Squamous Cell Carcinoma Antigen: more sensitive second ...

C ancer biomarkers
as published in CLI December 2004
Squamous Cell Carcinoma Antigen:
more sensitive second generation assays
By Dr. R. Einarsson
Squamous cell carcinoma antigen (SCCA) is a serological marker for squamous cell carcinomas
(SCC) of the uterine cervix, lung, head and neck and oesophagus. The antigen
is a group of cytoplasmic proteins found in normal squamous epithelia. The measurement
of the serum levels of SCCA in patients with cancer of the cervix of the uterus has
been reported to be useful for distinguishing between patients with and without a high
risk of developing lymph node metastases. In addition, SCCA has been shown to correlate
with the clinical course of the disease following treatment and to predict relapse
[1-4]. Deep stromal infiltration and positive lymph nodes significantly were associated
with increased SCCA level in many patients. However, about 30% to 50% of patients
with cervical carcinoma have been shown to have normal SCCA concentration. In particular,
more than 50% of patients in clinical FIGO stage I and II had normal SCCA levels.
Thus, the determination of SCCA alone is not suitable for clinical purposes. For this
reason several attempts have been made to try to overcome the limitation of SCCA on
its own by the concomitant use of additional tumour markers.
SCCA was first isolated by conventional protein purification methods from a cervical
squamous cell carcinoma. Biochemical characterisation of the original protein fraction
(TA-4), showed that it comprised a group of proteins with a molecular weight of
approximately 45 kDa [1]. This protein fraction was then used to raise polyclonal antibodies
for further characterisation and for the development of assays for serological
studies [1]. The TA-4 fraction could be further separated in two subfractions by isoelectric
focusing (acidic fraction pI6.25). Each of these subfractions
were shown to consist of at least ten different protein components [1].
Current assays of SCCA
The clinical utility of serum SCCA assays has been best documented when they were
being applied to the prognosis, monitoring therapy and follow-up of cervical cancer
patients. It has been suggested that in the tumour there might be differences in the
expression of the acidic and neutral protein fractions. Studies supported the hypothesis
that the acidic SCCA fraction was more associated with cancer than the neutral fraction.
Most clinical data on the utility of SCCA assays have been generated in studies
using radioimmunoassay or fluorescence polarisation immunoassay in Abbott’s IMx
SCC assay. Both SCCA1 and SCCA2 have been measured in these systems [1]. Serum
SCC antigen levels correlate with clinical stage and treatment outcome in various carcinomas.
High pre-treatment levels indicate extensive disease and poor prognosis for
patients with cervical cancer of the squamous cell carcinoma histotype, but not for the
adenocarcinomas. A good correlation has been reported between serum SCC antigen
levels and the extent of disease [3]. Most SCCA data have been reported for cervical cancer
patients, but due to low sensitivity and specificity SCCA has limited clinical utility in
early-stage disease. Similar results have also been reported for patients with lung cancer
of histological type squamous cell carcinoma and for patients with squamous cell carcinoma
of the head and neck. New methods have been developed to measure serum
SCCA1 or SCCA2 separately and by measuring recombinant SCCA preparations.
However it has been shown that recombinant SCCA2 is underestimated using the IMx
SCC assay [6]. In serum samples from cervical cancer patients, the SCC antigen consists
mainly of the SCCA2 isomer and this might be one of the reason for the limited sensitivity
of the available SCCA assays.
