The clinical implications of FDG-PET/CT differ according to histology in advanced gastric cancer

Background The prognostic impact of preoperative 18F-FDG PET/CT in advanced gastric cancer (AGC) remains a matter of debate. This study aims to evaluate the prognostic impact of SUVmax in preoperative 18F-FDG PET/CT of AGC according to histologic subtype, with a focus on the differences between tubular adenocarcinoma and signet ring cell (SRC) carcinoma. Methods As a discovery set, a total of 727 AGC patients from prospective database were analyzed according to histologic subtype with Cox proportional hazard model and p-spline curves. In addition, another 173 patients from an independent institution was assessed as an external validation set. Results In multivariate analysis, high SUVmax in preoperative 18F-FDG PET/CT of AGC was negatively correlated with disease-free survival (DFS) and overall survival (OS) in patients with diffuse type (DFS: HR 2.17, P < 0.001; OS: HR 2.47, P < 0.001) or SRC histology (DFS: HR 2.26, P = 0.005; OS: HR 2.61, P = 0.003). This negative prognostic impact was not observed in patients with intestinal type or well or moderately differentiated histology. These findings have been consistently confirmed in a validation set. The p-spline curves also showed a gradual increase in log HR as SUVmax rises only for SRC histology and for diffuse-type AGC. Finally, a novel predictive model for recurrence of AGC with diffuse type or SRC histology was generated and validated based on the preoperative SUVmax. Conclusions Preoperative high SUVmax of AGC is a poor prognostic factor in those with diffuse type or SRC histology. This study is the first to demonstrate the differential prognostic impact of preoperative PET/CT SUVmax in AGC according to histologic subtype and provide a clue to explain previous discrepancies in the prognostic impact of preoperative PET/CT in AGC. Prospective studies are required to validate the role of preoperative SUVmax in AGC. Electronic supplementary material The online version of this article (10.1007/s10120-018-0847-5) contains supplementary material, which is available to authorized users.


Introduction
F-18 fluoro-2-deoxyglucose positron emission tomography/computed tomography ( 18 F-FDG PET/CT) has become an indispensable method for the diagnosis, staging, and response evaluation of many malignancies [1][2][3]. In gastric cancer (GC), 18 F-FDG PET/CT is a useful tool for the diagnosis of recurrent disease after curative surgery [4][5][6][7]. However, the role of preoperative 18 F-FDG PET/CT is not yet fully established. While the National Comprehensive Cancer Network guidelines recommend the use of preoperative 18 F-FDG PET/CT in GC patients to rule out distant metastasis, the prognostic impact of preoperative 18 F-FDG PET/CT remains a matter of debate. Several reports suggest a potential prognostic role for preoperative 18 F-FDG PET/CT, while others argue against this [8][9][10]. These discrepancies may be Hong Jae Chon, Chan Kim, and Arthur Cho equally contribute to this study.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1012 0-018-0847-5) contains supplementary material, which is available to authorized users. due in part to small sample sizes and heterogeneous patient populations in different studies. Notably, 18 F-FDG PET/CT has low sensitivity in detecting early GC (EGC) and signet ring cell (SRC) GC, but many previous studies overlooked this, including heterogeneous populations in the studies and analyzing the clinical characteristics of the population as a whole. Because SRC GC is a unique histologic subtype of GC with a distinct tumor biology and bioenergetics, it should be analyzed separately [11][12][13][14]. The present study evaluated the prognostic impact of SUV max in preoperative 18 F-FDG PET/CT of advanced GC (AGC) according to histologic subtype, with a focus on the differences between tubular adenocarcinoma and SRC carcinoma.

