40
THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE ALKALINE PHOSPHATASES IN TUMOR BIOLOGY AND THEIR POTENTIAL USE IN CLINICAL PRACTICE AKADEMISK AVHANDLING som med vederbörligt tillstånd av Medicinska Fakulteten vid Umeå Universitet för avläggande av doktorsexamen i medicinsk vetenskap kommer att offentligen försvaras i sal B, LUO, Umeå Universitet, fredagen den 16 november 1984, kl 10.00. av ANNIKA JEPPSSON

THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

  • Upload
    lamhanh

  • View
    240

  • Download
    0

Embed Size (px)

Citation preview

Page 1: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE ALKALINE

PHOSPHATASES IN TUMOR BIOLOGY AND THEIR POTENTIAL USE IN

CLINICAL PRACTICE

AKADEMISK AVHANDLINGsom med vederbörligt tillstånd av Medicinska

Fakulteten vid Umeå Universitet för avläggande av doktorsexamen i medicinsk vetenskap

kommer att offentligen försvaras i sal B, LUO, Umeå Universitet,

fredagen den 16 november 1984, kl 10.00.

av ANNIKA JEPPSSON

Page 2: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

ABSTRACT

THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE ALKALINE PHOSPHATASES IN TUMOR BIOLOGY AND THEIR POTENTIAL USE IN CLINICAL PRACTICEAnnika Jeppsson, Department of Physiological Chemistry, University of Umeå, S-901 87 Umeå, Sweden

Placental alkaline phosphatase (PLAP) is a membrane bound enzyme normally synthesized by the syncytiotrophoblasts in the human placenta. Recent studies have indicated that trace amounts of placental-like a l ­kaline phosphatases also are present in several normal organs like testes and endocervix. PLAP and P LAP-1ike enzymes are furthermore synthesized by some tumors and can be detected in sera of approximately 12 % of pat i­ents with any type of cancer, more often in patients with genital tumors. This synthesis has been considered to be ectopic.

PLAP is known to be electrophoretically highly polymorphic. Both poly- and monoclonal antibodies were used to study this enzyme. One of the monoclonal antibodies was able to discriminate between different phenotypes of PLAP and thus immunochemical approaches to elucidate e n ­zyme polymorphism were established.

To evaluate the potential clinical use of PLAP as a tumor marker s e ­rum levels of the enzyme were measured by a radioimmunoassay in 100 pati­ents with the testicular tumor seminoma. Elevated levels of PLAP were found in 43 % of the patients with primary tumors and in 75 % of the pat i­ents with recurrent or metastatic disease. After successful treatment of seminoma the PLAP levels decreased. This indicates that measuring PLAP give useful information during follow up of treatment of seminomas.

The content of PLAP-like enzymes in seminoma tumors was determined in 13 typical seminomas. The levels, of enzyme found in the tumor tissue ranged from 870-13 404 ng/g wet weight, which should be compared to around 100 ng/g in normal testes. Analysis using monoclonal antibodies and enzyme inhibitors showed the PLAP-like enzymes present in seminomas to be similar to the enzymes in normal testes. This suggests that the increased expression of PLAP-like enzymes in seminomas results from an enhanced eutopic expression of enzymes found in normal testis.

A sensitive catalytic assay was used to quantify enzyme levels in sera from women with malignant gynaecological tumors. In the group of patients with cervical carcinoma 68 % had values exceeding the normal limit. For patients with ovarian cancer and carcinoma of the breast the percentages were 35 and 23 respectively.

Monoclonal and polyclonal antibodies against PLAP were evaluated for tumor immunolocalization of human PLAP-producing tumors in nude mice.The antibodies were labeled with "12 5 j a n d injected into mice with tumors. The distribution of 25j_an-ti-PLAP in various tissues showed that the labeled antibodies were enriched in the tumors, with a mean concentration ratio of 7. This indicates that there is a potential use of PLAP in lo­calizing tumors in humans.Key words: Alkaline phosphatase, seminoma, eutopic, gynaecological tumors,

immunolocalization

ISSN 0346-6612

Page 3: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

UMEÂ UNIVERSITY MEDICAL DISSERTATIONS New Series No 129 - ISSN 0346-6612

From the Department of Physiological Chemistry University of Umeå, Umeå, Sweden

THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE ALKALINE

PHOSPHATASES IN TUMOR BIOLO­GY

AND THEIR POTENTIAL USE IN CLINICAL PRACTICE

byANNIKA JEPPSSON

^ 3 ^ Umeå university

Umeå 1984

Page 4: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

2

TABLE OF CONTENTS

Abstract 3Publications 4Introduction 5Aims 14Results and discussion

PLAP as a tumor marker (I+II) 15The use of monoclonal antibodies against PLAP (III) 20Ectopic-eutopic expression of PLAP and PLAP-1 ike enzymes (V) 21Tumor localization by use of anti-PLAP antibodies (IV) 23

Acknowledgements 2 8References 2 9Paper I 39Paper II 45Paper III 5 9Paper IV 65Paper V 71

Page 5: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

3

ABSTRACT

THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE ALKALINE PHOSPHATASES IN TUMOR BIOLOGY AND THEIR POTENTIAL USE IN CLINICAL PRACTICEAnnika Jeppsson, Department of Physiological Chemistry, University of Umeå, S-901 87 Umeå, Sweden

Placental alkaline phosphatase (PLAP) is a membrane bound enzyme normally synthesized by the syncytiotrophoblasts in the human placenta. Recent studies have indicated that trace amounts of placental-like a l ­kaline phosphatases also are present in several normal organs like testes and endocervix. PLAP and PLAP-1ike enzymes are furthermore synthesized by some tumors and can be detected in sera of approximately 12 % of pat i­ents with any type of cancer, more often in patients with genital tumors. This synthesis has been considered to be ectopic.

PLAP is known to be electrophoretically highly polymorphic. Both poly- and monoclonal antibodies were used to study this enzyme. One of the monoclonal antibodies was able to discriminate between different phenotypes of PLAP and thus immunochemical approaches to elucidate e n ­zyme polymorphism were established.

To evaluate the potential clinical use of PLAP as a tumor marker s e ­rum levels of the enzyme were measured by a radioimmunoassay in 100 pati­ents with the testicular tumor seminoma. Elevated levels of PLAP were found in 43 % of the patients with primary tumors and in 75 % of the pat i­ents with recurrent or metastatic disease. After successful treatment of seminoma the PLAP levels decreased. This indicates that measuring PLAP give useful information during follow up of treatment of seminomas.

The content of PLAP-like enzymes in seminoma tumors was determined in 13 typical seminomas. The levels of enzyme found in the tumor tissue ranged from 870-13 404 ng/g wet weight, which should be compared to around 100 ng/g in normal testes. Analysis using monoclonal antibodies and enzyme inhibitors showed the PLAP-like enzymes present in seminomas to be similar to the enzymes in normal testes. This suggests that the increased expression of PLAP-like enzymes in seminomas results from anenhanced eutopic expression of enzymes found in normal testis.

A sensitive catalytic assay was used to quantify enzyme levels in sera from women with malignant gynaecological tumors. In the group of patients with cervical carcinoma 68 % had values exceeding the normal limit. For patients with ovarian cancer and carcinoma of the breast the percentages were 35 and 23 respectively.Monoclonal and polyclonal antibodies against PLAP were evaluated for tumor immunolocalization of human PLAP-producing tumors in nude mice.The antibodies were labeled with ^ 5 j ancj injected into mice with tumors. The distribution of ^ 5 i _ anti-PLAP in various tissues showed that the labeled antibodies were enriched in the tumors, with a mean concentration ratio of 7. This indicates that there is a potential use of PLAP in lo­calizing tumors in humans.Key words: Alkaline phosphatase, seminoma, eutopic, gynaecological tumors,

immunolocalization

ISSN 0346-6612

Page 6: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

4

This thesis is based on the following publications, which will bereferred to in the text by their Roman numerals:

I A. Jeppsson, B. Wahren, T. Stigbrand, F. Edsmyr and L. Andersson: A clinical evaluation of serum placental alkaline phosphatase in seminoma patients. Brit. J. Urology 55:73-78 (1983)

II T. Stigbrand, P.Å. Holmgren, A. Jeppsson, M.-G. Damber and B. von Schoultz: On the value of placental alkaline phosphatase as a marker for gynaecological malignancy. Acta Obst. Gyn. (in press)

III J.L. Millan, G. Beckman, A. Jeppsson and T. Stigbrand: Genetic variants of placental alkaline phosphatase as detected by a monoclonal antibody. Hum. Genet. 60:145-149 (1982)

IV A. Jeppsson, B. Wahren, J.L. Millan and T. Stigbrand: Tumor and cellular localization by use of monoclonal and polyclonal antibodies to placental alkaline phosphatase. Brit. J. Cancer 49:123-128 (1984)

V A. Jeppsson, B. Wahren, E. Brehmer-Andersson, C. Silfverswärd, T. Stigbrand and J.L. Millan: Eutopic expression of placental-like alkaline phosphatase in testicular tumors.Int. J. Cancer (in press)

Page 7: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

5

INTRODUCTION

The diagnosis of cancer is dependent upon the ability to dis­criminate between normal and neoplastic tissue. Today, this determination relies almost exclusively on the recognition of abnormal morphology associated with neoplasia. The utilisation of specific biochemical differences between normal and neoplastic tissue is another approach for early detection of tumors and for monitoring treatment. In addition, the advances in the field of immunochemical analysis have made it possible to search for tumor- specific and tumor-related antigens.

