Humoral immune responses of lung cancer patients against the Transmembrane Phosphatase with TEnsin homology (TPTE)

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Humoral immune responses of lung cancer patients against the Transmembrane Phosphatase with TEnsin homology (TPTE) Andreas Kuemmel a,∗,1 , Petra Simon a,b,1,2 , Andrea Breitkreuz a,b , Julia Röhlig a,2 , Ulrich Luxemburger a,b , Amelie Elsäßer c , Lars Henning Schmidt d , Martin Sebastian e , Ugur Sahin a,b , Özlem Türeci f , Roland Buhl a a

Department of Hematology, Medical Oncology & Pneumology, University Medical Center Mainz, 55131 Mainz, Germany TRON gGmbH, Translational Oncology at the University Medical Center, Johannes Gutenberg-University Mainz, 55131 Mainz, Germany c Institute of Medical Biostatistics, Epidemiology and Informatics, Johannes Gutenberg-University, 55101 Mainz, Germany d Department of Medicine A, University Medical Center Muenster, 48149 Muenster, Germany e Department of Medicine III, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany f Ganymed Pharmaceuticals AG, Freiligrathstr.12, 55131 Mainz, Germany b

a r t i c l e

i n f o

Article history: Received 2 March 2015 Received in revised form 19 July 2015 Accepted 25 July 2015 Keywords: TPTE NSCLC Serology Cancer/testis antigen Prognosis NY-ESO 1

a b s t r a c t Objective: The cancer/testis (C/T) antigen Transmembrane Phosphatase with TEnsin homology (TPTE) is aberrantly expressed in many tumors including lung cancer. In the present study, we analyzed TPTEauto-antibodies in lung cancer patients. Methods: Using a crude-lysate ELISA, we analyzed a large cohort of 307 sera from lung cancer patients and 47 healthy donors for TPTE-specific autoantibodies. Sero-reactivity was correlated with clinical parameters and patients’ survival. Results: TPTE-specific antibodies were detected in 41 of 307 (13.4%) sera from lung cancer patients. Based on an optimal cut-off value calculated by ROC curve analysis sensitivity for diagnosing lung cancer was 52% and specificity was 72%. TPTE sero-positivity was not associated with tumor stage, tumor histology, gender or age. Multivariate analysis indicated that TPTE sero-positivity is associated with prolonged survival in patients with lung cancer, but established prognostic factors for survival prediction such as stage and histology remain indispensable. Conclusion: Autoantibodies against TPTE occur spontaneously in lung cancer patients. TPTE sero-reactivity has moderate sensitivity and specificity for diagnosing lung cancer and is a positive prognostic marker. © 2015 Published by Elsevier Ireland Ltd.

1. Introduction TPTE (Transmembrane Phosphatase with TEnsin homology) is a germ cell-specific protein that is aberrantly transcribed in human cancers of the liver, the prostate and the lung [1,2]. Copies of the TPTE gene are located on chromosomes 13, 15, 21, 22, and Y, but only the copy on chromosome 21 seems to be expressed [3]. TPTE shares sequence homology with the ubiquitously expressed tumor suppressor PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) [4,5], but differs by an N-terminal extension com-

∗ Corresponding author at: Department of Hematology, Medical Oncology & Pneumology, University Medical Center Mainz, Langenbeck Strasse 1, 55131 Mainz, Germany. Fax: +49 6131 17 6628. E-mail address: [email protected] (A. Kuemmel). 1 Both authors contributed equally to this study. 2 Parts of this study are part of the author’s doctoral thesis.

prising three transmembrane regions. In contrast to PTEN, TPTE lacks phosphatase activity [4] and its biological function is unclear. TPTE is absent in healthy tissues [1,5] except for a stable expression in testis and therefore belongs to the group of cancer/testis (C/T) antigens such as more common antigens like NY-ESO-1 and MAGE-A3 which have been extensively studied in preclinical and clinical settings, particularly in lung cancer [6,7]. Many C/T antigens elicit spontaneous humoral and cellular immune responses in cancer patients [8]. Regarding TPTE there is only one study in HCC so far. In that study, TPTE was expressed in HCC-tissue in 24 out of 62 patients (38%) and TPTE- autoantibodies were found in the sera of 6 out of the 24 patients (25%) with TPTE positive tumors but never in the sera of patients without TPTE-expression in cancer tissue. [1]. Spontaneous autoantibody responses against a tumor antigen indicate strong immunogenicity as well as existence of a cognate CD4+ T cell response, which makes such antigens particularly

http://dx.doi.org/10.1016/j.lungcan.2015.07.012 0169-5002/© 2015 Published by Elsevier Ireland Ltd.

