Elevated Serum Megakaryocyte Potentiating Factor as a Predictor of Poor Survival in Patients with Mesothelioma and Primary Lung Cancer

Abstract

Background:

There is an urgent need for a companion assay to work with mesothelin-targeted therapeutic agents and for noninvasive and accurate prognostication of malignant mesothelioma (MM) patients. We report the development and validation of a blood-based assay for megakaryocyte potentiating factor (MPF) and the evaluation of its effectiveness for prognosis in MM and lung cancer patients.

Methods:

Using electrochemiluminescence technology, we developed a sensitive MPF assay and performed both analytical and clinical validations. Further, the effectiveness of the MPF assay in predicting prognosis was evaluated for 95 MM and 272 lung cancer patients.

Results:

We performed comprehensive analytical and clinical validation, including precision and accuracy, interference, preanalytical variables, sensitivity, and specificity for mesothelioma. In MM patients, increased serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (log-rank P = 0.003; n = 95). In refractory MM patients, increased MPF is a strong predictor of poor outcome with an HR = 6.12 (log-rank P = 0.0007; n = 57). In a lung cancer patient cohort, increased MPF is a predictor of poor survival, with an HR = 1.57 (log-rank P = 0.003; n = 272).

Conclusions:

The MPF assay has robust technical characteristics, with strong analytic and clinical validation. Clinical studies indicate that increased serum MPF is a predictor of poor survival for MM patients, throughout the course of the disease. Increased MPF is also associated with poor overall survival for patients with newly diagnosed lung cancer.

Background

Malignant mesothelioma (MM)⁵ is a highly aggressive disease with poor prognosis (1). In patients with this disease, the current first-line therapy is cisplatin and pemetrexed, which is associated with a median overall survival of 12.1 months (2). For patients that progress on first-line therapy, there is no approved second-line treatment, and responses to other treatments are rare (3). Thus, there is a strong need to develop novel therapies against MM. The mesothelin gene encodes a 69-kDa precursor protein, which is cleaved into 2 parts: a 31-kDa soluble megakaryocyte potentiating factor (MPF) and a membrane-bound 40-kDa mesothelin (4, 5). The membrane-bound version of mesothelin can also be shed into circulation as soluble mesothelin (SM) (6). Mesothelin is a plasma membrane glycoprotein originally identified as over-produced on ovarian cancer cells (4, 7). Subsequent work reveals that it is overproduced in several other cancers, including mesothelioma, pancreatic cancer, and adenocarcinoma of the lung (8–11). There has long been an interest in developing mesothelin-targeted therapies, some of which are now showing evidence of antitumor activity for MM (12–14).

As mentioned, mesothelin is also produced by lung cancer cells. By use of immunohistochemistry (IHC), mesothelin production was reported in 40%–50% of lung cancers (11, 15, 16). Two recent studies have shown high levels of mesothelin production, detected via IHC in 9% and 24% of lung cancer cases, is associated with poor survival (17, 18). Thus, mesothelin is also a potential therapeutic target in lung cancer.

The only US Food and Drug Administration–approved mesothelioma test was developed and validated as MesoMark™ (19). It detects increased SM in nearly 60% of MM patients and in much lower percentages of lung, ovarian, and pancreatic cancer patients (19). It was approved as a “humanitarian use” test for monitoring patients with epithelioid and biphasic MM for which no supporting effectiveness data was necessary (20). Since then, multiple studies have examined the correlation between baseline serum SM and overall survival with use of the mesothelin assay, some with inconsistent results (21) and others showing a lack of association (22, 23). Thus, more effectiveness data are needed.

Further, a biomarker assay to support the development of mesothelin-targeted agents is also needed. Because these antibody-based therapeutic agents bind to mesothelin, an SM test, such as MesoMark, will be subjected to interference by these agents. As many mesothelin-targeted agents are in clinical development (24, 25), a specific bio-marker assay is needed for treatment evaluation and patient monitoring.