These antigens are often referred to as oncofetal antigens, because of their appearance both in neoplastic and fetal tissue. Oncofetal antigens are products of an increased transcription of that part of the genome which is typical for the immature, undifferentiated cell, either it is a normal embryonal or a malignant cell. The study of transcription and translation of fetal and/or embryonic genes in tumor tissue may give valuable clues to the nature of the abnormal gene regulation occurring during tumor development.Eutopic synthesis is the term used to describe the normal case in which there is synthesis of proteins which are normally present in the cell or tissue. Ectopic synthesis implies the expression by neoplastic cells of new proteins for that cell-type occurring during tumor development.

Oncofetal antigens or ”tumor markers" as they are also referred to, have since the discovery of alphafetoprotein in the 1960*s been examined for their potential use in clinical practise (1). Although oncofetal antigens are not restricted to fetal or malignant tissues, their quantitative increase in such specimens, and in the blood of certain cancer patients, make them suitable for clinical application. The development of sensitive assays with ability to detect very low levels of protein in blood or other body fluids, have made it possible to determine the level and then correlate the

Page 8: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

6

amount with the disease status. The quantitation of car- cinoembryonic antigen (CEA) (2) and alphafetoprotein (AFP) (1) have been proven to provide useful and unique information for the management and evaluation of several malignant diseases (3,4).

Three other applications for tumor markers should be considered, and they are now being evaluated. The first is tumor detection and localization by means of photoscanning after administration of radiolabeled antibodies, the second is to use the presence of oncofetal antigens in tumors in clinical histopathology, and the third is cancer treatment with antibodies coupled to chemotherapeutic agents or isotopes.

ALKALINE PHOSPHATASES

The human alkaline phosphatases (ALPs) are membrane-bound glycoproteins that hydrolyze a wide variety of phosphate compounds at high pH. They are present in different forms in different tissues. High ALP activity has been demonstrated in many organs in humans including placenta, intestine, liver, bone and kidney. This indicates that ALP's have a vital biological role. Most other tissues have been found to contain low levels of the enzyme. Biochemical * genetic and immunological evidence indicates that at least three gene loci code for the protein moieties of the various ALPs (20). The proposed scheme of evolution for the human alkaline phosphatses is shown in figure 1 (reproduced with permission from Stigbrand and Fishman 1984). An additional group of enzymes related to the placental alkaline phosphatases have recently been iden­tified on catalytical and immunochemical basis. This new group of enzymes is referred to as placental alkaline phosphatase-like (PLAP-like) enzymes (72).

All alkaline phosphatases are active as dimers. In order to be2+ 2+ .active the ALP molecule has to contain Zn and Mg ions. If some

of these ions are removed the enzyme will lose its catalytical activity.

Page 9: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

7

Tissue unspecific AP precursor gene

Ancestral gene

Intestinal AP precursor gene

Intermediate Intestinal gene

Tissue unspecific AP gene

Tissue unspecific AP

Intestinal AP gene

Intestinal AP

PLAP gene

/1Allelic variants of placental AP

Variants of PLAP-like AP

Figure 1. A t e n t a t i v e model of the evo lut ion of the human a l k a l in e phosphatases.

Page 10: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

TADLE 1A P R OV I SIONAL CL AS S I F IC A T I ON S YSTEM FOR ALKA L I N E P H OS PHATASE ISOZYMES

Common Name A b br e v i at i o nT issue u n sp ecific a lkaline p h os phatase AP

Elect r o ph o r e ti c Subunit m i g r a t i o n ImmunologicCo mp o s i ti o n (PAGE or Starchgel) ReactivityS ingle band aAP

Cata l y t ic P r operties Heat S t a b i l ity A m in o Ac idat 65 C_ _ _ _ _ Inhibition

L - H o m o a rg

A dult intestinal alkaline p h os p hataseT e rm placental a lkaline p h osphatasePlacental alkaline phosph a ta s e - li k e enzymes (in normal tissues like testis, thymus)

IAP

PLAP

PLAP-1ike

S ingle band

PP(1-2) Polymorphic

PLPL(N)°

alAP(aPLAP)aPLAP(alAP)

aPLAPaspec i f lc

L- Ph e

L-Phe

L-Leu

T u mo r - a ss o c i at e d APPlacental alkaline phosphatase (Regan isozyme)Placental alkalinephosp h a ta s e - li k eenzymes(Nagao isozyme)Intestinal alkaline phosphatase-1 ike a lkaline phosphatases (KaSahara isozyme)

PLAP

PLAP-1ike

IAP-1ike

PP(T) Polymorphic

PLPL(T)

ILIL(T)

aPLAP9aspe c i f ic

aPLAP'aspec i f lcl-labs

alAP(aPLAP) (♦-)

L-Phe

L-Leul-Phe

L-Phe

a The subunit of term placental alkaline phosphatase has been denoted P. The dimer PP canbe specified in the Donald and Robson nomenclature (Ann. Hum. Genet. (Lond.) 1974, 37: 303-313.) to be PP(1), PP(l-2), PP(2), PP(3), PP(3-1) and PP(3-2) for the S, FS, F, I, SI and FI common phenotypes as well as the products of the rare alleles.The subunit for tissue un-specific (non-specific) and intestinal alkaline phosphatase has been denoted A and I, respectively.

b The subunit of the £lacental like alkaline phosphatase has been denoted PL, the dimerPLPL. The parenthesis (N) indicates normal tissue origin. The results from different groups working with monoclonals indicate the existence of polymorphism and the (N) can inthe future be changed to a system similar to what is used for the term placental AP. Thepotential existence of hybrid enzymes could be denoted, for example, PPL(T).

c The subunit composition of the first discovered tumour-associated AP is identical to theterm placental enzyme, but the Rumour origin is indicated by a (T). The polymorphism whenpossible to evaluate could be described in the same terms as for PLAP.

d The subunit of the tumour-associated PLAP-1ike enzyme is PLPL(T) and the nomenclaturecould cover the introduction of polymorphism when enough data have been generated on this point.

e ILIL(T) indicates the tumour origin of the enzyme.f The identification of the PLAP-1ike enzymes can be based on reactivity with specific

monoclonal antibodies and/or the characteristic amino acid inhibition (L-Leu inhibition).g The PP(T) form of the enzyme is defined by reactivity with monoclonal antibodies and/or

lack of L-Leu inhibition.

Page 11: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

9

Many cellular processes have been associated with the enzymatic activity of alkaline phosphatases, but their physiological role is still unknown. No single substrate has been identified to be the predominant ALP substrate. Most of the ALP is located on the plasma membranes where extensive transport is taking place, therefore there have been suggestions of involvement in transport mechanisms.

PLACENTAL AND PLACENTAL-LIKE ALKALINE PHOSPHATASES

Biological properties:Placental alkaline phosphatase (PLAP) is a tissue specific ALP. It is synthesized by the syncytiotrophoblasts of the placenta from the 12th week of pregnancy, and released into the maternal circulation. The pregnancy associated enzyme has in twin studies been shown to be controlled by the fetal genome (5,6). During the first trimester of pregnancy the maternal serum level of PLAP is very low. However from mid pregnancy there is a measurable increase and during the last trimester serum-PLAP rises exponentially to a value of around 250ng/ml at term (7,8,9). The serum level of PLAP correlates well with the size of the placenta (10) and attempts have been made to correlate pregnancy PLAP-levels with fetal status. In pregnant women with intrauterine growth retardation, preeclampsia or diabetes the PLAP-levels have been shown to be significantly lower, than in normal pregnancies (11,12). However the value of measuring serum-PLAP in pregnancy is still controversial.

Biochemical and immunological properties:Human placental alkaline phosphatase has been purified to homogeneity and has a molecular weight of approximately 120 000 (24,25). During pregnancy it is also present in the circulation as high molecular weight aggregates.