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interesting target candidates for immunotherapy [9]. Furthermore, autoantibody responses may be used as biomarkers for lung cancer screening or as prognostic factors for the disease or predictors for response to treatment [10–12]. Currently a lung cancer screening test combining multiple ELISAs for auto-antibodies against other C/T antigens like NY-ESO-1 und MAGE A4 has passed clinical trials [13]. In this study, we analyzed the TPTE autoantibody response in lung cancer patients by a crude lysate ELISA (CrELISA) [14] and its correlation with survival. 2. Methods 2.1. Study population We analyzed serum specimens of patients admitted to the Pulmonary Division of Gutenberg-University Medical Center (Mainz, Germany). The study was performed compliant to the rules and regulations of the state ethics committee, all subjects gave written consent. Medical records were reviewed for clinical data including histology, tumor markers (i.e., LDH, carcinoembryonic antigen, neuron-specific enolase and cytokeratin fragment 19), smoking history, clinical staging (according to IUCC/AJCC recommendations including clinical examination, CT scans, sonography, endoscopy, MRI, bone scan) and pathological staging if patients had surgery. As patients were diagnosed between 1998 and 2007, staging was based on UICC 6th TNM Edition. The study population included patients of stages IA to IV, both NSCLC and SCLC and various treatments including surgery, chemotherapy, radiotherapy, treatment with tyrosine kinase inhibitors, monoclonal antibodies or multimodal therapy. 45 (15%) patients had SCLC, and 22% of them had limited disease. Of the 260 (85%) patients with NSCLC, 8% were defined as stage I, 4% as stage II and 32% as stage III and 57% as stage IV. Out of patients with NSCLC, 134 (52%) had adenocarcinoma, 88 (34%) had squamous cell carcinoma and 6 (2%) had adenosquamous carcinoma. 8 (3%) had carcinoma with neuroendocrine differentiation and 8 (3%) were diagnosed with large cell carcinoma. 2 (1%) patients had carcinoid tumors and 14 (5%) had non-small cell lung cancer which could not be specified. 2 patients had carcinosarcoma and were neither specified NSCLC nor SCLC. 28 patients (9%) underwent surgical treatment of the primary pulmonary lesion only. 27 patients (9%) had neoadjuvant or adjuvant therapy including chemotherapy, radiotherapy or both. 160 patients (52%) received chemotherapy only and 8 (3%) had radiotherapy only. 43 patients (14%) were treated with combined radio-chemotherapy, 30 patients with TKIs or monoclonal antibodies or TKI/antibodies with chemotherapy (many patients in this group were treated in clinical trials). 11 (4%) patients received best supportive care. All patients had regular follow-up visits. Systematic restaging was performed after 3, 6, 12, 18, 24, 36, 48 etc. months or earlier if necessary. Restaging included clinical examination, chest X-ray, abdominal ultrasound scan and blood tests. CT scans were performed if progression was suspected. Survival time and progression-free survival time (PFS) were calculated from the date of histological diagnosis to death, progressive disease, or last contact with the patient, respectively. In the latter case the survival time was regarded as censored. 288 patients (84%) suffered from disease progression or recurrent disease, 2 had stable disease and 14 patients had complete remission or had no tumor recurrence after surgery. 2.2. TPTE-specific crude lysate ELISA (CrELISA) Crude lysates of bacteria expressing the recombinant TPTE protein were used as a source of the specific antigen. As TPTE is a

Table 1 Baseline characteristics of the study population. Parameter

Patients (n = 307)

%a

Age Sex Smoker or former smoker TPTE-Sero-positivity SCLC Carcinosarcoma NSCLC Adenocarcinoma and BAC Squamous cell carcinoma Adenosquamous carcinoma Large cell carcinoma Neuroendocrine differentiation Carcinoid NSCLC NOS Td T1 T2 T3 T4 Tx Nd N0 N1 N2 N3 Nx Md M0 M1 Mx Stage NSCLCd I II III IV Stage SCLCd Limited disease Extensive disease Therapy Surgery alone Neoadjuvant/adjuvant therapy Chemotherapy alone Radiotherapy alone Radio-chemotherapy TKI/antibody-therapy ± chemotherapy Best supportive care Survival time