MPF is a cleavage product released into the circulation during the maturation of mesothelin, and it is not bound by mesothelin-targeted agents. Thus, its serum level could directly reflect the tumor load and serve as a biomarker for mesothelin production in tumors. ELISA were previously developed against MPF and evaluated for detecting increased MPF in MM (26–28). The results indicated that MPF is increased in 91% and 74% of MM patients (26, 27) and that there is a correlation between MPF and mesothelin (27). However, these assays had limited analytical validation, which cannot meet the stringent criteria for integrated bio-markers in clinical trials for novel agents (29, 30), and the prognostic value of MPF is not known. Thus, we developed a significantly improved MPF assay by using an electrochemiluminescence immunoassay (ECLIA) for use as an integrated biomarker in the development of mesothelin-targeted therapies. Comprehensive analytical and clinical validation of the MPF assay was performed. Studies were also carried out to determine the effectiveness of a serum MPF assay for prognosis in MM or primary lung cancer patients.

MATERIALS AND METHODS

Mesothelioma cases and controls

All samples from mesothelioma patients were collected between June 2008 and January 2016 with Institutional Review Board-approved protocols at the NIH Clinical Center, with informed consent obtained from all patients. Twenty treatment-naïve mesothelioma patients from a previously reported study were included (31). All remaining 75 samples, 57 with prior chemotherapies, were prospectively collected between January 2014 and January 2016 on a natural history protocol at NCI. Survival time was calculated from the date of diagnosis to either the date of last follow-up (1/31/2017) or the date of death. Serum samples from 56 individual healthy donors were collected at the Blood Research Service NIH Clinical Center with signed informed consent.

Lung cancer cases

The patient samples were from self-identified African-American patients with lung cancer from the NCI-Maryland lung cancer study. The study accrual and eligibility criteria for the case-control study were previously described (32, 33). Institutional Review Board approval was obtained from all participating institutes and NIH. Tumor staging was based on pathology if the individual went to surgery and clinical staging if no surgery was performed. To obtain data on lung cancer-specific mortality, the date and cause of death were obtained from the National Death Index. The linkage process has been previously described (32). Lung cancer-specific death was defined as a case with lung cancer listed as the primary, secondary or tertiary cause of death or death due to another cancer within 2 years of the lung cancer diagnosis. Survival time was calculated from date of surgery when performed, or the date of diagnosis if not, to either date of last known follow-up (last National Death Index update on 12/31/2012) or date of death due to lung cancer.

MPF assay

The MPF assay uses our previously generated antibodies: the capture antibody MPF49 (γ2a, κ), which binds to MPF topographic epitope 3, and detection antibody MPF25 (γ1, κ), which binds to epitope 1 (26). MPF49 was biotinylated, and MPF25 was conjugated with Sulfo-Tag NHS-Ester (Meso-Scale Diagnostics). The assay was then optimized with a 96-well streptavidin plate (Meso-Scale Diagnostics).

All MPF tests were conducted in accordance with standard operating procedures. Briefly, the streptavidin assay plates were blocked with blocking buffer for 1 h at room temperature with constant shaking. The biotinylated capture antibody solution (25 μL) was added to the microplates. After incubation for 1 h, plates were washed and the serial diluted MPF calibrator, diluted patient samples, or reference samples (50 μL) were added to further incubate for 1h. After a wash step, the Sulfo-Tag detection antibody solution (25 μL) was added and incubated for 1 h. After washing, a 2X Read Buffer was added, which was read with a QuickPlex instrument (Meso-Scale Diagnostics) within 20 min. The data were analyzed with WorkBench 4.0 software (Meso-Scale Diagnostics), and concentration of the analytes was determined by use of the 5 Parameter Logistic nonlinear regression model.

Statistical analyses

GraphPad Prism 7.0 was used to conduct non-parametric ROC analyses comparing the MPF results in the mesothelioma patients to those of the healthy volunteers. The area under the curve (AUC) was also calculated. The 95% CI was determined for the AUCs. The upper limit of the reference interval was determined by use of healthy donor serum samples and was based on mean + 3SD.