PLAP is extremely heat-stable. It can be heated in 70°C for 30 minutes without loosing any enzymatic or immunological activity.This is in contrast to the other types of ALP which are

Page 12: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

10

heat-labile (16). L-phenylalanine is an un-competitive inhibitor to placental alkaline phosphatase which has been used to distinguish PLAP from tissue non-specific ALP. PLAP is inhibited to 75 % with L-phenylalanine as inhibitor, while tissue non-specific ALP is not inhibited (17,18,19). Direct evidence of differences in primary structures between placental and non-placental alkaline phos­phatases have come from comparisons of peptide maps of partly

32hydrolyzed phosphatases labeled at their active sites with P and of enzymes labeled with radioactive iodine (20,21,22,23). There is a significant difference in electrophoretic mobility between PLAP, the tissue non-specific and intestinal ALPs (14,15).

The immunochemical characteristics of placental alkaline phos­phatase are clearly different from the tissue non-specific form of ALP. PLAP and intestinal alkaline phosphatase share some antigenic determinants and a cross-reactivity is observed with unabsorbed antisera against PLAP and intestinal ALP. However, monospecific antisera for each form of the enzyme can be prepared by absorbtion with purified PLAP or intestinal ALP (13).

Genetic polymorphism:Placental alkaline phosphatase is highly polymorphic. It displaysthe highest degree of polymorphism of all human enzymes studied sofar (26). The existence of polymorphism for the enzyme was firstreported by Boyer (27). Three common alleles and many rare alleleshave been described at an autosomal locus (PI). The three commonalleles usually occur in heterozygote combinations with one of thecommon alleles (5,28,29). In an investigation of 5 000 placentae ofdifferent origin 44 phenotypes of PLAP were found (30). The three

s f icommon alleles were originally called PI , PI and PI . They give rise to the phenotypes S, F, I, FS, SI and FI, named after their mobility, S-slow, F-fast and I-intermediate, electrophoretic mobility on starch gel. One of the rare variants of PLAP is the D or 18-variant. It was first described by Boyer (27). The enzymatic activity of the D-variant is inhibited not only by L-phenylalanine but also by L-leucine (31).

Page 13: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

11

In 1974 Donald and Robson introduced a new nomenclature, where S, Fand I are called 1, 2 and 3 respectively, and the heterozygousphenotypes are called 2-1, 3-1 and 3-2. The alleles are conse-

1 2 3quently called PI , PI and PI (30). The gene frequencies showmarked differences between ethnic groups; the PI* allele is themost common in all ethnic groups, the highest frequency is among

2Africans (90 - 93 %), the PI is most common in Caucasian popula-3tions (24 - 34 %) and the PI among Orientals, especially Chinese

and Japanese (24 - 28 %) (30,32). The precise nature of the molecular differences for the variants are not yet known.

Placental alkaline phosphatase seems to be a late evolutionary product since no analogs can be identified either on structural or on catalytical basis in lower animals (104,105). PLAP has shown to be present in placentae of chimpanzee and orangutan, but not in the lowland gorilla and spider-monkey (106).

Fishman et al first demonstrated the occurence of placental alkaline phosphatase in serum from a cancer patient (107). He named the enzyme , Regan isoenzyme, after the patient with squamos cell carainoma of the lung where it was first found. The enzyme of the tumor was found to be indistinguishable from the placental ALP.

PLAP-like enzymes are similar to PLAP, but differ in the respect that they are inhibited not only by L-phenylalanine but also with L-leucine (43). The PLAP-like enzymes are identical to PLAP in heat stability, electrophoretic mobility, immunological properties with polyconal antisera and urea sensitivity.

UN-SPECIFIC ALKALINE PHOSPHATASE

The ALP predominantly found in human liver, bone and kidney is called tissue un-specific alkaline phosphatase. Using immunochemi­cal techniques with polyclonal antisera these ALPs are indistinguishable from each other (14). They also show the same pattern of inhibition with L-phenylalanine and L-homoarginine, 0 -

Page 14: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

12

10 % and 80 % respectively (17,33), The non-specific ALP is heat- sensitive, By use of polyacrylamide gel electrophoresis it is possible to distinguish liver, bone and kidney ALPs from each other (34), After desialylation the enzymes have identical isoelectric points (35). It is suggested that liver, bone and kidney ALPs are encoded by a single locus for alkaline phosphatase since they are similar in catalytic properties and antigenic specificity. The tissue-specific differences are probably introduced during the expression in different types of cells, by modifications of the glycosylation patterns. Human liver ALP has been purified and characterized. The enzyme has a molecular weight of 140 000 and exists exclusively as a dimer (21). The human kindney alkaline phosphatase has not yet been purified and the bone ALP has only been partially purified (36).

INTESTINAL ALKALINE PHOSPHATASE

Intestinal ALP is the third main form of human alkaline phosphatases. It is tissue-specific and found in the mucosa of the small intestine. The inhibition patterns of intestinal ALP with L-phenylalanine and L-homoarginine are the same as for PLAP, the inhibition being 75 % and 5 % respectively. The peptides L-phe-gly-gly and L-leu-gly-gly can be used as inhibitors to dis­tinguish the intestinal ALP from PLAP (37). Intestinal ALP is partially heat-sensitive.

Two forms of normal intestinal ALP have been recognized, the fetal and the adult form (38,39). They have similar thermostabilities and they are indistinguishable in their inhibition characteristics with aminoacid and peptide inhibitors. The forms differ in electrophoretic mobility, partly because of the presence of sialic acid residues on the fetal form and the absence of it on the adult form. Even after neuraminidase treatment there is a small but significant difference. Intestinal ALP has been purified both with conventional methods and with immunoaffinity chromatography (40,41). The enzymes are dimers with an approximate molecular

Page 15: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

13

weight of 140 000 for the fetal and 170 000 for the adult form (41).

Page 16: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

14

The aims of the present study have been:

-to evaluate the clinical usefulness of PLAP and PLAP-like enzymes as tumor markers in testicular and gynecological malignancies,

- to evalute the possibility of using anti-PLAP antibodies in radioimmunolocalization experiments with PLAP-producing tumors.

- to determine whether the synthesis of the PLAP-like enzyme in testicular tumors is an ectopic or eutopic production.

- to evaluate the possible use of monoclonal antibodies in the study of the polymorphism of PLAP

Page 17: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

15

Present investigation

PLAP AS A TUMOR MARKER (I+Il)

Tumor associated placental alkaline phosphatase, was first demonstrated in serum of a lung cancer patient in 1968 (42), The enzyme of the tumor was found to be indistinguishable from PLAP with respect to L-phenylalanine inhibition and heat-stability. No differences could be detected between the placental ALP and the tumor enzyme with immunological techniques (42,43). The NH^-

terminal amino acid sequence, peptide map, subunit molecular weight and isoelectric point were reported to be very similar for the placental and tumor associated enzymes (44). Therefore has the expression of PLAP by tumors during the last decade been considered to be an ectopic synthesis.

Another tumor associated alkaline phosphatase similar to PLAP was originally identified because it was inhibited by L-phenylalanine and L-leucine. This PLAP-like enzyme was first demonstrated in a patient with pleuritis carcinomatosa. It was called the Nagao isoenzyme after the patientsfname (43). With the new nomenclature the Nagao enzyme is called PLAP-like alkaline phosphatase. Today there is no explanation for the L-leucine sensitivity, but post- translational modifications occurring in tumor cells may result in the exposure of sites which could affect the inhibition pattern (45). On starch gel electrophoresis and in L-leucine sensitivity the PLAP-like enzyme very much resemble the rare D-variant of PLAP (27) and it has been suggested that they would be identical (31). However, from a genetic point of view this is unlikely since the frequency of the allele coding for the D-variant is low and the PLAP-like enzyme is not unusual among cancer patients (46).

Page 18: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

16

Both immunological and catalytical assays have been developed to measure placental and placental-like alkaline phosphatase content in tumors and sera of cancer patients. The early assays were not completely specific and not so sensitive. The assays have made use of the catalytical activity remaining after heat inactivation or they have been based on immunological methods not using specific antisera. Antiss et al. (47) were the first to describe a quantita­tive assay for PLAP and they have been followed by others: now there have been at least 15 different assays published (9,10,30,48,49,50,51,52,53,54,55,56,72). The variation in results between groups could in part be due to differences in specificity and sensitivity. Using a specific enzyme-linked immunosorbent assay with a sensitivity of 0.4 ng/ml Millan and Stigbrand found that 70 % of a healthy population had PLAP concentrations below the limit of detection, and all healthy individuals had concentrations of PLAP below 1.85 ng/ml (50). McLauglin et al. using a monoclonal antibody reactive only with placental-type alkaline phosphatase in a solid-phase enzyme immunoassay, found no detectable PLAP- concentrations in healthy non-pregnant individuals. The assay had a lower limit of detection, of 0.1 U/l (approximately 0.07 ng/ml) (54). The basal level of PLAP reported in healthy individuals could reflect the eutopic expression found in normal tissues, such as cervix, ovary, testis, lung, thymus and breast (57,58,59,60,61,73).