62 ± 10 years 221 males 250 41 45 2 260 134 88 6 8 8 2 14

72 96 13 15 1 85 52c 34c 2c 3c 3c 1c 5c

31 92 37 136 11

10 30 12 44 4

56 39 82 101 29

18 13 27 33 9

114 180 13

37 59 4

20 11 83 148

8 4 32 57

10 35

22e 78e

28 27 160 8 43 30 11 736 ± 819 daysf

9 9 52 3 14 10 4

b

a

Percent of non-missing values. Mean ± standard deviation. Percentage of NSCLC patients. d Based on clinical staging, pathological staging was used if available, of note: 4 patients had neo-adjuvant therapy. e Percentage of SCLC patients. f Median ± standard error. b

c

homologue of the ubiquitously expressed PTEN, but contains an extended N-terminal domain having four potential transmembrane motifs, we used only the N-terminal part of TPTE to exclusively detect TPTE-specific antibodies. As expression of membrane proteins often is challenging we also excluded the transmembrane regions and focused on TPTE aa 1-51. Escherichia coli bacteria were transformed either with pBK-pBK-CMV-TPTE-1-51 or with an empty pBK-CMV vector (will be referred to as “reference”) and grown in LB medium to A595 nm of 0.4-0.6 E. Protein expression was induced with 1 mM IPTG, and cells were allowed to grow for an additional 4 h at 37 ◦ C. Proper induction of protein expression and its kinetics were monitored by SDS gel analysis. Bacteria were spun down and resuspended in a small volume of PBS pH 7.2 containing 0.2 mM protease inhibitor AEBSF-hydrochloride. Cells were placed on ice and disrupted by sonication. TPTE and reference lysates were diluted to a total protein concentration of 2 mg/ml in PBS containing

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Fig. 1. Sero-reactivity against TPTE in healthy donors and lung cancer patients. Sera from 47 healthy controls (A) and 307 patients suffering from lung cancer (B) were tested by CrELISA for the prevalence of TPTE autoantibodies. A positive result was defined as an absorbance value exceeding the 99th percentile value of 47 healthy donors (indicated as dash line).

0.2 mM AEBSF and 20% (v/v) glycerol. Aliquots were shock-frozen in nitrogen and stored at −80 ◦ C until use. Crude lysates of bacteria recombinantly expressing the TPTE protein were used as a source of the specific antigen. Before use, the bacterial crude lysate containing TPTE antigen, as well as the reference lysate, were diluted in coating buffer (500 mM NaH2PO4, pH 6.0), then transferred to flat-bottom F96 Maxisorp microwell plates (50 ␮l/well) and immobilized for 2.5 h at 37 ◦ C. Plates were washed three times with washing buffer (50 mM Tris, 150 mM sodium chloride, pH 7.2). Human sera were diluted 1 + 100 in preincubation buffer (50 mM

MES, 150 mM NaCl, 0.05 % Tween, 5% milk powder, 50% reference lysate, pH 6.2) and incubated for 1.5 h to block E. coli-specific antibodies. 50 ␮l of the preincubated serum was added per well and incubated for 2 h on an orbital shaker at ambient temperature. Each serum sample was tested in triplicates in parallel on wells coated with TPTE-1-51 and reference lysate. Plates were washed again as described above and incubated for 1 h at room temperature with 50 ␮l/well of secondary antibody (goat anti-human IgG-AP) diluted 1:5000 in 50 mM HEPES (pH 7.4) containing 3% (w/v) milk powder. Plates were developed with 100 ␮l/ well of substrate solu-

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Fig. 2. ROC-curve of TPTE aa 1-51 by CrELISA. The blue line indicates sensitivity and 1-secificity of the CrELISA for every possible cut off value. The dash line indicates random chance. The arrow shows the maximum value of Youden’s index at the cut off value 0.0305 (sensitivity 52.1% and specificity 72.3%, dotted lines).

tion (2 mg 4-nitrophenyl phosphate disodium salt hexahydrate per ml developing buffer) for 30 min at room temperature. Enzymatic reaction was stopped by adding 100 ␮l/well 200 mM EDTA (pH 8.0) and absorbance values were immediately read at 405 nm on a microplate reader.

