Intratumoral Fusobacterium Nucleatum Levels Predict Therapeutic Response to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma

Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide (1). In spite of advances in multimodal therapies, including surgical removal of tumors, chemotherapy, radiotherapy and chemoradiotherapy, esophageal cancer remains a malignancy with high degree of fatality and overall 5-year survival rates of 15% to 20% (2, 3). Globally, esophageal squamous cell cancer (ESCC) is the predominant histologic subtype of esophageal cancer (4). In particular, ESCC cases make up to 80% of all esophageal cancers in developing countries (5). The standard treatment strategy for locally advanced esophageal cancer in Western and Asian countries comprises neoadjuvant chemoradiotherapy or chemotherapy (NAC), followed by surgery (6, 7). Previous studies have shown that patients who respond well to NAC often exhibit improved overall survival (8, 9). A combination of cisplatin and 5-fluorouracil (5-FU) is currently used as standard chemotherapeutic regimen for patients with esophageal cancer; however, the reported response rates remain relatively poor (10, 11). A recent study reported that addition of docetaxel to this regimen significantly improved the therapeutic response in patients with node-positive esophageal cancer (12). Nevertheless, most tumors acquire resistance to chemotherapeutic agents with subsequent treatment failure. Furthermore, there is currently no effective molecularly targeted therapy available for esophageal cancer, and the efficacy of immunotherapy in these patients remains unclear.

To improve treatment response in patients with esophageal cancer, it is of paramount importance to elucidate the underlying mechanism(s) that confer chemotherapeutic resistance in these patients. It has been postulated that cancer chemoresistance is attributed to complex interplay between gene regulation and external environmental factors. In this context, in recent years, gut microbiota has garnered a lot of attention in various malignancies, and it has been linked to both initiation and progression of gastrointestinal cancers through modulation of intestinal inflammation (13–15) and tumor-related signaling pathways (16). Recent studies have demonstrated that composition of the gut microbiome can significantly influence response to immunotherapy (17) and chemotherapy (18). Two recent independent studies identified an overabundance of Fusobacterium nucleatum (F. nucleatum) in colorectal cancer tissues using metagenomic analysis (19, 20) and a high prevalence of F. nucleatum in these tissues associated with worse overall survival (21). In line with these observations, we identified that even in patients with ESCC, the presence of intratumoral F. nucleatum in neoplastic tissues was significantly associated with poor patient survival (22). Interestingly, building upon this growing evidence, a recent study reported that patients with colorectal cancer who experienced increased incidence of tumor recurrence also possessed significantly higher burden of intratumoral F. nucleatum in their primary cancer tissues compared with those who did not exhibit tumor recurrence (23). In functional studies, F. nucleatum has been shown to enhance colorectal cancer chemoresistance through modulation of autophagy (23). In spite of the collective evidence highlighting the clinical importance of F. nucleatum in gastrointestinal cancers, whether changes in its expression levels contributes to patient prognosis and chemotherapeutic response in patients with ESCC has not yet been explored.

Accordingly, in this study, we report that increased levels of intratumoral F. nucleatum associate with advanced tumor stage and poor survival. We also observed that higher burden of this microorganism in ESCC tissues predicted recurrence-free survival (RFS), as well as associated with poor response to NAC in patients with ESCC.

This study analyzed a total of 551 cases with ESCC, which consisted of two independent clinical cohorts. A first patient cohort (training) included 207 patients with ESCC, who were surgically treated at the Nagoya University Hospital (Nagoya, Japan) between October 2001 and October 2015. The second patient cohort (validation) comprised 344 patients with ESCC who underwent surgical resections, including 316 with radical surgeries, at the Kumamoto University Hospital (Kumamoto, Japan) between 2005 and 2016. Furthermore, this cohort included 187 patients that experienced surgery alone, 41 who received neoadjuvant chemoradiation therapy and 116 patients with NAC. Among these 116 patients in the NAC group, 101 patients were treated with two cycles of docetaxel, cisplatin, and 5-FU (DCF) regimen. The study workflow is summarized in Supplementary Fig. S1. Tumor depth (clinical T1–3) and regional lymph node involvement without distant metastases (N1) were used as the selection criteria for selecting patients for NAC treatment. Recurrence-free survival (RFS) was defined as the time period between the date of surgery to the time of tumor recurrence or death. Our study was conducted in accordance with the Declaration of Helsinki. A written informed consent was obtained from each patient, and the institutional review boards of all participating institutions approved this study. The patient characteristics are summarized in Table 1. The median follow-up duration for all cases after surgery was 20.4 months in the training cohort and 31.5 months in the validation cohort. The pathologic diagnosis of all ESCC tumor tissue specimens was confirmed histologically, and the tumor–node–metastasis (TNM) staging was determined according to the American Joint Committee on Cancer staging handbook (7th edition; ref. 24), prior to and after surgery.