To test the magnitude of association between MPF markers, survival (in both mesothelioma and lung cancer patients, for which lung cancer-specific survival was assessed), hazard ratios (HRs) were estimated with use of univariable and multivariable Cox proportional hazards-regression modeling. Multivariable analyses were adjusted to control for the following potential confounding variables: age (continuous), sex (male/female), current smoking status (never/former/current), pack-years (continuous), stage (stage I, stage II, stage III, stage IV), and histology (adenocarcinoma, squamous cell carcinoma, other). Variables selected for adjustment were based on standard prognostic variables used in the literature. All statistical analyses were performed using STATA^® 13.0 (StataCorp). MPF levels were dichotomized into “high” and “low” with the use of a cutoff point of 1.2 ng/mL, which was determined from healthy donors (mean + 3SD). Analyses on mesothelioma patients were adjusted for age, sex, histology, and tumor site.

MPF assay: analytical and clinical validation

See information in the Data Supplement that accompanies the online version of this article at http://www.jalm.org/content/vol3/issue2.

RESULTS

Development of an ECLIA for MPF

We used ECLIA technology to develop an assay for MPF with nearly 100-fold improvement in both sensitivity and quantification range over previously reported ELISAs (26). The new ECLIA-based MPF assay has a detection limit of 7.2 pg/mL with a recombinant MPF isolated from HEK293 cells (see Fig. 1 in the online Data Supplement). The assay is performed with a 7-point calibration curve with recombinant MPF (Fig. 1A). After adjustment for sample dilution, the assay has a quantification range of 0.10–500 ng/mL (5000-fold), which is sufficient to quantify all mesothelioma patient and healthy donor samples tested.

Fig. 1. Validation of MPF test.

Results of panels A–C are shown as mean + SD. Calibration curves (A). Three consecutive 7-point calibration curves generated each with replicates. Dilution linearity analysis (B). An MM patient sample was diluted and subsequently tested by the MPF test. Test was done in triplicate with an average CV of 1.7% (range, 0.0–4.2%) for all samples across the entire dilution range. Preanalytical blood sample stability test (C). Healthy donor blood samples were obtained in serum collection tubes. They were stored for 0, 1, 4, and 7 days at room temperature (RT, n = 4) and 4 °C (n = 5). The sera were subsequently isolated and tested for MPF. The MPF test results of 95 mesothelioma patients and 56 healthy blood donor serum samples (D). The cutoff of 1.2 ng/mL was determined as mean + 3 SD with data from healthy donors. Data are expressed as mean with 95% CI. Comparison between serum MPF levels in peritoneal (n = 26) and pleural mesothelioma patients (n = 68) (E). Data are expressed as mean with 95% CI. ROC analysis with data from panel D (F). The ROC analysis showed that the test has an AUC of 87.3% (95% CI, 81.5–93.1%).

Analytic validation

With use of 3 test reference panels, the average imprecision (CV) within run is 2.8% (range, 1.8%–3.8%). The average day-to-day CV of the reference samples is 5.2% (n = 20; range, 4.4%–5.9%; see Table 1 in the online Data Supplement). With use of 10 mesothelioma patient samples, the average day-to-day CV is 5.5% (n = 3; range, 0.9%–12.8%; see Table 1 in the online Data Supplement). The total mean recovery of added antigen (10–80 ng/mL) in serum samples is 94.6%, and the individual recoveries were 80%–102% (see Table 2 in the online Data Supplement). Dilution linearity was demonstrated with 3 MM patient samples diluted across the reference interval to the lower limit of detection. Linear regression analyses of the diluted MM patient samples show that the goodness of fit R² ≥0.998 for all 3 samples (Fig. 1B; see Table 3 in the online Data Supplement).

The assay is resistant to interference from a range of native components, as well as exogenous chemotherapeutic agents. After the addition of potential interferents, mean results were 92%–96% of the expected values, shown in Table 4 in the online Data Supplement. Because the MPF assay will be used in patients on antimesothelin therapies, it was tested against both type I (naturally occurring) human antimouse antibody (HAMA) and type II HAMA (therapeutic antibody induced), which frequently affect mouse antibody-based immunoassays. The results show no detectable interference of HAMA type I/II to the test, with mean results of 101%–102% of the expected values (see Table 4 in the online Data Supplement).