It has recently been shown that smokers have a significantly higher level of PLAP or PLAP-like enzymes then non-smokers (52,62). It is possible that the PLAP-like enzyme observed in smokers is of lung origin. This could indicate that an inflammatory or premalignant change in the lung results in an enhanced expression of the enzyme normally produced in the lung.

Placental-like alkaline phosphatase has been shown to be syn­thesized by a number of different malignancies (94). Very high levels of PLAP have been described in seminomas and certain other testicular tumors (63) and in serum from patients with seminomas (63,64). Also, patients with ovarian cancer have been demonstrated

Page 19: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

17

to have increaced levels of PLAP-like enzyme in the serum (65,66,67,74).

TESTICULAR TUMORS

The two tumor markers AFP and HCG occur in a high frequence in the serum of patients with testicular tumors. AFP is present in the serum of patients with non-seminomatous tumors. It is elevated in about 75 % of the patients with teratocarcinoma, in 64 % of the patients with choriocarcinoma (71,108). AFP is considered absent in seminomas, if present, one should suspect the presence of a non- seminoma tous component (7 5). The serum-HCG level is raised in 100 % of the patients with choriocarcinoma and in 56 % of the patients with embryonal carcinoma (71,108). Around 15 % of seminoma patients have an elevated serum-HCG level. HCG may occur independently of PLAP in the sera of seminoma patients but is also like AFP a warn­ing that a non-suspected non-seminomatous component has metastasized (75). AFP and HCG when present are quite relyable for monitoring patient treatment. The improved chemotherapy and the possibility to follow the patient with tumor markers have improved the survival rate for patients with non-seminomatous tumors significantly.

The pure seminomas comprise around 40 % of testicular germ cell tumors (76). The survival rate for seminoma patients is high, but around 10 % of the patients pursue a fatal course which perhaps could be improved with a better method of monitoring the disease

PLAP has been shown to be present in seminoma tissue and in serum of patients with active disease (63). However the importance of PLAP in the management of testicular tumors, especially seminoma, has not been clear. We have investigated 100 patients with seminoma. Serum determinations of PLAP were performed with a specific double antibody solid phase radioimmunoassay (9). Polyclonal antibodies against PLAP were used in this assay which thus measures both PLAP and PLAP-like enzymes. The sensitivity of

Page 20: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

18

the assay was 12 ng/ml and values above that were considered to be abnormal in serum of males (77), In the group of patients where preoperative serum samples were available, 9 out of 21 had elevated levels of PLAP with a mean of 53 ng/ml. Twelve cases of recurrence or metastatic growth were found among the 100 patients. Seventy-five per cent of these patients had raised levels of PLAP, in conjunction with the clinical relapse. In the group of patients with no evidence of disease, 9 out of 68 had serum levels above the detection limit. It is not clear why some patients with no evidence of disease had raised levels of PLAP. A possible explanation for it could be the higher levels of PLAP in smokers compared to non- smokers. It is not known whether the patients in our study were smokers or not. Other investigators have also observed clinically healthy patients with increased levels of enzyme (Kjell Nustad, personal communication). These now false positive levels might indicate a higher risk of relaps, but this is yet too early to evaluate. Observation periods of years are needed to substantiate the clinical significance of this observation. In many but not all patients the serum-PLAP paralleled the changes in tumor burden. We could not see a difference in serum-PLAP level between the patients with stage I and II disease.

In the control group 2/51 had measurable levels of PLAP. The higher levels of PLAP in smokers could be an explanation for these in­creased values in apparently healthy individuals. The smoking habits of the control-group is unfortunately not known.

The period between operation and irradiation is of particular interest since the policy of treatment usually not includes lym- phadenoectomy or laparatomy. We found raised PLAP-levels in 4 out of 18 samples taken from patients after orchiectomy but before radiation treatment. This might indicate that the operation has not been radical and that staging could be improved by PLAP measurements•

Page 21: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

19

Lange et al. (64) have shown that in a group of 28 patients with seminoma 16 had elevated PLAP levels. Only four of 22 non- seminomatous patients had raised levels. They also showed that in six of ten serial studies PLAP provided additional information by reflecting or predicting tumor behavior. This information could have been used clinically.

It seems reasonable to conclude with the information available today that all seminoma tumors express PLAP or PLAP-like enzymes (I,63,64,V). The enzymes are sometimes released into the circula­tion in levels that can be measured. When present , PLAP or PLAP- like enzyme level give useful information to the clinician during follow up of treatment of seminoma patients.

GYNAECOLOGICAL TUMORS

Nathanson and Fishman described in 1971, that gynecological cancer also could be associated with elevated PLAP-levels (78). Several reports have confirmed this observation (65,66,67,74). The percent­ages of raised values of PLAP in gynaecological malignancies have however varied significantly in different reports and the clinical significance remains unclear. There is little agreement in which of the tumors the highest incidence of increased values could be found.

A sensitive catalytical assay with a detection limit of 0.1 U/l, which corresponds to 0.5 ng/ml, was used to investigate the level of PLAP or PLAP-like enzymes in gynaecological tumors (II). The assay was found not to be influenced by the level of intestinal ALP present in the serum samples.The intestinal ALP was catalytically eliminated by heat inactivation. A difference in enzyme level between men and women was observed in the control group of ap­parently healthy individuals. Women had slightly higher catalytic activity than men.

Page 22: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

20

The clinical material consisted of 83 serum samples from women with cervical carcinoma (25), ovarian carcinoma (23) and carcinoma of the breast (35). Sixty-eight per cent of the women with a diagnosis of cervical carcinoma had increased values of PLAP. The correspond­ing figures for ovarian and breat cancer were 35 % and 23 % respectively. In the control group 4.5 % had raised levels. A high incidence of increased values ( ) among patients with cervicalcarcinoma has also been described (74). The highest incidences ( )have been reported for ovarian cancer (67). These studies however have been performed with different assays which partly explain the discrepancies.

Due to the high sensitivity of the catalytical assay may the reported incidence appear high. In comparison to the levels found for patients with seminoma are the absolute values considerable lower. For those patients with gynaecological cancer presenting high levels prior to treatment may the measurements of PLAP be of clinical value.

THE USE OF MONOCLONAL ANTIBODIES AGAINST PLAP (ill)

Electrophoretic analysis has been used to study the polymorphism of PLAP. A potential alternative approach to elucidate polymorphism would be by use of immunochemical methods. With polyclonal an­tibodies the different phenotypes of PLAP are indistinguishable from each other (9,13). However, Wei and Doellgast have shown that they with careful and extensive absorption of polyclonal antibodies (with purified phenotypes of PLAP) can demonstrate differences in antigenic determinants between the phenotypes of PLAP (79). The introduction of hybridoma technology by Köhler and Milstein (82) has introduced new immunochemical tools in biochemistry. Monoclonal antibodies have made it possible to study proteins at the level of single antigenic determinants. Phenotypic specific monoclonal antibodies against PLAP have been shown to discriminate the products of the three common alleles and several rare alleles from each other (45,81).

Page 23: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

21

We have in III used the monoclonal antibody produced against

PLAP (45). It was shown to react with the products of the PI* and3 2PI alleles, but poorly with the product of the PI allele. The

Fj^-reactivity with phenotype 2-1 and 3-2 was intermediate.

The Fjj antibody was demonstrated (III) also to be reactive with

the products of the rare alleles Pl^ and PI*** .The Pl***is the rare D-variant of placental alkaline phosphatase. Antibody F^^was fur­

thermore non-reactive with the rare phenotypes 8-2 and 9-2, while it was intermediate reactive with 8-1 and 9-1.

One placental sample electrophoretically classified as ahomozygote seemed to represent a heterozygote phenotype of the

2product of the PI allele and a new variant of PLAP, as detected by the F ^ monoclonal antibody.

The present results add a new dimension to the study of PLAP variants and establish monoclonal antibodies as powerful tools in the study of enzyme polymorphism.

ECTOPIC-EUTOPIC EXPRESSION OF PLAP AND PLAP-LIKE ENZTMES (V)

Alkaline phosphatases with enzymatic and immunological properties similar to P1AP have been identified in trace amounts in normal testes (58,59,83). The enzymes found in normal testes display a reactivity pattern with monoclonal antibodies to PLAP that is different from the pattern seen with the placental enzyme (83).

The pattern of inhibition with L-phe and L-leu and the tripeptides L-phe-gly-gly and L-leu-gly-gly is also characteristically dif­ferent from that of PLAP (83). Millan and Stigbrand therefore suggested an allelic variation of the normal testicular PLAP-like enzyme, with at least four phenotypes. They described the following pattern of reactivity with five monoclonal antibodies.