The NAC regimen consisted of 2-hour intravenous administration of 60 mg/m² docetaxel beginning on day 1, a 24-hour continuous intravenous infusion of 350 mg/m² 5-FU from days 1 through 5, and 1-hour intravenous administration of 6 mg/m² cisplatin from days 1 through 5. Two scheduled courses of NAC regimen were administered 3 weeks apart prior to esophagectomy. Surgery was carried out within 4 to 6 weeks following the final treatment day of preoperative chemotherapy, when curative resection was considered feasible.

Genomic DNA from fresh frozen tissues in the training cohort were extracted using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Likewise, genomic DNA from the formalin-fixed paraffin-embedded (FFPE) tissues in the validation cohort were extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen). The amount of F. nucleatum DNA was quantified by use of a qPCR assay. The nus G gene of F. nucleatum and the reference human gene SLCO2A1 were amplified using custom TaqMan primer/probe sets (Applied Biosystems) in 384-well optical PCR plates, as described previously (22).

The response to chemotherapy was assessed in the validation cohort using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (25). Briefly, CT images were analyzed using with the following definitions: complete response (CR), as disappearance of all clinical and radiologic evidence of the tumor; partial response (PR), as decrease of 30% or more in the sum of longest diameters of all target measurable lesions; progressive disease (PD), as increase of more than 20% of the sum of longest diameters of all target measurable lesions or the appearance of new lesions; and stable disease (SD), as all other indications. Patients with CR or PR were defined as responders, while PD and SD were classified as nonresponders.

A total of 86 of 101 patients who received NAC also underwent PET/CT using a hybrid PET/CT imager, consisting of a dedicated GSO full-ring PET scanner and a 16-slice helical CT scanner (Gemini GXL16, Philips Medical Solutions). All patients fasted for a minimum of 5 hours prior to the examination. Emission scans were acquired in a 3D mode, with a 144 × 144 matrix, 60 minutes after intravenous injection of 185–300 MBq 18F-fluoro-deoxy-glucose (FDG), immediately after urination. PET/CT transmission data were acquired for the area defined from the base of the skull to the proximal thighs. Standardized uptake value (SUV_max) response was classified as follows (26): complete metabolic response (CMR), as complete resolution of FDG uptake within the measurable target lesion, with the appearance of no new lesion; partial metabolic response (PMR), with at least 30% reduction in SUV_max of FDG uptake; progressive metabolic disease (PMD), with more than 30% increase in the SUV_max of the FDG uptake or appearance of FDG avid new lesion/s that is/are morphologically typical of cancer; stable metabolic disease (SMD), as disease which did not qualify for CMR, PMR, or PMD. Patients with CMR or PMR were defined as responders.

The histopathologic response to NAC was classified into four categories according to the following criteria (27): grade 1, as no evidence of viable tumor cells; grade 2, with less than 10% viable tumor cells; grade 3, with 11%–50% viable tumor cells; and grade 4, with more than 50% viable tumor cells. Subsequently, grade 1–3 tumors were combined as the group of patients with response (TRG 1–3), while grade 4 tumors were classified as nonresponders (TRG 4).

All statistical analyses were carried out using JMP, version 10 (SAS Institute). Continuous variables were expressed as medians and were compared using a t test or Mann–Whitney U test. Categorical variables were compared using χ² or Fisher exact test. All P values were calculated using a two-sided test, and a P <0.05 was considered statistically significant. For time-to-event analyses, survival estimates were calculated using the Kaplan–Meier analysis, and the survival differences between groups were compared using the log-rank test. Associations between RFS and clinicopathologic features were evaluated by univariate Cox proportional hazards regression analysis. Parameters determined to be significant by univariate analysis were included in multivariate Cox proportional hazards regression analysis. Similarly, we analyzed associations between chemotherapeutic response and clinicopathologic features using by univariate and multivariate logistic regression analysis.

We first assessed the burden of F. nucleatum in ESCC tissues by a qPCR assay in two independent patient cohorts, where matched cancer and normal tissues were available. We observed that F. nucleatum DNA levels were significantly higher in cancer tissues compared with the paired adjacent normal tissues in both cohorts (training cohort; n = 45, P = 0.006; validation cohort; n = 48, P < 0.0001; Fig. 1A and B, respectively).

We next analyzed the abundance of F. nucleatum in the training (n = 207) and validation (n = 344) cohorts, based upon all tumor stages. Interestingly, we observed a marked enrichment of F. nucleatum in patients with ESCC with advanced (T2-T4) versus those with an earlier stage disease (T1), in both cohorts (P < 0.05; Fig. 1C and D).