Preanalytical studies were also performed. When whole blood samples were stored in serum collection tubes at 2–8 °C for 1, 4, and 7 days, the recovery efficiencies of MPF were 95.6%, 90.0%, and 81.8%, respectively (Fig. 1C). MPF was less stable with blood stored at room temperature (20–24 °C), with recovery efficiencies of 81.5%, 70.3%, and 62.0% for the same time points (Fig. 1C). In repeated freeze and thaw studies, the antigen could withstand 3 freeze/thaw cycles with a minimal loss (recovery at 96.8% ± 4.5%) (see Fig. 2 in the online Data Supplement). However, 5 freeze/thaw cycles resulted in a 14% reduction of MPF in donor sera (recovery at 86.0% ± 3.2%; see Fig. 2 in the online Data Supplement).

Clinical validation

The MPF assay was evaluated with serum samples from 95 mesothelioma patients against that of 56 healthy blood donors. MPF levels in mesothelioma patients are significantly higher than that of the healthy individuals. The mean of MPF is 10.5 ng/mL (95% CI, 5.75–15.27) in mesothelioma patients, compared with 0.51 ng/mL (95% CI, 0.45–0.57) in the reference group (P = 0.002; Fig. 1D). Levels of MPF in patients with peritoneal MM (n =26) are also higher than those with pleural MM (n =68), with mean levels at 21.8 ng/mL and 6.4 ng/mL, respectively (P = 0.004; Fig. 1E).

ROC analysis comparing sera from mesothelioma patients and healthy individuals yielded an area under curve (AUC) of 87.3% (95% CI, 81.5%–93.1%; Fig. 1F). This MPF assay result is similar to data reported of MesoMark that measures SM protein, with an AUC for ROC curve of 87.1% (19), and other studies on mesothelin-related peptides for mesothelioma diagnosis (34). Similar to Meso-Mark, the MPF assay would likely be inadequate for the diagnosis of mesothelioma. The cutoff MPF level was determined with the healthy donor samples at 1.2 ng/mL (mean + 3SD), corresponding to 38 pmol/L, which is about 40-fold lower than the MesoMark cutoff of 1.5 nmol/L for SM. At this cutoff value, the assay has a sensitivity of 66.3% and a selectivity of 98.2% in the MM patients tested (n = 95).

Survival analysis of MPF in MM patients

Survival data were collected for all 95 MM patients (Table 1). Median durations of follow-up are7.8 months (range, 0.4–92.3 months) and 17.5 months (range, 12.2–35.7 months) for deceased and censored patients, respectively. Survival analysis was performed with serum MPF results by use of the predetermined cutoff of 1.2 ng/mL, at which 63 patients were positive (66.3%). The results show that the MM patients with increased MPF have worse prognosis than those with low or “normal” MPF (log-rank P = 0.003; Fig. 2A). Median survival times are 24.0 and 92.3 months for the MM patients with high and low MPF, respectively (Fig. 2A). Univariable survival analysis estimated an HR of 2.46 (95% CI, 1.33–4.53; P = 0.004; Table 3). Following adjustment for age, sex, histology, and site of tumor, the relationship remained statistically significant (HR: 2.06; 95% CI, 1.01–4.20; P =0.046) (Table 3). As nearly 72% of the patients are pleural MM, survival analysis reveals that increased MPF is also a worse prognostic bio-marker for this patient population with an HR of2.19 (95% CI, 1.23–3.91; P = 0.014; see Fig. 3 in the online Data Supplement).

Table 1.

Characteristics of the malignant mesothelioma patient population.

