The prognostic value of serum CA 19-9 for patients with advanced lung adenocarcinoma

We retrospectively analyzed 433 patients diagnosed advanced (stage IIIB or IV) lung adenocarcinoma at Kobe City Medical Center General Hospital between March 2006 and December 2012. We excluded patients who received no chemotherapy (n = 71), or for whom no data on tumor markers (CEA, CYFRA 21-1 and CA 19-9) before receiving chemotherapy was available (n = 116). Patients who reported never having smoked were defined as never-smokers, those who had smoked within 1 year of the diagnosis were categorized as current smokers, and the rest were considered to be former smokers. All patients were classified by clinical stage according to the 7^th edition TNM (tumor, node, metastasis) classification [18]. OS was measured from the diagnosis of lung cancer until death from any cause or the end of the follow-up. We isolated tumor DNA from various specimens and analyzed epidermal growth factor receptor gene (EGFR) mutation status at exons 18–21, using the peptide nucleic acid-locked nucleic acid PCR clamp methods, as described previously [19]. We retrospectively analyzed the presence of intestinal pneumonia, non-tuberculous mycobacteriosis (NTM) infection, bronchiectasis, and diffuse panbronchiolitis by reviewing patients’ charts and radiological records.

Serum samples were obtained to determine tumor markers CEA, CYFRA 21-1 and CA 19-9 as a part of routine evaluations within 28 days before starting chemotherapy. The concentration of each tumor makers was measured using LumiPulse Presto kit (Fujirebio Inc., Tokyo, Japan). It utilizes the chemiluminescent enzyme immunoassay (CLEIA) principle and is a fully automated assay [20]. The CLEIA method uses 1.6 ng/mL as the upper limit of normal (ULN) serum CYFRA 21-1 level in healthy individuals [21]. For this study, we set the cutoff value for CYFRA 21-1 at 2.2 ng/mL, which was used in a previous study that showed the prognostic impact of CYFRA 21-1 in patients with ALAD, and is the mean value for healthy subjects +3 SD (standard deviation) [4]. The cutoff values for serum CEA and CA 19-9 were set at 5.0 ng/mL and 37.0 U/mL, which are their respective ULNs [22, 23]. This testing was performed at the Department of Laboratory Medicine at our hospital.

Additionally, we retrospectively analyzed post-operative specimens from patients with clinical stage I lung adenocarcinoma who underwent surgery at our hospital between January 2008 and May 2010. We evaluated the presence of vessel invasion, pleural invasion, lymph node metastasis and postoperative pathological stage. The presence of mucin was also assessed using diastase-resistant periodic acid Schiff (D-PAS) staining in all samples with 5 % increments (Fig. 1a, b) [24, 25]. We tested CA 19-9 expression, immunohistochemically (IHC), using the 116-NS-19-9 antibody (Covance Inc., Princeton, USA). We applied the expression score, which was described previously [26‐28]. Percentages of CA 19-9⁺ tumor cells (proportion score) was scored as 0: none (0 %); 1: 1–10 %; 2: 11–30 %; 3: 31–50 %; 4: 51–70 %; and 5: 71–100 % of each tumor sample. The intensity of staining (intensity score) was scored as 0: no staining; 1: weak staining; 2: moderate staining; and 3: strong staining in >10 % of cancer cells (Fig. 1c–f). The proportion score and intensity score were added to yield a total expression score of 0–8; samples that scored ≥ 3 were defined as CA 19-9⁺. We defined cases with at least one of the pathologic invasive factors—vessel invasion, pleural invasion, or lymph node metastasis—as positive for cancer invasive factors [29]. All pathological analyses were evaluated by two experienced pathologists who were unaware of the patients’ conditions.

Continuous variables were analyzed using Student’s t-test. Dichotomous variables were analyzed using Fisher’s exact test. Correlations between CA 19-9 levels and CYFRA 21-1 levels were assessed using Spearman’s rank-based correlation test. The Kaplan − Meier method was used to estimate survival outcomes; groups were compared using the log-rank test. Cox’s proportional hazard models were fitted to determine associations between patient characteristics and survival outcomes. A multivariate Cox proportional hazard model was developed on all clinically important factors (age, sex, smoking status, ECOG PS, EGFR status, stage, positivity of serum CEA, CYFRA 21-1 and CA 19-9, and platinum doublet therapy administration). The results are expressed as hazard ratios (HRs) with 95 % confidence intervals (CI). All tests were two-tailed. A value of P < 0.05 was considered to indicate significance. We conducted statistical analyses on JMP software (11^th version; SAS Institute, Cary, NC, USA).

We included 246 patients with ALAD in the study (Fig. 2). Patient characteristics and comparison of clinical profiles of CA 19-9⁺ and CA 19-9⁻ patients are shown in Table 1. Their median age was 67 years (interquartile range, 61–75 years); 184 (75 %) patients had Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1; and 26 (11 %) patients had stage IIIB disease. Of the 246 patients, 100 (41 %) had EGFR mutations in their specimens, and 22 (9 %) had chronic lung inflammatory diseases (16 with interstitial pneumonia, 3 with NTM infection, and 3 with bronchiectasis). We found 163 (66 %) were CEA⁺ (>5.0 ng/ml), 155 (63 %) were CYFRA 21-1⁺ (>2.2 ng/ml) and 76 (31 %) were CA 19-9⁺ (>37.0 U/mL). Chemotherapy regimens of patients who did not receive platinum doublet therapy were tyrosine kinase inhibitors: n = 34; pemetrexed: n = 18; TS-1: n = 6; paclitaxel: n = 5; vinorelbine: n = 4; gemcitabine: n = 4; docetaxel: n = 3; and gemcitabine/vinorelbine therapy: n = 2.