Molecular diagnostics
Molecular cloning has demonstrated that SCCA is produced by two almost identical
genes named SCCA1 and SCCA2 [7]. The two SCC genes are tandemly arranged
[Figure 1] and located on chromosome band 18q21.3. The gene products have been
characterised as serine and cysteine protease inhibitors [6,7]. There is a high degree of
homology (98% at the nucleotide level and 92% at the amino acid level) between the
encoded proteins SCCA1 and SCCA2. SCCA1 corresponds to the neutral protein fraction
and SCCA2 to the acidic protein fraction and both belong to the family of serine
proteinase inhibitors (serpins). The specificity of SCCA1 and SCCA2 is due to small
differences in the amino acid composition (4-6 amino acid residues) in the reactive site
loop located on exon 8 [9]. SCCA1 is an inhibitor of papain-like cysteine proteases, such
Figure 1. A. Serpin cluster on chromosome 18q21.3 B. Mechanism for the formation of SCCA1/A2
and SCCA2/A1 fusion genes by homologous crossover. Adapted from Röijer, et al (8).
as cathepsin K, L and S, whereas SCCA2 inhibits chymotrypsin-like serine proteases,
cathepsin G and mast cell chymase [9]. SCCA1 and SCCA2 may exist in free form and
also in complexes with serine and cysteine proteases. The use of immunohistochemistry
and RT-PCR has shown that SCCA1 and SCCA2 can be found in normal tissues as well
as well in carcinomas [10]. Studies of the mRNA levels of SCCA1 and SCCA2 have indicated
that the relative expression of SCCA2 was definitely higher in cancer cell lines, in
cervical cancer tissues and in head and neck cancer as compared to SCCA1.
Monoclonal antibodies specific for SCCA1 and SCCA2 antigens
The discovery of two distinct SCCA related serine proteinase inhibitors and the hypothetical
differences in cancer specificity of SCCA1 and SCCA2 underscores the need for
specific assays that can discriminate between SCCA1 and SCCA2 and further recognise
all serological forms of SCCA with similar sensitivity. Monoclonal antibodies specific
for different forms of SCC antigens were produced and MAbs purified by Protein A
affinity chromatography. The reactivity of the monoclonal antibodies and their epitope
specificities were established by determination of the reactivity with free and complexed
SCC antigens, reduced SCC antigens and different fusion transcripts positioned
between exon 2-7 and exon 8 of SCCA1 and SCCA2. Determination of dose-response
curves for different sandwich immunoassays was the final confirmation of the assay
specificity. Despite the high degree of sequence homology of the gene products SCCA1
Table 1. Summary of CanAg SCCA monoclonal antibodies. Group A1a, A2 and A3a react preferentially
with total¨ SCC; Group A1b and A3b react preferentially with "Free" SCC;Group B1 and
B2 recognise Total SCCA1; Group C1a and C2a recognise Total SCCA2; Group C1b and C2b recognise
only Free SCCA2.
and SCCA2, it has been possible to establish monoclonal antibodies specific for SCCA1
and SCCA2 (differences in the reactive site loop of each inhibitor). Table 1 is a summary
of SCCA monoclonal antibody reactivity (different epitope groups) and by combining
these reagents sensitive and discriminatory immunoassays could be developed.
Selection of monoclonal antibodies for specific immunoassays
The established monoclonal antibodies [Table 1] made it possible to design new sensitive
and specific immunoassays for measuring different serological forms of SCCA.
However, only by the application of such assays to clinical samples can it be proven if
any of these MAb combinations have a greater clinical utility than the present assay.
A. Total SCC antigen assay
Two antibodies both recognising SCCA1 and SCCA2 are necessary for a sandwich assay

C ancer biomarkers
as published in CLI December 2004
measuring total or Pan SCC. Optimal reactivity in the ELISA assay for measuring total
SCC was obtained with the antibody pair SCC140 (catcher antibody) and SCC107
(detector antibody).
B. Total SCCA1 assay
Combination of monoclonal antibodies from Group B and monoclonal antibodies
from Group A permitted the design of specific immunoassays for SCCA1 antigen. A
monoclonal antibody characterised by high sensitivity for SCCA1 antigen and low
cross-reactivity with SCCA2 antigen was selected. Optimal reactivity in the immunoassay
for measuring total SCCA1 was obtained with the antibody pair SCC111 (catcher
antibody) and SCC107 (detector antibody).