Next, we determined the associations between F. nucleatum burden and various clinicopathologic features in two independent ESCC patient cohorts (training cohort; n = 207 and validation cohort; n = 344). The cut-off thresholds to categorize tumors into the high and low groups were determined using ROC analysis and Youden index, based on the level of F. nucleatum that provided the highest sensitivity and specificity to predict ESCC recurrence in the training cohort. The same cut-off values were then applied to the patient in the validation cohort to evaluate survival. We observed that there was no effect of age (P = 0.26), gender (P = 0.62), tumor location (P = 0.29), or N stage (P = 0.88) on the expression of expression of F. nucleatum in the cancer tissues within the training cohort. Similar results were noted in the validation cohort for age (P = 0.82), gender (P > 0.99), location (P = 0.42), and N stage (P = 0.12).

However, in the training cohort, high intratumoral F. nucleatum levels were associated with higher T category (P = 0.03) and in patients who received preoperative treatment (P = 0.03). Similarly, high levels of intratumoral F. nucleatum were significantly associated with larger tumor size (P = 0.004), higher T category (P < 0.001), higher TNM stage (P = 0.03), and in patients who underwent preoperative treatment (P = 0.01) in the validation cohort (Table 1). Collectively, our results indicate that high levels of F. nucleatum associate with an invasion depth in patients with ESCC.

Considering that presence of F. nucleatum in cancer cells is associated with advanced disease, we were curious to interrogate its relationship with tumor recurrence in patients with ESCC. Therefore, we determined the relationship between the F. nucleatum levels and cancer recurrence in the training cohort of 207 patients (87 patients with recurrence and 120 without recurrence), wherein we observed a significant association for higher F. nucleatum levels in patients with recurrence (P = 0.04; Fig. 2A). Likewise, these findings were subsequently confirmed in the validation cohort of 316 patients with ESCC, which included 91 patients without recurrence and 225 with recurrence. Here again, we noted that the overall levels of F. nucleatum were significantly higher in neoplastic tissues in ESCC patients with recurrence versus those without recurrence (P = 0.01; Fig. 2B).

To determine whether intratumoral F. nucleatum burden in patients with ESCC is associated with RFS, we performed Kaplan–Meier analysis in both cohorts. Interestingly, patients in the training cohort with high versus low intratumoral F. nucleatum levels exhibited a significantly poor RFS (log-rank P = 0.02; Fig. 2C); a finding which was also true when interrogated in the independent validation cohort (log-rank P = 0.003; Fig. 2D).

Because we observed a higher burden of F. nucleatum in patients with advanced ESCC (T2–4 vs. T1), we investigated whether the presence of this bacterium had any effect on patient survival, even in early ESCC. Patients with advanced ESCC stratified by the T category alone (T2–4 vs. T1) exhibited poor prognosis in both patient cohorts (Supplementary Fig. S2A and S2B). Importantly, however, when the T category was combined together with the F. nucleatum levels, we observed that even patients with early-stage T1 ESCC with high levels of this bacterium exhibited a worse RFS, which was similar to the one noted for patients with advanced disease, in both cohorts (training cohort: log-rank P = 0.002 and validation cohort: log-rank P = 0.009, Fig. 2E and F, respectively). These findings highlight that presence of high levels of F. nucleatum indicate an important prognostic biomarker potential for this bacterium in patients with ESCC.

Next, we were curious to investigate the clinical significance of F. nucleatum levels in term of patient survival in the context of other clinicopathologic features, using univariate and multivariate analysis, in both patient cohorts. In the training cohort, univariate Cox regression analysis revealed that patients with proximal location of tumors (HR = 2.04; 95% CI, 1.10–3.50; P = 0.02), and those with higher TNM stages (III/IV vs. I/II; HR = 3.32; 95% CI, 2.05–5.63; P < 0.0001), and those with high levels of F. nucleatum were associated with poor RFS (HR = 1.61; 95% CI, 1.06–2.52; P = 0.03; Table 2). These findings were further evaluated in a multivariate Cox model adjusted for various clinicopathologic features, which were in agreement with our univariate analysis and demonstrated that proximal location of tumors (HR = 3.09; 95% CI, 1.64–5.45; P = 0.001), and those with higher TNM stages (III/IV vs. I/II; HR = 3.78; 95% CI, 2.30–6.46; P < 0.0001), and those with high levels of F. nucleatum were associated with poor RFS (HR = 1.72; 95% CI, 1.12–2.70; P = 0.01), suggesting that this bacterium was indeed an independent risk factor for predicting poor RFS in the patients within the training cohort.