Comparison of clinical profiles of CA 19-9⁻ and CA 19-9⁺ patients showed the CA 19-9⁻ group included a significantly higher percentage of patients with ECOG PS 0 or 1 status (P < 0.001), serum CYFRA 21-1⁻ (P = 0.002), and platinum doublet therapy administration (P = 0.007). EGFR status (P = 0.888) and presence of inflammatory lung disease (P = 0.147) did not statistically differ between the two groups.

Clinical profiles of CYFRA 21-1⁻ and CYFRA 21-1⁺ patients are compared in (Additional file 1: Table S1). The CYFRA 21-1⁻ group had a significantly higher percentage of patients with ECOG PS 0 or 1 status (P = 0.002), inflammatory lung disease (P = 0.020), serum CA 19-9⁻ (P = 0.002), and platinum doublet therapy administration (P = 0.046). EGFR status (P = 0.502) did not statistically differ between the two groups.

Test of correlation between CA 19-9 levels and CYFRA 21-1 levels showed no significant relationship between these tumor markers (r = 0.006). The scatter plot is shown in (Additional file 2: Figure S1).

The OS of patients included in this study was 21.4 months (interquartile range, 18.9 − 25.0 months; Table 2). Overall survival curves of CA 19-9⁺ and CA 19-9⁻ patients are shown in Fig. 3a. The OS of serum CA 19-9⁺ patients (n = 170, 69 %) was 12.5 months (95 % CI [CI]: 9.7 − 16.9 months); that of serum CA 19-9⁻ patients (n = 76, 31 %) was 26.2 months (CI: 21.8 − 28.8 months; P < 0.001). The OS of serum CYFRA 21-1⁺ patients (n = 155, 63 %) was 16.9 months (CI: 12.6 − 19.7 months), and that of serum CYFRA 21-1⁻ patients (n = 91, 37 %) was 31.8 months (CI: 26.2 − 43.9 months; P < 0.001).

Cox’s multivariate regression analysis of the influence of clinical characteristics on survival outcomes indicated that ECOG PS 0 or 1 (HR: 0.42, CI: 0.29 − 0.62, P < 0.001), mutated EGFR status (HR: 0.41, CI: 0.29 − 0.57, P < 0.001), stage IIIB (HR: 0.38, CI: 0.21 − 0.64, P < 0.001), serum CYFRA 21-1⁻ (HR: 0.47, CI: 0.32 − 0.66, P < 0.001), serum CA 19-9⁻ (HR: 0.60, CI: 0.43 − 0.85, P = 0.005), and administration of platinum doublet therapy (HR: 0.64, CI: 0.42 − 0.97, P = 0.034) independently predicted longer OS. To clarify the relationships of chemotherapy regimens to OS, we analyzed the platinum doublet subcohorts with regard to OS; we found that CA 19-9⁻ independently predicted longer OS (HR: 0.54, CI:0.35 − 0.84, P = 0.007).

We initially divided the patients into 4 groups by serum CA 19-9 and CYFRA 21-1 level: (a) both CA 19-9⁺ and CYFRA 21-1⁺ (59 patients, 24 %; median survival time [MST]: 10.0 months [CI: 8.8 − 13.4 months]), (b) CA 19-9⁺ and CYFRA 21-1⁻ (17 patients, 7 %; MST: 26.7 months [CI: 12.5 − * months]), (c) CA 19-9⁻ and CYFRA 21-1⁺ (96 patients, 39 %; MST: 22.5 months [CI: 16.9 − 26.6 months]), and (d) both CA 19-9⁻ and CYFRA 21-1⁻ (74 patients, 30 %; MST: 31.8 months [CI: 25.0 − 58.5 months]). Although the MSTs of (a) and (b) (P = 0.001) and that of (c) and (d) (P = 0.003) differed statistically, the difference between the two single-positive groups, (b) and (c), was not significant (P = 0.14). Therefore, we combined groups (b) and (c) into one single positive group. We also defined group (a) as double positive and group (d) as double negative (Fig. 2). Survival curves for these 3 groups are shown in Fig. 3b. Their MSTs were double positive: 10.0 months (CI: 8.8 − 13.4 months); single positive: 23.2 months (CI: 19.3 − 26.7 months); and double negative: 31.8 months (CI: 25.0 − 58.5 months; P < 0.001).

We pathologically evaluated 116 consecutive surgically resected specimens from patients with clinical stage I lung adenocarcinoma. Of those, 54 (47 %) were CA 19-9⁺. Relationships between CA 19-9 expression and patients’ clinicopathological characteristics are summarized in Table 3. Comparison of clinical profiles of CA 19-9⁺ and CA 19-9⁻ patients showed the CA 19-9⁺ group to include significantly higher proportions of patients with vessel invasion (P = 0.032), pleural invasion (P = 0.023), cancer invasive factors (P = 0.005) and positive PAS stain (P = 0.001). CA 19-9⁺ patients had significantly shorter recurrence-free survival than CA 19-9⁻ patients (P = 0.030). The Kaplan-Meier curve is shown in Fig. 4. We also investigated the association between EGFR status and survival in the 116 patients with stage I lung adenocarcinoma; for whom 110 (95 %) had EGFR mutation analyses available, which showed 61 (53 %) to have EGFR mutations. Log-lank analysis revealed that EGFR status had no prognostic effect on recurrence-free survival (P = 0.569), or OS (P = 0.171).