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Autoantibody biomarkers for early detection and prognosis of gastric cancer

Karina Silina1*, Pavel Zayakin1, Guntis Ancans2, Zane Kalnina1, Irena Meistere1, Angelina Pismennaya1, Marcis Leja2 and Aija Line1
  • 1 Latvian Biomedical Research and Study Centre, Latvia
  • 2 Riga Eastern Clinical University hospital, Latvian Oncology Center, Latvia

Gastric cancer (GC) is one of the most common reasons of cancer-related death worldwide. The high mortality rate is due to the lack of symptoms in early stages hence over 80% of GC cases are diagnosed at stage IIIA-IV, when the treatment efficiency is poor and the estimated 5-year survival rate ranges from 4 to 20% (1). Currently, diagnosis is based on endoscopic examination followed by the histological analysis of gastric biopsy, which is a highly labour-intensive and invasive technique. To increase the proportion of cases diagnosed at early stages and improve the management of this disease, it is necessary to develop novel methods that would be suitable for regular screening of asymptomatic patients.
Cancer autoantibodies (AAb) are attractive biomarkers for early diagnosis of cancer as they are specific and stable molecules in an easily accessible blood sample that are formed early in disease progression even several years before clinical manifestation of cancer (2). However several drawbacks have hampered the elaboration of autoantibody-based tests for cancer diagnosis: (i) antigen repertoires differ markedly among tumours, (ii) autoantibody repertoires in patients are heterogeneous - the frequency of antibodies against any individual cancer antigen is generally low ranging from 1 to ~15%, and (iii) autoantibody repertoires to some extent resemble the response to tissue damage by viral infections or autoimmune diseases (3, 4). This could be overcome by identifying a comprehensive repertoire of cancer-associated antigens and selecting the most specific and sensitive biomarkers by screening large cohorts of cancer and inflammatory disease patients and healthy donors using multiplex autoantibody detection assays. Based on this principle, recently a multiplex AAb-based test for lung cancer detection “EarlyCDT®-Lung” (5) has been released in market and is used in a population screening trial in Scotland.
By using the T7 phage display based SEREX approach we have identified >1000 B cell antigens in gastric, breast, prostate cancer and melanoma patients and used them to generate custom antigen microarrays (6). Initially, screening of sera from 100 GC patients and 100 cancer-free controls (HD) revealed that 360 antigens were GC-associated. Next, 86 most specific antigens in GC were selected for the production of a focused validation array. Among these there were known cancer-testis (CT) antigens such as CTAG2, DDX53, MAGEC1, as well as previously uncharacterized antigens and a number of novel peptides (patent pending). This array was screened with an independent set of sera from 235 GC, 154 peptic ulcer and atrophic gastritis patients and 213 HD. A rank value was assigned to each antigen according to its frequency of reactivity and signal intensity in GC and HD. A “serum score” for each serum was calculated by summing up the reactive AAb signals that were adjusted by the corresponding antigen rank value. The calculated serum score value was used as the output of the AAb test (7).
By using the backward elimination approach a minimal set of 45 antigens was selected that showed very similar diagnostic performance as the whole 86 antigen set. The Receiver Operating Characteristic curve analysis showed that the 45 AAb serum score could discriminate GC from HD with a 90% specificity and 59% sensitivity (Area Under the Curve (AUC) 0.79, p=2e-31), thereto early stage GC was detected with the same precision as advanced GC. The AUC was similar for the discrimination of GC from gastric ulcer, but the precision for the discrimination between GC and gastritis patients was lower (AUC 0.64, p=5e-5) (7). Atrophic gastritis is a chronic inflammatory condition that has been demonstrated as a premalignant lesion for gastric cancer (8) and thus this cohort might represent a heterogeneous group of patients that potentially contains early undiagnosed cancer cases. The follow-up information could be obtained for one gastric ulcer and two gastritis patients that showed reactivity to known CT antigens, and strikingly it showed that all of these patients were diagnosed with cancer up to five months after the serum sample had been collected (Table 1). Hence it is tempting to speculate that the low precision of discrimination between these two cohorts, at least in the case of CT antigen reactivity, is due to the presence of nascent cancer cells in these patients that are detected by cancer-specific AAbs, and not due to gastric inflammation-associated AAbs among the set of 45 AAbs. Further research is underway to clarify this question.
Next we looked whether AAbs could also be useful as prognostic biomarkers applicable in the management of late stage patients. A preliminary prognostic analysis of serum scores was performed on 33 stage III and IV patients and showed that the 45 AAb signature has a significant correlation to shorter survival (<1 year) after surgery (Figure 1A). Further analysis was done to define the minimal set of short survival-associated AAbs as well as to see whether there might be AAbs associated to longer survival which are masked in this large set of 45AAbs. Indeed, two distinct signatures of seven AAbs showed a significant association to shorter (Figure 1B) and longer (>1 year) (Figure 1C) survival after surgery. These AAbs were also determined as the most significant independent prognostic factors for these patients by the multivariate Cox regression analysis (Table 2). It is currently unknown what is the function of the corresponding antigens. The reason of the prognostic significance of the detected AAb response is also elusive, but two options exist: (i) the survival outcome is underlined by the molecular alterations of the corresponding antigens that generate untolerised epitopes and thus elicit the detected AAbs. which then act as mere indications of these molecular changes, or (ii) the formation of AAbs against these antigens itself can have a functional significance in the tumour microenvironment affecting the immunological processes and influencing survival. In the latter case, these AAbs could serve as indicators for the existence of a protecting or tumour-promoting immunological context in the patients tumour and could be useful for therapeutic decisions.
We show here that a signature of 45 cancer AAbs can be used for early diagnosis of GC. Further work is in progress to increase the sensitivity of the test, to validate its diagnostic performance in geographically distinct populations, to characterise its predictive potential in risk population of atrophic gastritis patients, and to validate the prognostic value of AAbs in late stage patients.

Figure legend
Figure 1. Kaplan-Meier survival curve on 33 stage III and IV GC patients for (A) 45 AAb signature, (B) 7 autoantibody signature associated with shorter survival and (C) 7 autoantibody signature associated with longer survival. Green line indicates patients with serum score value 0 (lack of autoantibodies), blue line indicates autoantibody-positive patients with serum score >0. Numbers by the colour code indicate how many patients fall in each group. Dashed lines indicate CI of 95%. X axis represents survival after surgery in days.

Tables
Table 1. Follow-up information on three gastric inflammatory disease patients showing sero-reactivity to CT antigens.
Table 2. Multivariate Cox regression analysis for serum scores calculated from the sets of 7 shorter and 7 longer survival-associated autoantibodies.

Figure 1
Image

Acknowledgements

This work has been financed by the ERDF project number 2010/0231/2DP/2.1.1.1.0/10/APIA/VIAA/044.
The authors thank Dr. Thomas Wex and Prof. Peter Malfertheiner from Otto-von-Guericke University Magdeburg, Clinic of Gastroenterology, Hepatology and Infectious Diseases for clinical samples.

References

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Keywords: gastric cancer diagnosis, Cancer antigens, cancer autoantibodies, autoantibody biomarkers, serological screening, gastric cancer prognosis

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Adaptive Immunity

Citation: Silina K, Zayakin P, Ancans G, Kalnina Z, Meistere I, Pismennaya A, Leja M and Line A (2013). Autoantibody biomarkers for early detection and prognosis of gastric cancer. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01082

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Received: 30 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Dr. Karina Silina, Latvian Biomedical Research and Study Centre, Riga, Riga, LV1067, Latvia, karina@biomed.lu.lv