Patient selection
Between January 2006 and December 2013, patients with GC who underwent 18 F-FDG-PET/CT and subsequent curative surgical resection at Yonsei Cancer Center, Severance Hospital, Seoul, Korea, were enrolled in the study. A predesigned data collection format was utilized to extract data from a prospectively maintained database. The main eligibility criteria were as follows: (1) pathologically confirmed AGC of tubular adenocarcinoma or SRC-histologic subtype; (2) available documented information regarding the primary tumor site, postoperative pathological stage, surgery, recurrence, and survival; and (3) patients who received curative resection including those who presented with enlarged paraaortic lymph nodes having radical surgical resection with a curative aim accompanied by paraaortic lymph node dissection. The main exclusion criteria were as follows: (1) patients with EGC; (2) patients who received neoadjuvant chemo-or radio-therapy; and (3) patients with multiple primary cancers. After applying these criteria, 727 of the original 1605 patients were included in the final analysis (Fig. S1). The pathological stage was classified according to the American Joint Committee on Cancer (AJCC) staging manual (7th edition). This study was approved by the Institutional Review Board of Severance Hospital (#2015-1751-001). For a validation cohort, AGC patients who underwent preoperative 18 F-FDG-PET/CT and curative surgical resection at CHA Bundang Medical Center, Seongnam, Korea, between March 2007 and February 2014, were enrolled in the study. Data acquisition and analysis were adopted identically as in the aforementioned institution.
The WHO and the Lauren classifications were used for the histopathological evaluation of surgical specimens. Tubular adenocarcinoma was additionally classified as being well, moderately, or poorly differentiated according to the WHO classification. Accordingly, we divided the patients into three groups for further analyses: well or moderately differentiated (WMD), poorly differentiated (PD), and SRC. In terms of the Lauren classification, the tumors were classified as intestinal, diffuse, or mixed type.

F-FDG PET/CT and image analyses
All 18 F-FDG PET/CT were performed with either the Discovery STe PET/CT (GE Healthcare, Milwaukee, WI, USA) or the Biograph TruePoint 40 PET/CT (Siemens Healthcare, Erlangen, Germany). All patients fasted for at least 6 h before the scan, and the glucose level in the peripheral blood of all patients was confirmed to be 140 mg/dL or less before 18 F-FDG injection. Approximately, 5.5 MBq 18 F-FDG/kg body weight was administered intravenously 1 hour before image acquisition. After the initial low-dose computed tomography (CT) (Discovery STe: 30 mA, 140 kVp, Biograph TruePoint: 36 mA, 120 kVp), standard PET/CT imaging was performed from the neck to the proximal thighs with acquisition times of 2.5 min/bed position for the Biograph Truepoint 40 PET/CT and 3 min/bed position for the Discovery STe scanner in three-dimensional mode. Images were then reconstructed using ordered subset expectation maximization (2 iterations, 20 subsets).
The images were retrospectively reviewed on a GE AW 4.0 workstation by two experienced nuclear medicine specialists (A.C. and M.Y.) who were unaware of the patients' clinical information, except for the diagnosis of GC. The evaluation of 18 F-FDG PET/CT images was performed in two steps. First, 18 F-FDG PET/CT images of all patients were visually assessed and the patients were classified as positive or negative with respect to 18 F-FDG uptake in the primary tumor. Lesions showing focally increased 18 F-FDG uptake that exceeded the uptake in the surrounding stomach wall and corresponding cancer lesions as observed by contrast-enhanced CT images and gastroduodenoscopy were classified as positive 18 F-FDG uptake. Focally or diffusely increased 18 F-FDG that was indistinguishable from physiological gastric wall uptake was judged to be negative 18 F-FDG uptake. After the visual assessment, the maximum standardized uptake value (SUV max ) of the primary lesion was obtained and recorded for semi-quantitative analysis.
For the validation cohort, the PET/CT imaging for 173 AGC patients from CHA Bundang Medical Center was performed with Biograph mCT 128 scanner (Siemens Medical Solutions, Knoxville, TN, USA). Initial low-dose CT scans for attenuation correction (120 kV, 120 mA, 3 mm section width, 3 mm collimation) and PET/CT scans of same area with three-dimensional mode were acquired consecutively. Images were reconstructed on 400 × 400 matrices using the TrueX algorithm plus time-of-flight (TOF) reconstruction and analyzed using a dedicated workstation and software (Syngo.via, Siemens Medical Solutions, Knoxville, TN, USA). Unless otherwise stated, all other methods applied for image acquisition and data analysis were adopted identically as in the aforementioned institution.