Page 24: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

22

Testis ALP % Relative activity

phenotype n Fn D10 C2 H7 B10

type I 26 16- 4 56±9 8Ì5 98-3 40±12

type II 3 83-10 101±8 75-6 100^1 81Ì3

type III 2 60 60 55 100 73

type IV 1 18 85 2 100 39

n number of samples% relative reactivity (mean - SD) with respect to the purified PLAP (phenotype 1) considered as 100 % reactive (reproduced with permission from Millån and Stigbrand (81)).

Very high levels of PLAP or PLAP-like enzymes have been described in seminoma tissue (63) and in serum of seminoma patients (63,64,1). Therefore it was interesting to examine whether the enzyme found in testicular tumors, especially seminomas, resembled PLAP or the normal testicular PLAP-like ALP. In other words to test if the synthesis is ectopic or eutopic. Using a sandwich ELISA (50) we determined the content of PLAP-like enzyme to be in the range 870 - 13404 ng/g tissue wet weight with a mean of 5 684 ng/g in seminomas. This should be compared with the level in normal testis, which have been reported to be around 100 ng/g tissue wet weight (83). None of the 12 typical seminomas examined contained less than 870 ng/g. In the five mixed tumors with seminoma components studied, tumors with a large seminoma component had an increased PLAP-like ALP content. The variation in enzyme content between typical seminomas were considerable. The reason for this variability is not clear. A true heterogeneity between seminomas may occur; a difference not detectable neither macroscopically nor microscopically. Storage for longer periods of time could reduce the content of immunoreactive enzyme.

Page 25: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

23

The reactivity of the PLAP-like enzyme produced by typical semi­nomas was investigated with a panel of five monoclonal antibodies. These were the same antibodies used in the experiments with normal testes. Seven out of seven seminoma samples contained enzyme characterized with low reactivity with the BIO, C2 and Fll an­tibodies, intermediate reactivity with the DIO antibody, and high reactivity with the H7 antibody. These tumors were interpreted to belong to the suggested phenotype I of the normal testes ALP (83). One seminoma tumor contained PLAP-like ALP that showed high reac­tivity with all five antibodies, thus resembling testis phenotype II and placental phenotype 1 and 3. We have assayed 30 serum samples from patients with testicular tumors for reactivity with the antibodies, among these patients we have found 22 type I, 2 type II, 4 type IV and 2 type V. The phenotype V has not been described before and is reactive only with the C2 antibody (data to be published (84)).It is not known whether the testicular PLAP-like enzyme have an­tigenic determinants that are unique. Monoclonal antibodies specific for the testicular enzyme will give an answer to this.

Enzyme inhibitors can be used to differentiate tumor-PLAP-like enzymes from PLAP (85,86). Also, in this respect the seminoma enzymes differed from PLAP, and resembled what can be seen in normal testis (83). PLAP was preferentially inhibited by L-phe-gly- gly, while the tumor enzymes were preferentially inhibited by L- leu. In the case of L-phe there was no significant differencebetween PLAP and the tumor enzyme. The testis phenotypes I and IIshowed the same pattern of inhibition. These results strongly suggest that the expression of PLAP-like enzyme in seminomasresults from the enhanced eutopic expression of the enzyme found innormal testis.

TUMOR LOCALIZATION BY USE OF ANTI-PLAP ANTIBODIES (IV)

The perspective of using antitumor antibodies to deliver toxic substances selectively to tumor cells was already expressed in the

Page 26: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

24

beginning of this century (87). In 1953 attempts to localize tumors in mice with radio-labeled antibodies was first reported (88). The reproducibility of these early results were limited because of the use of antibodies against poorly defined tumor antigens. The dis­covery of the oncofetal antigens, which are now well characterized proteins purified from human carcinomas, offered the first real possibility to use defined antigens as targets for tumor localiza­tion for radiolabeled antibodies.

Both polyclonal and monoclonal antibodies have been used in radioimmunolocalization experiments with satisfactory results. The advantages with monoclonals are the specificities for the antigen and the homogeneity of the antibody preparations. The difference in specificity of antibodies could be a reason for the discrepancy in results between different groups when using immunolocalization with polyclonal antibodies in patients. Mach et al. (93,98) utilizing polyclonal anti-CEA antibodies obatined positive external photos­cans in only about 50 % of known tumor sites in patients with colorectal carcinoma. Goldenberg et al. also using CEA as target antigen detected 70 - 90 % of known tumors in pateients with colorectal, ovarian, cervical and lung cancer (92,99). Monoclonal antibodies have been demonstrated to give comparable results to those obtained by using polyclonal antibodies (100).

A model system with animals bearing heterograft of human carcinomas was early developed. Nude mice and hamsters are the two animals most widely used (89,90).

Several radioactive isotopes have been used in the tumor localiza­tion experiments, the most common in use have been iodine-125 and iodine-131. The coupling reaction with the antibody is usually not difficult to perform and the biological half life of three daysmake it convenient to work with. The high energy gamma-ray activity

13-1of I would be an advantage if it was to be used for tumor 123therapy. I has also been tried (91). Its energy is very suitable

Page 27: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

25

for the Ö-camera. It is, however, expensive and has a short half- life (13 h).

A general problem in ”in vivo" tumor localization has been the highbackground radioactivity from blood and tissues. Goldenberg et al.

131(92) solved this problem by combining the I-labeled affinity purified goat-anti CEA that they used, with a second radiolabel that remained in the general circulation (99m Tc04 and 99m Tc labeled albumin). The second radioalabel was distinguishable from the first by two channel gamma-ray scintigraphy, its image could therefore be subtracted from the of the labeled anti-CEA antibody to provide correction for unbound and metabolized antibody. This technique has limitations, for example is the differences in the absorption coefficients of the photons from the radiolabels used large enough to reduce even large tumor/non-tumor uptake ratios to marginally detectable levels. Even from higher ratios (>5) the variation of counts from the tumor with depth is very marked and small errors in the back-ground correction can result either in false negatives or false positives through the production of ar­tefacts (94).

Indium-111 (^^In) has been suggested as an alternative radiolabel (101) because of better detectable gamma energies by covnvential gamma cameras, chemical stability within the cells, and a suitable half life (2.8 days). But problems with the labeling have made it hard to use. However, recent reports show that by covalently couple the antibodies to diethylenetriaminepenta-acetic acid (DTPA) and then label the conjugate by addition of indium-111 it is possible to obtain antibodies with high specific activity (95,96),

Placental alkaline phosphatase is normally bound to the outer surface of the cytoplasmic membrane of the placenta. The enzyme also appears in tumors to be localized primarily to the plasma membrane (102). With this location it theoretically appears to be a perfect target for radioimmunodetection. In the present investiga­tion (IV) the nude mouse model was used forimmunolocalization

Page 28: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

26

radioiimnunodetection. In the present investigation (IV) the nude mouse model was used for immunolocalization experiments. The tumor used was Hep 2, a subclone from a HeLa cell-line derived from a cervix carcinoma. The cells (5x10^) were inoculated subcutaneously behind the front leg. After 6 - 1 0 days the tumors had grown to a weight of about 0.1 - 0.5 g. The antibodies used were polyclonal rabbit anti-PLAP antibodies purified on protein A-Sepharose and a monoclonal against PLAP that was affinity purified on a PLAP-

Sepharose column. The antibodies were labeled with iodine-125 using the chloramine-T-method and, after labeling, 80 % of the antibody preparation was reactive with PLAP. The distribution of radiolabeled antibodies in nude mice was expressed as a concentra­tion ratios (103) calculated as follows:

cmp/g specified tissueCone, ratio = — -------------------------

cmp/g whole mouse

To be able to decide when to sacrifice the animals we investigated the distribution of labeled anti-PLAP antibodies in different tissues as a function of time after injection of antibody. The concentration ratios for the different tissues remained rather constant at days 2, 4 and 6, but the tumor ratio was maximal at day 4. Therefore the fourth day after injection with labeled antibody was chosen for the subsequent experiments.

Both the rabbit polyclonal antibodies and the monoclonal an­

tibodies were shown to successfully localize the Hep 2 tumor. The125specific tumor concentration ratios obtained by the I-labled

antibodies to Hep 2 tumors were for the polyclonal and monoclonal antibodies 7.1 £ 1.2 and 6.8 ~ 3.4 respectively. There was no statistical difference between the antibodies. The control tumors had a concentration ratio of the rabbit anti-PLAP around 1.5.

Page 29: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

27

The amount of labeled antibody bound to the tumor showed a positive (r = 0.66), although low, correlation with the PLAP concentration in the tumor. Between tumor weight and concentration ratio the relation was inverse (r = -0.50).