Characteristic	MM cases (n = 95)	MPF ng/mL (mean + SD)
Age (mean ± SD)	57.6 ± 14.6	10.5 ±23.4
Sex (%)
Male	70 (74%)	12.5 ± 26.6
Female	25 (26%)	5.6 ± 8.7
Tumor site (%)
Peritoneal	26 (27.4%)	21.8 ± 40.0
Pleural	68 (71.6%)	6.4 ± 10.2
Testicular	1 (1.0%)	0.6
Histology
Biphasic	3 (3.2%)	1.3 ± 0.9
Epithelioid	86 (90.5%)	11.4 ± 24.4
Sarcomatoid	2 (2.1%)	1.1 ± 0.6
Missing	4 (4.2%)	2.7± 3.9
Survival (median years, IQR)	2.4 (1.2–7.7)

IQR, interquartile range.

Fig. 2. Increased MPF as a predictor of poor survival in mesothelioma patients, including those with prior therapies.

Kaplan–Meier survival analysis based on the levels of serum MPF by use of the predefined cutoff for all 95 mesothelioma patients (A). Increased MPF was a predictor of poor survival (log-rank P = 0.003). Similar survival analysis of 57 previously treated mesothelioma patients with a median time span of 21 months before blood sampling and testing (B). Increased MPF was a predictor of poor survival in this previously treated population (log-rank P = 0.0007). All survival days start from the time of diagnosis.

Table 3.

Survival analysis of MPF in patients with malignant mesothelioma and in African-Americans with newly diagnosed lung cancer. Univariable and multivariable logistic regression analysis of MPF in patients with malignant mesothelioma and newly diagnosed lung cancer.

		Univariable			Multivariable
Patient groups	N	HR	95% Cl	P	HR	95% Cl	P
All MM patients	95	2.46	1.33–4.53	0.004	2.06	1.01–4.20	0.046
MM patients with prior therapies	57	6.12	1.86–20.07	0.003	3.97	0.88–17.91	0.073
					Adjusted for age at diagnosis, sex, histology, and site
African-Americans lung cancer	314	1.56	1.14–2.15	0.006	1.49	1.03–2.16	0.035
					Adjusted for age, sex, smoking, stage, and histology

HR, hazard ratio.

Fifty-seven of the MM patients had been previously treated elsewhere, and all of them were prospectively enrolled at our institute between January 2014 to January 2016. The median time from diagnosis to the first serum sampling at our institute is 21.6 months (range, 1.0–131.9 months). Of these MM patients with previous therapies, 40 were positive for serum MPF (70.2%), with levels not statistically different from that of the treatment-naïve patients (not shown). Survival analysis of these previously treated MM patients shows that increased MPF continues to be a predictor of poor survival with log-rank P = 0.0007 (n = 57; Fig. 2B). Univariable analysis shows that the patients with increased MPF have an increased hazard of death; HR = 6.12 (95% CI, 1.86–20.07; P = 0.003; Table 3). Multivariable analysis indicates that the patients with prior therapies and increased MPF have an HR of 3.97 (95% CI, 0.88–17.91; P = 0.073; Table 3). Although the multivariable analysis is of borderline significance, this is likely due to the small sample size. Collectively, these data indicate that increased serum MPF is a predictor of poor prognosis in MM patients, regardless of the time at which patients are tested.

Survival analysis of MPF in primary lung cancer patients

We next evaluated the relationship between serum MPF levels and prognosis in a cohort of 272 African-Americans with lung cancer (Table 2). We focused on African-Americans because previous work demonstrated that tumor mesothelin positivity was significantly associated with triple-negative breast cancer (the subtype most common among African-American women) and poor clinical outcomes (35).

Table 2.

Characteristics of the African-American lung cancer patient population.

Characteristic	Cases(n = 272)
Age (mean ± SD)	64.6 ±9.9
Sex (%)
Male	151 (56%)
Female	121 (44%)
Race (%)
African-American	272 (100%)
Smoking Status (%)
Never	20 (7%)
Former	100 (37%)
Current	147 (55%)
Missing	5 (1 %)
Pack-years (mean ± SD)	37.2 ± 30.3
Stage
I	71 (33%)
II	15 (7%)
III	64 (30%)
IV	62 (29%)
Missing	60
Histology
Adenocarcinoma	110 (44%)
Squamous cell carcinoma	76 (31%)
Large cell carcinoma	52 (21%)
Othera	11 (4%)
Missing	23
Survival (median years, IQR)	1.8 (0.9–3.7)

IQR, interquartile range.