9 Luke C et al. Biochemistry 2000; 39: 7081.
10. Cataltepe S et al. J Histochem Cytochem 2000; 48: 113.
The author
R. Einarsson, Ph. D.,
Biotech Division,
CanAg Diagnostics AB
SE 41455 Gothenburg
Sweden
Fax + 46 31 85 70 40
E mail roland.einarsson@canag.se
C. Total SCCA2 assay
Different antibodies from Group A and Group C [Table 1] can be selected for measuring
total SCCA2 antigen. Three monoclonal antibodies recognising free SCCA2 and
two monoclonal antibodies recognising total SCCA2 antigen were established. Optimal
reactivity for measuring total SCCA2 antigen (Free SCCA2 and SCCA2 in complex
with proteases) was obtained with the antibody pair SCC103 (catcher antibody) and
SCC107 (detector antibody).
D. Free SCCA2 assay
Three antibodies from Group C (SCC104, SCC 109 and SCC161) were found to be specific
for free SCCA2 antigen. All antibodies demonstrated low cross-reactivity with
SCCA1 antigen. The preferred configuration for measuring free SCCA2 antigen was
based upon the pair SCC 104 (catcher antibody) and SCC107 (detector antibody).
Clinical evidence for the role of CanAg SCCA specific assays
SCCA is the most useful serological marker for cervical cancer and has demonstrated
its clinical value for therapy monitoring and for establishing prognosis. However, this
biomarker is not sensitive enough for the early diagnosis of cervical cancer. Using the
newly developed ELISA assay for measuring the different serological forms of SCCA, it
was shown that SCCA2 was the dominating serological form of SCCA in healthy subjects
and in most cervical cancer patients (FIGO stage II-IV) [5]. However, both SCCA1
and SCCA2 followed the clinical course of the disease, and could be used for monitoring
treatment outcome in cervical cancer patients and also to predict progressive disease.
In most patients SCCA2 antigen showed the most pronounced elevation of the
SCCA antigens in clinically confirmed progressive disease. In some patients however the
SCCA1 antigen was the earliest predictor of progressive disease. Optimal clinical sensitivity
and specificity would be obtained using an assay measuring all serological forms
of SCCA.
Future Prospects for SCCA1 and SCCA2
The SCC antigen is an established biomarker for patient management in cervical cancer,
lung cancer and head and neck cancer patients. Levels of the antigen can provide
information on prognosis, treatment outcome and the prediction of recurrence.
However there is a great need for cancer markers that will further improve these aspects.
In this respect, the second generation SCCA assays such as those described above may
be useful for the early detection of disease and prediction of progressive disease. Studies
with SCCA1 and SCCA2 have shown that for optimal clinical sensitivity, an assay recognising
both SCCA1 and SCCA2 with similar sensitivity is to be preferred. The specific
determination of SCCA1 and SCCA2 antigen levels may provide additional clinical
information compared to the determination of Total SCC. In conclusion, it might be
possible to increase the sensitivity and specificity of the measurement of SCCA in early
cancer diseases such cervical, lung and head and neck cancer by applying the new second
generation monoclonal antibodies. The new antibodies will also be useful to characterise
the native proteases associated with SCCA1 and SCCA2 in squamous cell carcinoma
and normal tissues.
References
1. Kato H in Serological Cancer Markers (S. Sell ed.), Totowa, Humana Press, pp. 437-
451 (1992).
2. Snyderman CH et al. Arch Otalaryngol Head Neck Surg 1995; 121: 1294.
3. de Bruijn HWA et al. Tumour Biol 1998; 19: 505.
4.Takeda A et al. Biol Chem 2002; 383: 1231.
5. de Bruijn HWA et al. Tumour Biol 2003; 24: 83.
6.Cataltepe S et al. Clin Chim Acta 2000; 295: 107.
7.Suminami Y et al. Tumour Biol 1998; 19: 488.
8. Röijer E et al. Tumour Biol 2003; 24: 46.