The amount of antibodies in the mouse localizing to other tissues than the tumor ranged from a mean concentration ratio of 0.7 ± 0.2 in the muscle to 2.2 £ 0.5 in the kidney. The concentration ratios for the blood were as high as those for the tumors. This has also been shown for CEA containing tumors and anti-CEA antibodies (89,103). The blood concentration ratio declined with time. The initially high ratio could be due to circulating anti-PLAP, par­tially degraded labeled antibody and perhaps free iodine from metabolized radioactive antibody. The PLAP-concentration in blood, however, was non-measurable, so there should not be too much bind­ing of labeled antibody to circulating antigen. The control antibodies used were anti-CEA and the distribution for these an­tibodies in the various tissues was different from the specific antibodies. The highest ratio was found in blood and there was no significant difference between the Hep 2 tumors and the other organs.

The PLAP-content in the Hep 2 tumors was 3 110 Ì 2 689 ng/g and the content of PLAP in Hep 2 cells in tissue culture is about 56 000 ng/g, indicating that modifications in gene expressions may take place following transfer from an in vitro to an in vivo situation.

The positive results obtained here are encouraging. The PLAP producing tumors were localized both by polyclonal and monoclonal antibodies. The fact that smaller tumors had somewhat higher mean concentration ratios would be an advantage if the method was to be used in a clinical situation, in which small tumors might be of crucial importance.

Page 30: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

28

Acknowledgements

There are two persons I especially want to thank for their con­tribution to this thesis:

Torgny Stigbrand, who made me start with the tumor marker business. With his extensive knowledge and never-ending enthusiasm he has helped me avoid all the pitfalls in research.

Britta Wahren at National Bacteriological Laboratory in Stockholm, without her exceptional knowledge, extreme patience with me, stimulation and trust in me, this work would never have been finished.

I am grateful to both of you, you have complemented each other in a perfect way!

I would also like to express my warm thanks to:

Karl Gustav Paul and Thomas Olivecrona heads of the Department of Physiological Chemistry for their valuable support.

Ulla Domar for all the discussions about PLAP and life.

Jose Luis Millan, who thought me more about PLAP than anyone else.

Kerstin Hjortsberg, Maria Lindberg and Elisabeth Näslund for skil­ful technical assistance.

Everybone at VTU, who made the work done in Stockholm a pleasure.

Ulla-Britt Mattsson and Marianne Lundberg for excellent secretarial assistance•

All other members of the staff at the Department of Physiological Chemistry, who have supported and assisted me in many ways.

Page 31: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

29

REFERENCES1. Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A.,

and Irlin, I. S. Production of embryonal a-globulin by transplantable mouse hepatomas. Transplantation (1963) 1:174-180.

2. Gold, P. and Freedman, S. 0. Specific carcinoembryonic antigens of the human digestive system. J. Exp. Med. (1965) 122:467-481.

3. Goldenberg, D. M., Neville, A. M., Carter, A. C., Go, V. L. W., Holyoke, E. D., Isselbacher, K. J., Shein, P. S., and Swartz, M.CEA (Carcinoembryonic antigen): its role as a marker in the m a n a g e ­ment of cancer. J. Cancer Res. Clin. Oncol. (1981) 101:239-242.

4. Ruoslahti, E. and Seppälä, M. Alpha-fetoprotein in cancer and fetal development. Adv. Cancer Res. (1979) 29:275-346.

5. Robson, E. B. and Harris, H. Genetics of the alkaline phosphatase polymorphism of the human placenta. Nature (1965) 207:1257-1259.

6. Beckman, G. Genetics of human placental phosphatases. Thesis 1970, Umeå.

7. Fishman, W. H., Anstiss, C. L., Pirnik, M. P. and Driscoli, S. G.The exponential growth curve for the placental isoenzyme of alkaline phosphatase in sera of normal and diabetic pregnancies. Amer. J.Clin. Path. (1973) 60:353-358.

8. Fishman, W. H., Bardawil, W. A., Habib, H. G., Anstiss, C. L. and Green, S. The placental isoenzyme of alkaline phosphatase in sera of normal pregnancy. Amer. J. Clin. Path. (1972) 57:65-74.

9. Holmgren, P. Å., Stigbrand, T., Damber, M.-G., von Schoultz, B.A double antibody solid phase radioimmunoassay for placental alkaline phosphatase. Clin. Chim. Acta (1978) 83:205-210.

10. Sussman, H. H., Bowman, M. and Lewis, J. L. Placental alkaline phosphatase in maternal serum during normal and abnormal pregnancy. Nature (1968) 218:359-360.

11. Holmgren, P. A., Stigbrand, T., Damber, M.-G. and von Schoultz, B. Serum levels of placental alkaline phosphatase in highrisk p r e g ­nancies. Obstet. Gynaecol. (1979) 54:631-634.

12. Yamaguchi, R. and Shimozato, N. Diagnosis of placental function by prediction curves for heat-stable alkaline phosphatase (HSAP). Tohoku J. Exp. Med. (1978) 124:73-82.

13. Lehmann, F.-G. Immunological relationship between human placental and intestinal alkaline phosphatase. Clin. Chim. Acta (1975) 65: 257-269.

Page 32: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

30

14. Boyer, S. H. Human organ alkaline phosphatases. Discrimination by several means including starch gel electrophoresis of anti- -enzyme-enzyme supernatant fluid. Ann. N. Y. Acad. Sci. (1963) 103:938-950.

15. Moss, D. W., Campbell, D. M. Ana g nos t o u - K a k a r a s , E. and King,E. J. Characterization of tissue alkaline phosphatases and their partial purification by starch gel electrophoresis. Biochem. J. (1961) 81:441-447.

16. Neale, F. C., Clubb, J. S., Hotchkis, D. and Posen, S. Heat stabil­ity of human placental alkaline phosphatase. J. Clin. Pathol. (1965) 18:359-363.

17. Fishman, W. H., Green, S. and Inglis, N. R. L-Phenylalanine:An organ specific stereospecific inhibition of human intestinal alkaline phosphatase. Nature (1963) 198:685-687.

18. Fishman, W. H. and Ghosh, N. K. Isoenzymes of human alkalinephosphatase. Adv. Clin. Chem. (1967) 10:255-370.

19. Fishman, W. H., Inglis, N. R. and Ghosh, N. K. Distinctionsbetween intestinal and placental isoenzymes of alkaline pho sphat­ase. Clin. Chim. Acta (1968) 19: 71-79.

20. Seargeant, L. E. and Stinson, R. A. Evidence that three structuralgenes code for human alkaline phosphatases. Nature (1979) 281:152-154.

21. Badger, K. S. and Sussman, H. H. Structural evidence that human liver and placental alkaline phosphatase isoenzymes are coded bydifferent genes. Proc. Natl. Acad. Sci. USA (1976) 73:2201-2205.

22. McKenna, M. J., Hamilton, T. A. and Sussman, H. H. Comparison ofhuman alkaline phosphatase isoenzymes. Structural evidence for three protein classes. Biochem. J. (1979) 181:67-73.

23. Whitaker, K. B. and Moss, D. W. Comparison of radioactive peptidesobtained from specifically labelled human renal and placental alkaline phosphatases. Biochem. J. (1979) 183:189-192.

24. Ghosh, N. K. and Fishman, W. H. Purification and properties ofmolecular-weight variants of human placental alkaline phosphatase.Biochem. J. (1968) 108:779-792.

25. Gottlieb, A. J. and Sussman, H. H. Human placental alkaline pho s­phatase: Molecular weight and subunit structure. Biochim. Biophys. Acta (1968) 160:167-171.

26. Harris, H., Hopkinson, D. A. and Robson, E. B. The incidence ofrare alleles determining electrophoretic variants: data on 43 enzymeloci in man. Ann. Hum. Genet. (London) (1974) 37:237-253.

Page 33: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

31

27. Boyer, S. H. Alkaline phosphatase in human sera and placentae. Science (1961) 134:1002-1004.

28. Beckman, L., Björling, G. and C h r i s tod ou l o u , C. Pregnancy enzymes and placental p oi vmorphism. 1. Alkaline phosphatase. Acta Genet. Basel (1966) 16:59-73.

29. Robson, E. B. and Harris, H. Further studies on the genetics ofplacental alkaline phosphatase. Ann. hum. Genet.(Lond) (1967) 30:219-232.

30. Donald L. J. and Robson, E. B. Rare variants of placental alkaline phosphatase. Ann. Hum. Genet (Lond) (1974) 37:303-313.

31. Inglis, N. R., Kirley, S. Stolbach, L. L. and Fishman, W. H.Phenotypes of the Regan isoenzyme and identity between the placentalD-variant and the Nagao isoenzyme. Cancer Res. (1973) 33:1657-1661.

32. Beckman, G. and Beckman, L. The placental alkaline phosphatase polymorphism. Variations in Hawaiian subpopulations. Hum. Hered. (1969) 19:524-529.