^aIncludes designation NSCLC and SCLC.

Using 1.2 ng/mL as the cutpoint, as indicated by previous analyses, we assessed the relationship between MPF levels and lung cancer survival. Eighty-three patients had increased serum MPF levels (30.5%). Patients with high MPF at the time of their diagnosis had a worse outcome than patients with low levels (Fig. 3; log-rank P = 0.003). Univariable analysis shows an HR = 1.57 (95% CI, 1.14–2.17; P = 0.005; Table 3). After adjustment for potential confounding factors, including age, sex, smoking status, pack-years of smoking, tumor stage, and tumor histology, the relationship remained significant (HR = 1.50; 95% CI, 1.04–2.18; P = 0.029; Table 3), suggesting that increased MPF may be an independent predictor of worse overall survival in African-Americans with lung cancer. As shown in Table 2, the population included individuals with heterogenous histological subtypes. As adenocarcinoma and squamous cell carcinoma are the main forms of lung cancer, we then performed stratified analyses by histology. Increased MPF detection was associated with a poor prognosis in both adenocarcinoma (HR = 1.39; 95% CI, 1.05–1.85; P = 0.020) and squamous cell carcinoma (HR = 1.44; 95% CI, 1.04–2.00; P =0.029) following adjustment for age, sex, stage, smoking status, and pack-years of smoking.

Fig. 3. Increased MPF as a prognostics bio-marker for poor survival in patients with newly diagnosed lung cancer.

Survival analysis based on the levels of serum MPF for African-American lung cancer patients (n = 272). Survival days start from the time of diagnosis. Increased MPF was associated with poor survival (log-rank P = 0.005).

DISCUSSION

MPF is a secreted protein product of the mesothelin gene that does not bind to therapeutic antibodies under development for mesothelin-producing cancers. In this study, we describe the development, and analytical and clinical validation of an assay for MPF as a biomarker for mesothelin-targeted agents. Our results further indicate that increased MPF predicts significantly worse overall survival throughout the course of the disease for MM. Our data also provide the first association between increased serum MPF and worse survival in patients with primary lung cancer. Thus, serum MPF analyses identify high-risk patients with mesothelioma and lung cancer, and could serve as a biomarker for the development of mesothelin-targeted therapies.

In this study, we used ECLIA technology to develop an MPF serum assay with higher sensitivity and wider signal dynamic range. With this MPF assay, there is a nearly 100-fold improvement in sensitivity and quantification range when compared with the previously reported ELISA (26). The increased sensitivity is important because it allows more precise quantification of MPF levels in cancer patients with lower levels of this tumor antigen. Further, the ECLIA-based MPF assay has a quantification range between 0.1–500 ng/mL, necessary to quantify MM patients with MPF levels ranging from 0.19–146.5 ng/mL.

This report provides both preanalytical and analytical validation that includes the limit of detection determination, imprecision, dilution linearity, and interference analysis, all of which are necessary for an integrated biomarker in clinical trials (29, 30). The analytical studies indicate that the MPF assay is robust and free of the interference of a range of factors, including both endogenous and exogenous chemotherapeutic agents. In addition, it is resistant to interference from HAMA, both naturally occurring type I and antibody therapy-induced type II. The average interday imprecision is 5.2% for reference samples and 5.5% for mesothelioma patient samples. Both the spike-and-recovery and dilution linearity studies showed high recovery and excellent linearity (r ≥ 0.998). Thus, the results strongly support the analytical validation of the assay.

The clinical evaluation of the MPF assay in mesothelioma patients shows that it has good discriminatory performance over the healthy control samples with an AUC for ROC analysis of 87.3% (n = 95). This is highly comparable to the reported data of MesoMark (19). Neither MesoMark nor the MPF assay would be adequate for the diagnosis of mesothelioma. The reference interval was determined to be 0–1.2 ng/mL for serum MPF.