2.3. Statistical analysis Standard descriptive statistics such as frequencies, mean, standard deviation, median and standard error were calculated to describe the study population. One of the principal aims of this study was to show the prognostic value of TPTE-serology on patients’ survival. Additionally, results of serologic testing were compared with clinical parameters such as sex, age, tumor markers, histology, TNM, stage, and response to therapy using Fisher’s exact test or Mann–Whitney U-test if appropriate. As TPTE testing was done at individual time points during the course of disease, we tried to rule out that the time between tumor diagnosis and TPTE-sero-reactivity testing confounds the results. Median time from tumor diagnosis to TPTE testing was 39 days (minimum 51 days before diagnosis, maximum 2265 days after diagnosis). There was no association of TPTE sero-positivity with time between diagnosis and serological testing (p = 0.47). To investigate the prognostic value of TPTE, we conducted several multivariable analyses using Cox proportional hazards regression. Cox regression model A included all patients. To analyze both SCLC and NSCLC patients TNM-Staging was included instead of stage grouping (Mountain [15] or Veterans’association [16], n = 307). Because of the different biology and clinical behavior of NSCLC and SCLC discrete models were analyzed for each entity (model B for NSCLC and model C for SCLC). The 2 patients with carcinosarcoma of the lung were not included in models B (n = 260)

and C (n = 45). Features considered as potential prognostic factors (reference category underlined) were: sex (male vs. female), age (as a continuous variable), stage (1 vs. 2 vs. 3 vs. 4 or limited vs. extensive disease), histology (NSCLC vs. SCLC vs. carcinosarcoma), NSCLC sub-type [17,18] (squamous cell carcinoma vs. other NSCLC, adenocarcinoma vs. other NSCLC, large cell carcinoma vs. other NSCLC), and TPTE-sero-reactivity (negative vs. positive). To analyze the prognostic value of the potential explanatory factors, a Cox proportional hazards model was applied using a forward stepwise selection (inclusion criteria: p value of the Score test ≤0.05, exclusion criterion: p value of the likelihood ratio test ≥0.1). Similarly, a Cox regression analysis with a stepwise backward selection (inclusion criteria: p value of the Score test ≤0.05, exclusion criterion: p value of the likelihood ratio test ≥0.1) was performed. If patients could not be included because of missing values, we repeated Cox regression analysis with the factors selected by the first and second Cox regression model using forward and backward selection models to analyze more cases. To visualize the results, univariate Kaplan–Meier charts were calculated for the factory selected by the Cox regression models. All analyses were performed using SPSS® 21 software (IBM) and were regarded as explorative, therefore, a global or local level of significance was not determined.

3. Results 3.1. Sero-reactivity against TPTE in healthy donors To establish a cut-off value for positive antibody reactivity against TPTE 47 sera from healthy donors were analyzed for serum concentrations of IgG auto-antibodies against TPTE aa 1-51 by CrELISA. Generally speaking, absorbances of sera were low and in

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Table 2 Overall survival: explanatory prognostic factors in a Cox proportional hazards model. Model Aa (n = 307)

FWDb

Agef TPTE sero-positivityg TNM T descriptor T1 vs- T2 T2 vs. T3 T3 vs. T4 T4 vs. Tx TNM M descriptor M0 vs. M1 M1 vs. Mx histology NSCLC vs. SCLC SCLC vs. carcinosarcoma Model Bh (n = 260)

HRc

95% CId

pe

1.01 0.68

1.00–1.03 0.47–0.99

0.046 0.035 0.0002

1.63 3.24 2.21 2.25 4.12 2.73 1.68 0.51 1.40 18.8

1.02–2.62 1.87–5.63 1.40–3.51 1.03–4.93 2.05–8.28 2.09–3.57 0.85–3.32 0.28–0.94 1.01–1.95 4.28–82.3

<0.0001

0.004

FWDb

i

Adenocarcinoma Large cell carcinomaj Stage I vs. II II vs. III III vs. IV Model Ck (n = 45)

