. W., Rugg, C. A., and Musgrove, E. A. Method for analysis of cellular DNA content of paraffin embedded pathological material using flow cytometry, J. Hitochem. Cytochem. 31, 1333-1335 (1983). Watson, J. V., Sikora, K. E., and Evan, G. E. A simultaneous flow cytometric assay for c-myc oncoprotein and cellular DNA in nuclei from paraffin embedded material, J. Zmmunol. Methods 83, 179-192 (1985). Evan, G. E., and Hancock, D. C. Studies on the interaction of the human c-myc protein with cell nuclei: pt2’.“” as a member of a discrete subset of nuclear proteins, Cell 43, 253-261 (1985). Watson, J. V., Stewart, J., Evan, G., Ritson, A., and Sikora, K. The clinical significance of flow cytometric c-myc oncoprotein quantitation in testicular cancer, Brit. .I. Cancer 53, 331-337 (1986). Watson, J. V., Curling, 0. M., Munn, C. F., and Hudson, C. N. Oncogene expression on ovarian cancer: A pilot study of c-myc oncoprotein in serous papillary ovarian cancer, Gynecol. Oncol. 28, 137-150 (1988). Haeder, M., Rotsch, M., Bepler, G., Henning, C., Havemann, K., and Heimann, B. Epidermal growth factor receptor expression in human lung cancer cell lines, Cancer Res. 48, 11321136 (1988). Sainbury, J. R. C., Nicholson, S., Angus, B., Farndon, J. R., Malcolm, A. J., and Harris, A. L. Epidermal growth factor re-

MARKERS

204.

205.

206.

207.

208.

209.

210.

211.

212.

15

ceptor status of histological sub-types of breast cancer, Brit. .Z. Cancer 58, 458-k (1988). Zajchowski, D., Band, V., Pauzie, N., Tager, A., Stampfer, M., and Sager, R. Expression of growth factors and oncogenes in normal and tumour-derived human mammary epithelial cells, Cancer Res. 48, 7041-7047 (1988). Battaglia, F., Polizzi, G., Scambia, G., Rossi, S., Panici, P. B., and Jacobelli, S. Receptors for epidermal growth factors and steroid hormones in human breast cancer, Oncology 45,424-427 (1988). Bauknecht, T., Runge, M., Schwall, M., and Ptleiderer, A. Occurrence of epidermal growth factor receptors in human adnexal tumours and their prognostic value in advanced ovarian carcinomas, Gynecol. Oncol. 29, 147-157 (1988). Hollstein, M. C., Smits, A. M., Galiana, C., Yamasaki, H., Bos, J. L., and Mandard, A. Amplification of epidermal growth factor receptor gene but no evidence of ras mutations in primary human esophageal cancers, Cancer Res. 48, 5119-5123 (1988). Seo, M. K., Lynch, K. E., and Podolscky, D. K. Multiplicity of transforming growth factors in human malignant effusions, Cancer Res. 48, 1792-1797 (1988). Rakawicz-Szulczynska, E. M., Herlyn, M., and Koprowski, H. Nerve growth factor receptors in chromatin of melanoma cells, proliferating melanocytes, and colorectal carcinoma cells in vitro, Cancer Res. 48, 7200-7206 (1988). Steck, P. A., Lee, P., Hung, M. C., and Yung, W. K. A. Expression of an altered epidermal growth factor receptor by human glioblastoma cells, Cancer Res. 48, 5433-5439 (1988). Fraizer, G. C., Bowen-Pope, D. F., Seifert, R. A., and Vogel, A. M. Association of increased platelet-derived growth factor secretion and retroviral expression in transformed mouse and human cells, Cancer Res. 48, 4874-4880 (1988). Murphy, L. C., Murphy, L. J., Dubik, D., Bell, G. I., and Shiu, R. P. Epidermal growth factor expression in human breast cancer cells: Regulation of expression of progestins, Cancer Res. 48, 4555-4560 (1988).