Statistical analysis
The cut-off date was December 31, 2015. The mean SUV max was compared according to the patients' basic demographic and clinical characteristics using independent sample t tests or analysis of variance. For pairwise comparisons of each level of categorical variables, the statistical significance was adjusted for inflated type I errors from multiple comparisons using the Bonferroni method.
Relapse-free survival (RFS) was measured from the time of surgery to initial tumor relapse (either local or distant) or death from any cause, and overall survival (OS) was calculated as the time from surgery to death from any cause or to the last follow-up date. Survival outcomes of the group with high SUV max were compared with survival outcomes of the group with low SUV max based on the median SUV max for each histologic subgroup using the log-rank test. The Cox proportional hazards model was used for multivariable analysis of prognostic factors, including age at diagnosis, sex, stage, and PET/CT SUV max . To determine additional associations between SUV max and survival outcomes, we examined the Cox regression model using the penalized spline smoothing method as described previously [15]. The performance of prognostic models was measured by Harrell's c-index. We assessed model calibration by plotting the model-predicted-and actual observed 3-and 5-year RFS probabilities as calculated using the Kaplan-Meier method. The bootstrapping method with 1000 re-samples was used for adjusting bias and checking the interval validation. Statistical significance was set as P < 0.05 for all analyses. All statistical analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA), SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and R package, version 3.2.4 (http:// www.R-proje ct.org).

Patient characteristics
The baseline characteristics of the patients are summarized in Table 1. A total of 727 patients with pathologically confirmed AGC were analyzed. The majority of patients were male (68.0%), and the median age at diagnosis of AGC was 60 years (range 26-94) years. All patients underwent radical gastrectomy; 6.9% were pathological stage I, 26.3% were stage II, 57.4% were stage III, and 9.5% were stage IV. This study included 63 stage IV patients who presented with enlarged paraaortic lymph nodes that were observed by either preoperative PET/CT or CT. These patients underwent radical surgical resection with a curative aim accompanied by paraaortic lymph node dissection. Regarding the WHO classification, 36.9% had WMD histology, 48.0% had PD histology, and the remaining 15.1% had SRC histology. When patients were classified according to the Lauren classification, 46.8% had intestinal-type AGC, 44.6% had diffuse type, and 8.7% had mixed type. Adjuvant chemotherapy was given in 88% of patients, excluding stage I patients (50 patients), those with poor performance after surgery (16 patients), and 21 patients who refused chemotherapy. All patients who had adjuvant chemotherapy received fluorouracil-based chemotherapy.

SUV max and histologic subtype
This study only included patients with AGC, and 80% of all patients showed a positive 18 F-FDG uptake (Supplementary Figure 1). In terms of WHO classification, 86% of WMD, 81% of PD, and 68% of SRC showed positive 18 F-FDG uptake. According to the Lauren classification, 84% of intestinal type and 76% of diffuse type GC showed positive 18 F-FDG uptake. In terms of stage, 72% of stage I, 75% of stage II, 82% of stage III, and 94% of stage IV showed positive 18 F-FDG uptake. Table 1 shows SUV max according to various clinicopathologic variables. Notably, SUV max was significantly correlated with the histologic type of AGC by both the WHO and Lauren classifications (Fig. 1a). The mean SUV max of AGC patients with SRC histology was 51% lower than that of AGC patients with WMD histology (4.5 ± 1.9 vs. 9.2 ± 6.1, P < 0.001). The majority of patients that had AGC with SRC histology had SUV max less than 5, whereas the majority of patients that had AGC with WMD histology had SUV max greater than 5. When the SUV max was analyzed according to the Lauren classification, patients with diffuse-type AGC, which mostly had SRC histology, had 33% lower SUV max than those with intestinal-type AGC, which mostly had WMD histology, (6.2 ± 4.0 vs. 9.2 ± 6.2, P < 0.001) (Fig. 1a). Moreover, the SUV max also correlated with the progression of stage, especially T stage (P < 0.001), but not nodal (N) stage (P = 0.427). Intriguingly, the SUV max correlated well with the maximal size of the tumor mass in AGC with WMD histology or intestinal type (Fig. 1b). However, the degree of correlation between SUV max and maximal tumor size was relatively weak in AGC with SRC histology or diffuse type. Collectively, these findings demonstrate the distinct tumor biology of AGC with WMD or SRC histology, especially in terms of glucose metabolism.