HRc

95% CId

pe

0.69 0.3

0.52–0.90 0.12–0.74

0.007 0.002 <0.0001

2.1 3.02 7.34

0.9–5.12 1.62–5.63 3.95–13.6

FWDb HRc

95% CId

pe

l

No factor accepted a b c d e f g h i j k l

Including all patients, other factors not accepted by Cox regression model: sex, TNM N descriptor. Forward likelihood ratio model, all models are confirmed using a backward likelihood ratio model. Hazard ratio: HR <1 suggests improved survival. Confidence interval. P-value according to the likelihood ratio test. Per anno. Negative vs. positive. Including only NSCLC patients, other factor not accepted by Cox regression model: sex, age, TPTE-Sero-positivity, histology: squamous cell carcinoma. Other NSCLC vs. adenocarcinoma. Other NSCLC vs. large cell carcinoma. Including only SCLC patients. Factors not accepted by Cox regression model: sex, age, TPTE sero-positivity, stage (limited vs. extensive disease).

the range of not detectable to 0.148. Based on this data set, a positive sero-reactivity against TPTE was defined as an OD value of a 1 + 100 diluted serum that exceeded the 99th percentile value of 47 healthy donors and was calculated as 0.139 (Fig. 1).

3.2. Sero-reactivity against TPTE in lung cancer patients In the cohort of 307 lung cancer patients (see Table 1), screened for TPTE-specific autoantibodies by CrELISA absorbance values were found to range between 0 and 0.82 and thus, to have a higher dynamic range as observed for healthy donor sera. 41 out of 307 sera resulted in absorbance values exceeding the deliberately set cut off value of 0.139 corresponding to a frequency of 13.4% (Fig, 1).

3.3. TPTE aa 1-51 by CrELISA as a diagnostic tool in lung cancer The results of the two CrELISA-tests, performed with the sera of healthy donors and lung cancer patients were pooled (n = 354). Based on the cut off value of 0.139 (corresponding to the 99% percentile in healthy donors) sensitivity of the CrELISA to detect lung cancer was 13% and specificity was 97.9%. Using ROC curve analysis and Youden’s index calculations the optimal cut off value for diagnosing lung cancer was 0.0305 with a sensitivity of 52% and a specificity of 72% (Fig. 2).

3.4. Correlation of TPTE sero-positivity with clinical parameters There was no difference in the prevalence of TPTE sero-reactivity between SCLC and NSCLC patients. A trend was observed towards more frequent TPTE sero-positivity among patients with large cell carcinoma (38% vs. 13%, p = 0.084), although this result is limited by the small number of cases (n = 8). There was no association of TPTE sero-reactivity and EGFR mutation status. There was a trend towards more frequent TPTE sero-positivity among patients without metastasis in general (19% in M0-patients vs. 10% in M1-patients, p = 0.061) and among patients without lung metastasis in particular (15% TPTE sero-positivity in patients without vs. 6% in patients with lung metastasis (p = 0.091)). There was a strong association with TPTE sero-positivity in patients with bone metastasis (16% TPTE sero-positivity in patients with vs. 2% in patients without bone metastasis, p = 0.002). In contrast, no association with other metastatic sites such as liver, pleura, skin, pericardium, brain, extrathoracic lymphatic nodes or adrenal gland was observed. Of note, neither in SCLC patients nor in NSCLC patients TPTE sero-reactivity was associated with stage grouping (Mountain or Veterans’ association). TPTE sero-reactivity was not correlated with pathologic serum levels of LDH, NSE, Cyfra or CEA or with sex, smoking habit and age. TPTE sero-reactivity was not a predictor for the outcome of chemotherapy.

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Fig. 3. Survival analysis. Kaplan-Meier estimates according to TPTE sero-positivity (A) TPTE sero-positivity indicates a better survival in all patients (n = 307, p = 0.018). (B) In NSCLC patients TPTE sero-positivity indicates a better survival (n = 260, p = 0.043). (C) In SCLC patients TPTE sero-positivity shows a trend towards better survival (n = 45, p = 0.133).