33. Kellen, I. A. and Lustig, V. Pattern of amino acid inhibition ofalkaline phosphatases as an aid in cancer diagnosis. Oncology (1971) 25:239-245.

34. Smith, I., Perry, J. D. and Lighstone, P. J. Disc electrophoresis of alkaline phosphatase: mobility changes caused by neuraminidase. Clin. Chim. Acta (1973) 45:219-223.

35. Stinson, R. A. and Seargeant, L. E. Comparative studies of purealkaline phosphatases from five human tissues. Clin. Chim. Acta(1981) 110:261-272.

36. Farley, J. R., Ivey, J. L. and Baylink, D. J. Human skeletalalkaline phosphatase. Kinetic studies including pH dependenceand inhibition by theophylline. J. Biol. Chem. (1980) 255:4680-4686.

37. Doellgast, G. J. and Meis, P. J. Use of specific inhibitors to discriminate alkaline phosphatase isoenzymes originating from human liver, placenta and intestine: Absence of meconial alkaline p h o s ­phatase in maternal serum. Clin. Chem. (1979) 25:1230-1233.

38. Mulivor, R. A., Hanning, V. L. and Harris, H. Developmental changein human intestinal alkaline phosphatase. Proc. Natl. Acad. Sci.,USA (1978) 75:3909-3912.

39. Komoda, T. and Sakagishi, Y. The function of carbohydrate moietyand alteration of carbohydrate composition in human alkalinephosphatase isoenzymes. Biochim. Biophys. Acta (1978) 523:395-406.

Page 34: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

32

40. Sugiura, M., Isobe, M., Hirano, K., lino, S., Suzuki, H. and Oda, T. Purification of human intestinal alkaline phosphatase.Chem. Pharm. Bull. (1975) 23:1537-1541.

41. Vockley, J. and Harris, H. Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography. Biochem. J. (1984) 217:535-541.

42. Fishman, W. H., Inglis, N. R., Green, S., Anstiss, C. L., Ghosh,N. K., Reif, A. E., Rustigan, R., Krant, M. J. and Stolbach, L. L. Immunology and biochemistry of Regan isoenzyme of alkaline phosphatase in human cancer. Nature (1968) 219:697-699.

43. Nakayama, T., Yoshida, M. and Kitamura, M. L-leucine sensitive, heat-stable al kaline-phosphatase isoenzyme detected in a patient with pleuritis carcinomatosa. Clin. Chim. Acta (1970) 30:546-548.

44. Greene, P. J. and Sussman, H. H. Structural comparison of ectopic and normal placental alkaline phosphatase. Proc. Nat. Acad. Sci.USA (1973) 70:2936-2940.

45. Millan, J. L., Stigbrand, T., Ruoslahti, E. and Fishman, W. H. Characterization and use of an allotype-specific monoclonal an t i ­body to placental alkaline phosphatase in the study of cancer- -related phosphatase polymorphism. Cancer Res. (1982) 42:2444-2449.

46. Stigbrand, T. and Engvall, E. Placental proteins as tumor markers.Sell, S. and Wahren B. (eds) In Human Tumor Markers. The Humana Press, Clifton, New J e r s e y ,(1982) 275-301.

47. Anstiss, C. L., Green, S. and Fishman, W. H. An automated tech­nique for segregating populations with a high incidence of Regan isoenzyme in serum. Clin. Chim. Acta (1971) 33:279-286.

48. Usategui-Gomez, M., Yeager, F. M. and Fernandez-de Castro, A.A sensitive immunochemical method for the determination of the Regan isoenzyme in serum. Cancer Res. (1973) 33:1574-1577.

49. Jacoby, B. and Bagshawe, K. D. A radioimmunoassay for placental- -type alkaline phosphatase. Cancer Res. (1972) 32:2413-2420.

50. Millän, J. L. and Stigbrand, T. "Sandwich" enzyme immunoassay for placental alkaline phosphatase. Clin. Chem. (1981) 27:2014-2018.

51. Haije, W. G., Meerwaldt, J. H., Talerman, A., Kuipers, Tj., Baggerman, L., Teeuw, A. H., van der Pompe, W. B. and van Driel , J.The value of a sensitive assay of carcino-placental alkaline phosphatase (CPAP) in the follow-up of gynecological cancers.Int. J. Cancer (1979) 24:288-293.

52. Tonik, S. E., Ortmeyer, A. E., Shindelman, J. E. and Sussman, H. H. Elevation of serum placental alkaline phosphatase levels in cigarette smokers. Int. J. Cancer (1983) 31:51-53.

Page 35: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

33

53. Chang, C. H., Raam, S., Angellis, D., Doellgast, G. J. and Fishman,W. H. A simple radioimmunoassay of human placental alkaline p ho s­phatase (Regan isoenzyme) using specific antibody polymers.Cancer Res. (1975) 35:1706-1712.

54. Me Laughlin, P. J., Gee, H. and Johnson, P. M. Placental-type alkaline phosphatase in pregnancy and malignancy plasma: specific estimation using a monoclonal antibody in a solid phase enzymeimmunoassay. Clin. Chim. Acta (1983) 130:199-209.

55. lino, S., Abek, K., Oda, T., Suzuki, H. and Sugiura, M. A new methodof radioimmunoassay for human placental alkaline phosphatase. Clin.Chim. Acta (1972) 42:161-165.

56. Doellgast, G. J. Immunoquantitation of human placental alkalinephosphatase using radial immunodiffusion. Anal. Biochem. (1977) 82:278-288.

57. Goldstein, D. J., Rogers, C. and Harris, H. A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus. Clin. Chim. Acta (1982) 125:63-75.

58. Chang, C. H., Angellis, D. and Fishman, W. H. Presence of therare D-variant heat-stable, placental-type alkaline phosphatasein normal human testis. Cancer Res. (1980) 40:1506-1510.

59. Millan, J. L., Eriksson, A. and Stigbrand, T. A possible newlocus of alkaline phosphatase expressed in human testis. Hum. Genet.(1982) 62:293-295.

60. Goldstein, D. J., Blaco, L. and Harris, H. Placental alkalinephosphatase in n o n m a l ignant human cervix. Proc. Natl. Acad. Sci.USA (1980) 77:4226-4229.

61. Nozawa, S., Ohta, H., Izumi, S. Hayashi, S. Tsutsni, F., Kurihara, S. and Watanabe, K. Heat-stable alkaline phosphatase in the normal femal genital organ -with special reference to the histochemical heat-stability test and L-phenylalanine inhibition test. Acta Histochem. Cytochem. (1980) 13:521-530.

62. Maslow, W. C., Muensch, H. A., Azama, F. and Schneider, A. S.Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and non smokers. Clin.Chem. (1983) 29:260-263.

63. Wahren, B., Holmgren, P. A. and Stigbrand, T. Placental alkaline phosphatase, alphafetoprotein and carcinoembryonic antigen in testicular tumors. Tissue typing by means of cytologic smears.Int. J. Cancer (1979) 24:749-753.

64. Lange, P. H., Millän, J. L., Stigbrand, T., Vesella, R. L., Ruoslahti,E. and Fishman, W. H. Placental alkaline phosphatase as a tumor marker for seminoma. Cancer Res. (1982) 42:3244-3247.

Page 36: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

34

65. Cadeau, B. J., Blackstein, M. E., Malkin, A. Increased incidenceof placenta-like alkaline phosphatase activity in breast and g e n ito­urinary cancer. Cancer Res. (1974) 34:729-732.

66. Benham, F. J., Povey, M. S. and Harris, H. Placental-like alkaline phosphatase in malignant and benign ovarian tumors. Clin. Chim. Acta (1978) 86:201-215.

67. Doellgast, G. J. and Homesley, H. D. Placental-type alkaline phosphatase in ovarian cancer fluids and tissues. Obstet.G y n e c o l .(1984) 63:324-329.

68. Rasmuson, T., Björk, G. R., Damber, L., Holm, S. E., Jacobsson, L.,Jeppsson, A., Littbrand, B., Stigbrand, T. and Westman, G.An evaluation of carcinoembryonic antigen, tissue polypeptide antigen, placental alkaline phosphatase and modified nucleosides as biological markers in malignant lymphomas. J. Cancer Res. Clin. Oncol. (1983) 84:331-343.

69. Rasmuson, T., Björk, G. R., Damber, L., Holm, S. E., Jacobsson, L., Jeppsson, A., Stigbrand, T. and Westman, G. Tumor markers in b r o n c h o ­genic carcinoma. An evaluation of carcinoembryonic antigen, tissue polypeptide antigen, placental alkaline phosphatase and pseudouridine. Acta Radiol. Oncol. (1983) 22:209-214.