The prognostic impact of SUV max according to histologic subtype
With the median follow-up duration of 32.5 months, 357 (49%) patients recurred and 301 (49%) died. To evaluate the prognostic impact of each histologic subtype, the survival outcomes were compared between the high-and low-SUV max groups. The cut-off value was the median SUV max of each histologic group (Table 1). In terms of DFS, AGC patients with high SUV max had significantly shorter DFS if they had diffuse-type AGC or SRC histology (P < 0.001 and P < 0.001, respectively), while there were no differences in the DFS of AGC patients with intestinal type or WMD histology (Fig. 2a, b). This was also true for OS; high SUV max only had a negative prognostic impact in AGC with diffuse type or SRC histology (P < 0.001 and P < 0.001, respectively; Fig. 2c, d).
In the Cox proportional hazard model, which was adjusted for sex, age, T stage and N stage (Table 2), high SUV max was also negatively correlated with DFS and OS in AGC patients with SRC histology (DFS: HR 2.26, P = 0.005; OS: HR 2.61, P = 0.003). Moreover, high SUV max was negatively correlated with DFS and OS in AGC patients with diffuse-type AGC (DFS: HR 2.17, P < 0.001; OS: HR 2.47, P < 0.001). This negative prognostic impact was not observed in AGC patients with WMD histology or intestinal type. In addition, even when the Cox regression model was applied with the exception of 16 stage IV patients, high SUV max was still a poor prognostic factor in SRC and diffuse-type gastric cancer (Supplementary Table 2).
To externally validate these findings, we also analyzed data from an independent institution. The same results were consistently observed in this validation cohort: (1) diffuse-type AGC with SRC histology had lower SUV max compared with intestinal-type AGC with WMD histology (Table S1); and (2) higher preoperative SUV max indicated poorer prognosis in AGC with SRC histology and diffuse type, but not in AGC patients with WMD histology and intestinal type (Fig. S2).
Taken together, these data confirmed that high SUV max has an independent negative prognostic role in AGC patients with SRC or diffuse-type AGC.

Prognostic implications of SUV max as a continuous variable (p-spline curve)
To further investigate the role of SUV max as a continuous variable in survival analysis, p-spline curves for DFS were generated with the R program as described previously [15] after adjusting for sex, age, T stage and N stage (Fig. 3). The results were consistent with those from the Cox regression analysis with dichotomous variables. The p-spline curves showed a gradual increase in log HR as SUV max rises only for SRC histology (Fig. 3a, right) and for diffuse type (Fig. 3b, right). There was no definite trend for WMD and PD histology (Fig. 3a, left) or intestinal type (Fig. 3b, left). This confirmed that SUV max is a continuous variable that can predict DFS in AGC patients with SRC histology or diffuse-type AGC.

Generating a predictive model for recurrence probability based on preoperative SUV max in SRC or diffuse-type AGC
To predict recurrence after curative surgery more precisely for AGC, we tried to develop a novel predictive Fig. 2 Kaplan-Meier survival curves comparing the high-and low-SUV max groups in each histologic subtype. a, b High SUV max only had a negative prognostic impact on disease-free survival (DFS) in AGC with SRC histology or diffuse type. c, d High SUV max only had a negative prognostic impact on overall survival (OS) in AGC with SRC histology or diffuse type model based on preoperative SUV max . Recurrence-free probabilities at 1, 3, and 5 years were calculated for AGC with SRC histology (Fig. 4a) or diffuse type (Fig. 4b) after adjusting for sex, age, T stage and N stage. The RFS rate gradually decreased as SUV max increased, and the 5-year RFS rate was less than 20% when the SUV max was greater than 5. To evaluate the performance of our predictive model, we generated calibration curves (Fig. 5) that showed good agreement between the predicted and actual RFS; the bootstrap-corrected c-indices of the model were 0.751 (95% CI 0.675-0.827) for AGC with SRC histology and 0.687 (95% CI 0.644-0.730) for diffuse-type AGC. Thus, we were able to generate and internally validate our novel predictive model for recurrence in AGC with SRC histology or diffuse-type AGC.