3.5. Prognostic relevance of TPTE sero-reactivity To evaluate the prognostic value of TPTE sero-reactivity regarding patients’ survival, a Cox proportional hazards model was used for comparisons with established prognostic factors. Cox regression model A includes all patients. Forward likelihood ratio analysis revealed age, TNM T descriptor, TNM M descriptor, tumor histology and TPTE sero-reactivity as explanatory prognostic factors, confirmed by backward regression model analysis (Table 2). Model B included only NSCLC patients. Using this model adenocarcinoma and large cell carcinoma histology were found to be prognostic factors along with stage. In model C, which focuses on

SCLC patients, none of the factors analyzed was accepted by the regression model. By univariate Kaplan-Meier analysis of the entire patient population, TPTE sero-reactivity was a prognostic factor regarding survival if all patients were analyzed (n = 307, p = 0.018). Median survival time was 422 ± 32 days for TPTE sero-negative and 624 ± 193 days for TPTE sero-positive patients (five-year survival 10% vs. 17%). Also in the subset of NSCLC patients TPTE seroreactivity was found to be a prognostic factor (n = 260, p = 0.043). Median survival time was 442 ± 29 days for TPTE sero-negative and 728 ± 283 days for TPTE sero-positive patients (five-year survival 12% vs. 23%). In SCLC patients TPTE sero-reactivity was not found to be a prognostic factor in an univariate analysis (Fig. 3A–C).

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3.6. TPTE and autoantibodies against other C/T-antigens In a subsequent yet unpublished analysis, we screened NSCLC patients for autoantibodies against NY-ESO 1 using a similar CrELISA. 88 patients participated in both studies. In that cohort, 6 patients (7%) were positive for TPTE-antibodies and 4 patients (5%) were positive for NY-ESO 1-antibodies. Notably, all patients with NY-ESO 1-antibodies also had antibodies against TPTE.

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TPTE sero-reactivity does not vanish over the observed period of time. We have no appropriate anti-TPTE antibody available for the detection of TPTE-antigen in tumor tissue to correlate antigenexpression and sero-reactivity, but according to a previous report spontaneous occurrence of TPTE-antibodies without antigen expression in cancer is unlikely [1]. 5. Conclusion

4. Discussion This study investigates for the first time the prevalence of spontaneously occurring IgG responses against TPTE in sera of patients with lung cancer. 13.4% of 307 lung cancer patients were seropositive for the cancer/testis antigen TPTE without any difference between NSCLC and SCLC patients. TPTE sero-positivity was neither associated with tumor stage, histology, gender nor age. However, multivariate analysis indicated that TPTE sero-positivity was associated with increased survival and seemed to be an independent prognostic marker. Only one study has ever investigated TPTE auto-antibodies in cancer patients. Dong et al. [1] demonstrated that HCC patients have an overall TPTE sero-prevalence of 10% (6/62 patients) respectively of 25% in the subset of patients pre-selected for TPTE antigen positivity of their tumors. In the same study, the authors also address lung cancer and report TPTE to be expressed in the tumor tissue of 36% of lung cancer patients. They hypothesized, the TPTE antigen could be involved in cancer cell survival [1]. So far, it is unknown if TPTE expression in tumor influences survival in lung cancer patients, but regarding C/T antigens in general, antigen expression is usually associated with a worse prognosis [6,21–24]. Recently a study in myeloma showed that C/T autoantibodies can activate complement factors and influence C/T antigen procession by antigen-presenting cells, therefore C/T antibodies might be regarded to be functional [19]. This is supported by another study in malignant melanoma showing that NY-ESO-1 autoantibody expression prior to immunotherapy correlates with better survival [20] and a case report of a long surviving lung cancer patient with high autoantibody titers against NY-ESO-1 whose disease progressed after years when the titers decreased. Taking together both hypotheses, a functional auto-antibody against TPTE should counteract cancer cell survival and therefore, the occurrence of TPTE auto-antibodies might indicate a better prognosis in cancer patients. Consistently, we observed a trend of TPTE sero-positivity to be associated with lower frequency of metastasis (p = 0.061), a clear association of TPTE sero-negativity with bone metastasis (p = 0.002) and a prolonged survival of TPTE seropositive lung cancer patients. Regarding the use of a TPTE-specific CrELISA as a tool to screen for lung cancer from patients’ sera, we figured out that at the cutoff derived from the 99% percentile in healthy donors a convenient specificity of 97.9% is achieved but sensitivity remains weak at 13%. In fact, at an optimal cut-off calculated using ROC curve analysis TPTE sero-reactivity has only moderate specificity (72%) and sensitivity (52%) for diagnosing lung cancer. Nevertheless, the test for TPTE-specific autoantibodies may still be useful as one component in a panel of tests for cancer associated autoantibodies, increasing sensitivity and specificity by combination to screen for and diagnose (not only) lung cancer. Our study has the limitation that patients were not uniformly analyzed for TPTE sero-reactivity at the time of diagnosis, but at any time during the course of the disease. Yet, some patients had multiple serum analyses and no patient had a conversion of TPTE sero-reactivity (data not shown). As the analyzed TPTE-specific antibodies were immunoglobulin IgG class, it is safe to assume that