70. Rasmuson, T., Björk, G. R., Damber, L., Holm, S. E., Jacobsson, L.,Jeppsson, A., Stigbrand, T. and Westman, G. Tumor markers in c o l o ­rectal carcinoma. An evaluation of carcinoembryonic antigen, tissue polypeptide antigen, placental alkaline phosphatase and pse u d o ­uridine. Acta Radiol. Oncol. (1984) 23:27-32.

71. Waldmann, T. A. and Mc Intire, K. R. The use of sensitive assays for alphafetoprotein in monitoring the treatment of malignancy. In Immunodiagnosis of Cancer, Eds.: R. B. Herberman, K. R., Me Intire, Marcel Dekker Inc. 1979.

72. Stigbrand T. In Human Alkaline Phosphatases Eds Stigbrand T andFishman H. Alan R. Liss Inc. New York 1984.

73. Me Laughlin, P. J., Travers, P. J., Me Dicken, I. W. and Johnson, P. M.Demonstration of placental and placental-1 ike alkaline phosphatase using monoclonal antibodies in an enzyme immunoassay. Clin. Chim.Acta (1984) 137:341-348.

74. Kell an, J. A., Bush, R. S. and Malkin, A. Placenta-like alkalinephosphatase in gynecological cancers. Cancer Res. (1976) 36:269-271.

75. Lange, P. Testicular cancer markers. In Cancer Markers. Eds. Sell, S.and Wahren, B. Clifton, New York: Humana Press (1982)

76. Mostofi, F. K. and Price, E. B. Jr. Testicular tumors of the malegenital system. In Atlas of Tumor Pathology. Second Series. Fascile 8. Pp. 1-84. Washington Armed Forces Institute of Pathology.

Page 37: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

35

77. Holmgren, P. Å., Stigbrand, T., Damber, M.-G., von Schoultz, B. and Wahren, B. Determination of placental alkaline phosphatase- Regan isoenzyme in cancer sera by a sensitive radioimmunoassay. Scand. J. Immunol. (1978) (Suppl. 8) 8:515-518.

78. Nathanson, L. and Fishman, W. H. New observations on the Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer (1971) 27:1388-1397.

79. Wei, S. C. and Doellgast, G. J. Specificity of allozyme-absorbed antisera to human placental alkaline phosphatase for individual phenotypes. Biochem. G e n e t . (1980) 18:1097-1107.

80. Harris, H. The principles of human biochemical genetics. Third revised edition. Elsevier/North-Holland, Biomedical Press. The Netherlands (1980).

81. Slaughter, C. A., Coseo, M. C., Cancro, M. P. and Harris, H.Detection of enzyme polymorphism by using monoclonal antibodies.Proc. Natl. Acad. Sci. USA (1981) 78:1124-1128.

82. Kohler, G. and Milstein, C. Continuous cultures of fused cells secreting antibody of predifined specificity. Nature (1975) 256:495-497.

83. Millan, J. L. and Stigbrand, T. Antigenic determinants of human placental and testicular placental-like alkaline phosphatasesas mapped by monoclonal antibodies. Eur. J. Biochem. (1983)136:1-7.

84. Wahren, B., Jeppsson, A. and Millan, J. L. Typing of serumsamples from testicular tumor patients with five monoclonal antibodies (Manuscript in p r e p a ra t ion ).

85. Doellgast, G. J. and Fishman, W. H. Inhibition of human placental- -type alkaline phosphatase variants by peptides containing L- -leucine. Clin. Chim. Acta (1977) 75:449-454.

86. Mullivor, R. A., Plotkin, L.I. and Harris, H. Differentialinhibition of the products of the human alkaline phosphataseloci. Ann. Human Genet. (1978) 42:1-13.

87. Erlish, P. In Collected studies in immunity, pp. 441-442. NewYork, John Wiley and Sons, 1906.

88. Pressman, D. andKorngold, L. The in vivo localization of anti- -Wagner-osteogenic-sarcoma antibodies. Cancer (1953) 6:619-623.

89. Mach, J. P., Carrel, S., Merenda, C., Sordat, B. and Cerottini, J. C.In vivo localization of radiolabelled antibodies to carcino-embryonic antigen in human colon carcinoma grafted into nudemice. Nature (1974) 248:704-706.

Page 38: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

36

90. Goldenberg, D. M. and Hansen, H. J. Carcinoembryonic antigen present in human colonic neoplasins serially propagated in hamsters. Science (1972) 175: 1117-1118.

91. Epenetos, A. A., Britton, K. E., Mather et al. Targeting of iodine-123 labelled tumour associated monoclonal antibodiesto ovarian, breast and gastrointestinal tumours. Lancet (1982) ii: 999-1006.

92. Goldenberg, D. M., Kim, E. E., De Land, F. H., Bennet, S. and Primus, F. J. Radioimmunodetection of cancer with rad io­active antibodies to carcinoembryonic antigen. Cancer Res.(1980) 40: 2984-2992.

93. Mach, J. P., Carrel, S., Form, M., Ritschard, J., Donath, A. and Alberto, P. Tumor localisation of radiolabeled antibodies against carcinoembryonic antigen in patients with carcinoma.New England J. of Med. (1980) 303:5-10.

94. Ott, R. J. et al. The limitations of the dual radionuclide subtraction technique for the external detection of tumours by radioiodine-labelled antibodies. Brit. J. Radiology (1983) 56:101-108.

95. Fairweather, D. S., B r a d w e l l , A. R., Dykes, P. W., Vaughan, A. T.,Watson-James, S. F., Chandler, S. Improved tumour localisationusing indium-111 labelled antibodies. British Medical Journal(1983) 287:167-170.

96. Rainsbury, R. and Westwood, J. Tumour localisation with monoclonalantibody radioactivity labelled with metal chelate rather thaniodine. Lancet (1982) Dec. 11, 1347-1348.

97. Haskell, C. M., Buchegger, F. Schreyer, M., Carrel , S. and Mach,J. P. Monoclonal antibodies to carcinoembryonic antigen: ionic strength as a factor in the selection of antibodies for immuno- scintigraphy. Cancer Res. (1983) 43: 3857-3864.

98. Mach, J. P., Forni, M., Ritschard, J., Buchegger, F., Carrel , S.,Widgren, S., Donath A. and Alberto, P. Use and limitations ofradiolabeled anti-CEA antibodies and their fragments for p h o t o ­scanning detection of human colorectal carcinomas. O n c o d e v e l . b i o l . Med. (1980) 1:49-69.

99. Goldenberg, D. M., De Land, F. H., Kim, E., Bennet, S., Primus,F. J., van Nagel 1, Jr. J., Estes, N., de Simone, P. and Rayburn, P.Use of radiolabelled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning. N. Engl. J. Med. (1978) 298:1384-1388.

100. Mach, J. P. et al. Tumor localization in patients by radiolabeled monoclonal antibodies against colon carcinoma. Cancer Res. (1983) 43:5593-5600.

Page 39: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

37

101. Ecklman, W. C., Park, C. H. and Reba, R. C. Radiolabell ing of antibodies. Cancer Res. (1980) 40:3036-3042.

102. Uchida, T., Shimoda, T., Miyata, H., Shikata, T. lino, S.,Suzuki, H., Oda, T., Hirano, K. and Sugiura, M. Immuno­peroxidase study of alkaline phosphatase in testicular tumor. Cancer (1981) 48:1455-1462.

103. Hedin, A., Wahren, B. and Hammarström, S. Tumor localization of CEA-containing human tumors in nude mice by means of m o n o ­clonal anti-CEA antibodies. Int. J. Cancer (1982) 30:547-552.

104. Manning, J. P., Inglis, N. R., Green, S. and Fishman, W. H. Characterization of placental alkaline phosphatase from the rabbit, guinea pig, mouse and hamster. Enzvmologia (1970). 39:307-318.

105. Goldstein, D. J. and Harris, H. Human placental alkaline p ho s­phatase differs from that of other species. Nature (1979) 280:602-605.

106. Doellgast, G. J. and Benirshke, K. Placental alkaline phosphatasein hominidae. Nature (1979) 280:601-602.

107. Fishman, W. H., Inglis, N. R., Stolbach, L. L. and Krant, M. J.A serum alkaline phosphatase isoenzyme of human neoplastic cell origin. Cancer Res. (1968) 28:150-154.

108. Scardino, P. T., Cox, D., Waldmann, T. A., Mclntire, K. R.,Mittemeyer, B. and Javadpour, N. The value of serum tumor markers in the staging and prognosis of germ cell tumors of thetestis. J. Urol. (1977) 118:994-999.

Page 40: THE SIGNIFICANCE OF PLACENTAL AND PLACENTAL-LIKE …umu.diva-portal.org/smash/get/diva2:923311/FULLTEXT01.pdf · the significance of placental and placental-like alkaline phosphatases

38