Discussion
Gastric cancer is increasingly recognized as a heterogeneous disease [11,14,16,17]. Classically, it is classified according to its histology, i.e., as intestinal type or diffuse type [18]. Intestinal-type GC is more predominant in older people and in men, whereas diffuse-type GC is more frequently found in younger women. Recently, genomic data is widely utilized to develop molecular classification systems for GC. The TCGA Research Network proposed a classification system to distinguish GC into four subtypes: (1) Epstein-Barr virus (EBV)-positive, with the highest DNA methylation levels; (2) microsatellite instability (MSI), characterized by hypermutated tumors;  (3) genomic stability (GS), which represents 20% of GC and comprises the majority of diffuse-type GC, has the most abundant CDH1 mutations and also shows increased RHOA mutations and CLDN18-ARHGAP fusions; and (4) chromosomal instability (CIN), which accounts for 50% of patients and is characterized by frequent TP53 mutations and high percentage of intestinal-type GC [16,19]. However, current clinical practice does not take this heterogeneity into account; rather, GC is regarded as a single type of malignancy, and a one-size-fits-all approach is applied. Although some previous studies have shown that the SUV max in PET/CT can differ markedly according to the histologic subtype, most studies still evaluate the prognostic impact of SUV max by considering GC to be a single disease entity rather than categorizing it into the various histologic subtypes. Not surprisingly, this has resulted in inconsistencies between studies. Furthermore, despite the large number of studies that have already observed poor 18 F-FDG PET uptake in patients with EGC only having mucosal or submucosal invasion, most studies still enroll patients with EGC. The largest preoperative study to date was reported by Lee et al., who evaluated the prognostic impact of PET/ CT in 271 GC patients [8]. Because approximately half of the enrolled patients had EGC, 45% of the patients had no detectable 18 F-FDG uptake, and so only the remaining 149 patients were available for further analysis. Consequently, the subgroup analysis was limited by the small sample size.
To overcome the limitations of previous studies, the present study prospectively collected data from more than 700 patients with AGC while excluding patients with EGC. Moreover, to avoid oversimplification, the patients were divided and analyzed according to their histologic subtypes. Furthermore, the prognostic impact of SUV max was evaluated not only as a dichotomous variable as determined by the median SUV max but also as a continuous variable by analyzing p-spline curves. As a result, we were able to reveal the distinct prognostic impact of SUV max in 18 F-FDG PET/CT according to histologic subtype.
First, diffuse-type AGC with SRC histology had lower SUV max compared with intestinal-type AGC with WMD histology, which is in agreement with previous studies. Second, although the SUV max of diffuse-type AGC was lower than that of intestinal type, it had a significant prognostic impact in terms of survival outcome. On the other hand, the SUV max of intestinal-type AGC was more directly correlated with primary tumor size than diffuse-type AGC, but it did not have any prognostic impact. These finding provide a clue to explaining previous discrepancies regarding the prognostic impact of 18 F-FDG PET/CT in GC patients. Finally, we established and validated a novel model that utilizes the preoperative SUV max to predict tumor recurrence after surgery in patients with SRC or diffuse type, which may be a useful tool for clinical application.
Each histologic subtype of GC differs in its biology, especially in its metabolic profiles, which leads to different 18 F-FDG uptake patterns [20]. Among the various histologic types of GC, SRC stands out as a unique subtype due to its distinct molecular and metabolic features. In terms of the glucose transporter GLUT-1, SRC is reported to express GLUT-1 at lower levels than WMD adenocarcinoma, leading to reduced 18 F-FDG uptake [21,22]. In addition, SRC has lower levels of the pyruvate kinase M2 isoform (PKM2) compared with other histologic subtypes; PKM2 is responsible for ATP production in the last step of glycolysis [12]. Furthermore, PKM2 expression is correlated with poor prognosis in SRC, while other subtypes are not. These metabolic characteristics help explain the different patterns and prognostic values of 18 F-FDG PET/CT in different histologic subtypes of GC, and our data highlight the importance of dividing GC into different histologic subtypes before PET/ CT analysis.
The main limitation of our study is the retrospective nature of data collection. Although we verified our findings in two independent cancer centers in Korea, more studies are needed to validate our findings in a prospective cohort. Especially, this study did not include patients who underwent preoperative treatment. Therefore, it is necessary to evaluate the role of PET/CT in Western patients who received preoperative treatment with other studies. In addition, this study only included patients with AGC, thus most patients received adjuvant chemotherapy. There are limitations in analysis of the effect of adjuvant chemotherapy on the prognostic remodeling. Second, volumetric PET parameters were not measured due to the large number of cases. Additional studies are needed to further evaluate the value of volumetric PET parameters rather than SUV max for predicting clinical outcomes in intestinal-type AGC.
In conclusion, this study demonstrated the differential patterns and prognostic impact of preoperative PET/CT SUV max in AGC according to histologic type. Although the SUV max did not have significant prognostic impact in WMDand intestinal-type AGC, higher preoperative SUV max indicated poorer prognosis in SRC and diffuse-type AGC. Novel predictive models for recurrence probability can be provided based on the preoperative SUV max in patients with SRC or diffuse-type AGC. To validate these findings, we are preparing a prospective trial. If the results of this study are confirmed in a prospective trial, SRC or diffuse-type gastric cancer patients with high SUV max should be stratified in adjuvant chemotherapy. Ultimately, a clinical trial in which novel or more intensive therapy approaches are applied to SRC and diffuse-type AGC patients with high SUV max should be performed.