This is the first study showing that a subset of patients with lung cancer, NSCLC as well as SCLC, have auto-antibodies against TPTE. Our data reveals that sero-positivity is associated with prolonged survival in lung cancer patients, although established factors such as stage and histology remain the crucial prognostic factors. Sensitivity and specificity of TPTE antibody testing are not sufficient for screening or reliably diagnosing lung cancer from patients’ sera, but TPTE may be of interest as component in a multi-antigen diagnostic test. CD8+ and CD4+ T-cell responses against an antigen are known to correlate with measurable titers of circulating IgG antibodies [21,22]. In a subsequent study, we selected patients based on their TPTE seropositivity and were able to detect TPTE-specific CD4+ and CD8+ T cell responses [23]. Based on its cancer-selective expression and its confirmed immunogenicity TPTE was selected as target for clinical trial of a liposome formulated RNA vaccine that has been recently initiated (Lipo-MERIT, NCT02410733). Conflict of interest The authors declare no conflict of interest. Acknowledgment The authors confirm that they did not receive third party funding. References [1] X.Y. Dong, Y.R. Su, X.P. Qian, X.A. Yang, X.W. Pang, H.Y. Wu, W.F. Chen, Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients, Br. J. Cancer 89 (2003) 291–297. [2] A.P. Singh, S. Bafna, K. Chaudhary, G. Venkatraman, L. Smith, J.D. Eudy, S.L. Johansson, M.F. Lin, S.K. Batra, Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgen-independent prostate cancer cells, Cancer Lett. 259 (2008) 28–38. [3] M. Guipponi, M.L. Yaspo, L. Riesselman, H. Chen, A. De Sario, G. Roizes, S.E. Antonarakis, Genomic structure of a copy of the human TPTE gene which encompasses 87kb on the short arm of chromosome 21, Hum. Genet. 107 (2000) 127–131. [4] N.R. Leslie, X. Yang, C.P. Downes, C.J. Weijer, PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration, Curr. Biol. 17 (2007) 115–125. [5] Y. Wu, D. Dowbenko, M.T. Pisabarro, L. Dillard-Telm, H. Koeppen, L.A. Lasky, PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase, J. Biol. Chem. 276 (2001) 21745–21753. [6] Y. Shigematsu, T. Hanagiri, H. Shiota, K. Kuroda, T. Baba, M. Mizukami, T. So, Y. Ichiki, M. Yasuda, M. Takenoyama, K. Yasumoto, Clinical significance of cancer/testis antigens expression in patients with non-small cell lung cancer, Lung Cancer 68 (2010) 105–110. [7] K. Tajima, Y. Obata, H. Tamaki, M. Yoshida, Y.T. Chen, M.J. Scanlan, L.J. Old, H. Kuwano, T. Takahashi, T. Mitsudomi, Expression of cancer/testis (CT) antigens in lung cancer, Lung Cancer 42 (2003) 23–33. [8] M.J. Scanlan, A.J. Simpson, L.J. Old, The cancer/testis genes: review, standardization, and commentary, Cancer Immun. 4 (2004) 1. [9] J. Vansteenkiste, M. Zielinski, A. Linder, J. Dahabreh, E.E. Gonzalez, W. Malinowski, M. Lopez-Brea, T. Vanakesa, J. Jassem, H. Kalofonos, J. Perdeus, R. Bonnet, J. Basko, R. Janilionis, B. Passlick, T. Treasure, M. Gillet, F.F. Lehmann, V.G. Brichard, Adjuvant MAGE-A3 immunotherapy in resected non-small-cell lung cancer: phase II randomized study results, J. Clin. Oncol. 31 (2013) 2396–2403.

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Please cite this article in press as: A. Kuemmel, et al., Humoral immune responses of lung cancer patients against the Transmembrane Phosphatase with TEnsin homology (TPTE), Lung Cancer (2015), http://dx.doi.org/10.1016/j.lungcan.2015.07.012