Given the recent development of agents to target the mesothelin protein, there is a strong need for noninvasive and accurate prognostication for MM (36). A previous report showed that with use of median levels (733 ng/mL) as a cutoff, mesothelioma patients with high pleural effusion fibulin-3 have poor survival (37). However, there was a poor correlation between plasma and pleural effusion fibulin-3 (37), therefore a blood-based prognostic test was not considered for fibulin-3. In our study, we used blood samples from MM patients. They were tested for serum MPF, with survival analysis as the primary objective. The MPF test was thoroughly validated, both analytically and clinically. For data analyses, a predetermined cutoff value established with the control group was used, without any patient exclusion. With the predefined cutoff of 1.2 ng/mL, our data suggest that mesothelioma patients with increased serum MPF levels have over 2-fold increased risk of mortality than those with lower levels. The association also holds when the analysis is restricted to patients with prior therapy, although it is of borderline significance in the multivariable model, which is likely due to the small sample size, i.e., 57 patients. Further study with an independent MM sample set would be desired to reconfirm the association between increased MPF level and poor prognosis.

MPF is a unique tumor antigen because it provides information about the response to agents that target mesothelin without being affected by the therapeutic antibodies in blood. The fact that increased serum MPF is associated with worse clinical outcome emphasizes the need for the therapeutic targeting of mesothelin in MM patients, and identifies a high-risk population likely to benefit from these therapies.

Our data indicating increased serum MPF levels are associated with poor survival in African-Americans with primary lung cancer is consistent with 2 previous studies that used IHC analysis of mesothelin in both early-stage and advanced lung adenocarcinoma. In these studies, strong mesothelin staining was associated with poor survival (17, 18). While mesothelin was detected by IHC in more than half of lung cancer in these 2 studies, only a small percentage of patients with the strongest mesothelin staining, 9% for early-stage and 24% for advanced lung cancer, were associated with poor survival (17, 18). A blood-based diagnostic test has many advantages over IHC analysis: it is more quantitative, easier to perform, noninvasive, and can be used to follow patients over time. A confirmatory study will be necessary to establish the value of serum MPF as a predictor of survival for lung cancer patients and to confirm its utility for both adenocarcinoma and squamous cell carcinoma, as reported here. While an advantage of our analysis was the analysis of a single population, its utility in populations of European and Asian descent should also be tested.

In summary, we developed an MPF test and conducted comprehensive analytical and clinical validation for clinical use. This work extends previous work in 2 ways. First, the test herein is much more sensitive with a greater quantification range than previous ELISA-based methods for detecting MPF, and has significant validation. Second, the detection of MPF, rather than SM, is a superior choice of biomarker for monitoring patient response as it does not have interference from mesothelin-targeted agents. Our data show that increased serum MPF is a predictor of poor overall survival for MM patients, throughout the course of the disease. The results further suggest an association between increased serum MPF and poor overall survival in patients with primary lung cancer. Thus, this MPF test could have a key role in identifying high-risk patients and in supporting the clinical development of mesothelin-targeted agents to improve their outcome.

IMPACT STATEMENT.

To support the development of mesothelin-targeted agents, we developed a blood assay for megakaryocyte potentiating factor (MPF), a cleavage polypeptide during mesothelin maturation, and conducted comprehensive validation of it. Our results demonstrate that increased serum MPF is a predictor of poor survival for mesothelioma, throughout the course of disease. Increased serum MPF is further associated with poor overall survival in African-Americans with newly diagnosed lung cancer. Thus, our study establishes a prognostic test for mesothelioma, identifies higher risk patient populations with either mesothelioma or lung cancer for investigational mesothelin-targeted therapies, and provides a biomarker for their development.

Nonstandard abbreviations:

MM

malignant mesothelioma

MPF

megakaryocyte potentiating factor

SM

soluble mesothelin

IHC

immunohistochemistry

ECLIA

electrochemiluminescence immunoassay

AUC

area under curve

HR

hazard ratio