We subsequently confirmed our findings in an independent validation cohort, wherein, once again we observed that in univariate analysis, preoperative therapy (HR = 1.96; 95% CI, 1.29–3.00; P = 0.002), TNM stages (HR = 3.08; 95% CI, 2.03–4.72; P < 0.0001), and higher burden of F. nucleatum (HR = 1.96; 95% CI, 1.23–3.04; P = 0.004) was significantly associated with worse RFS. Similarly, in multivariate analysis, TNM stages (HR = 3.21; 95% CI, 1.81–5.70; P < 0.0001) and high levels of F. nucleatum (HR = 1.70; 95% CI, 1.06–2.65; P = 0.03) were significantly associated with poor RFS. Collectively, these data demonstrate that high levels of intratumoral F. nucleatum are an independent risk factor for poor RFS in patients with ESCC.

We examined whether higher burden of intratumoral F. nucleatum have any correlation with response to NAC in patients with ESCC. We first investigated this association in the context of imaging data available to us from the CT scans. Of the 101 patients who underwent NAC treatment in the validation cohort, the F. nucleatum-high group had a significantly lower number of responders [i.e., patients with CR or PR; 42.9% (12/28) vs. 67.1% (49/73) in the F. nucleatum-low group; P = 0.04; Fig. 3A and B].

Next, we interrogated this correlation as determined by the metabolic response rates determined by SUV_max values obtained from PET/CT imaging. Reassuringly, these analyses also revealed that patients with higher burden of F. nucleatum had significantly fewer responders [i.e., patients with CMR or PMR; 47.6% (10/21) vs. 87.7% (57/65) in the low F. nucleatum group; P = 0.0004; Fig. 3C and D].

Finally, we performed the pathologic assessment of all patients based upon tumor regression grade (TRG) analysis. In these analyses, we noted that F. nucleatum levels were significantly higher in patients with ESCC with a low versus high pathologic response (TRG 4 vs. TRG 1, 2 and 3; P = 0.003; Fig. 3E and F). Taken together, these results illustrate that patients with high intratumoral levels of F. nucleatum appear to have greater resistance to NAC treatment.

Next, we analyzed the results of CT (RECIST), PET/CT, and TRG in univariate and multivariate settings to determine the clinical significance of F. nucleatum as a potential biomarker of chemotherapeutic response in patients with ESCC belonging to the validation cohort. The univariate logistic regression analysis revealed that higher levels of F. nucleatum associated with an overall poor chemotherapeutic response to NAC in all three approaches [RECIST: (OR), 2.72; 95% CI 1.12–6.78, P = 0.03; PET/CT: OR, 7.84; 95% CI 2.58–25.4, P = 0.0003; and TRG: OR, 11.6; 95% CI 2.25–214, P = 0.001; Table 3].

Likewise, multivariate analysis also revealed that high levels of intratumoral F. nucleatum burden was an independent risk factor for poor response to NAC in all three criteria (RECIST: OR, 2.97; 95% CI 1.19–7.73, P = 0.02; PET/CT: OR, 7.66, 95% CI 2.37–26.8, P = 0.0006; and TRG: OR, 10.3; 95% CI 1.96–190, P = 0.003). Collectively, these results illustrate that F. nucleatum is an important independent risk factor and a potential biomarker for predicting response to NAC in patients with ESCC.

With the growing recognition for the role of microbiome in human disease, over the last decade, one such organism, F. nucleatum, has been identified as an important bacterium linked to the pathogenesis of multiple human cancers. In this study, we for the first time, interrogated the clinical significance of F. nucleatum as a potential prognostic and predictive biomarker of response to neoadjuvant chemotherapy in large, multiple, independent cohort of patients with ESCC, and for the first time, investigated F. nucleatum as a potential predictive biomarker of response to neoadjuvant chemotherapy. In this study, we make several novel observations. First, we demonstrate that F. nucleatum burden is significantly higher in patients with ESCC with advanced disease stage. Second, we describe that higher levels of this bacterium are present in patients with recurrence, and are an independent risk factor for predicting poor RFS in ESCC. Third, we illustrate that using RECIST, PET/CT, and TRG analysis, higher burden of F. nucleatum predicts poor response to neoadjuvant chemotherapy (NAC) in patients with ESCC; collectively highlighting the potential possibility of its prognostic and predictive biomarker utility, as well as suggest the possibility of using an antibiotic intervention to target this bacterium for improving the therapeutic response rates to chemotherapy in patients with ESCC. In addition, as we developed a PCR-based cut-off to measure the F. nucleatum levels in the training cohort patients, and subsequently applied these in an independent validation cohort, we are enthused that our findings can be further validated in prospective settings for response prediction to neoadjuvant chemotherapy.

It has been recognized that F. nucleatum is frequently present in the human oral cavity, and acts as a pathogen in periodontal disease (28). Recently, several studies have reported that high F. nucleatum burden correlates with poor prognosis in colorectal cancer (21, 29, 30). Moreover, we previously reported a similar positive correlation between high F. nucleatum levels and poor overall and cancer-specific survival in patients with ESCC (22). The potential role of F. nucleatum in gastrointestinal cancers is poorly understood. Experimental evidence in colorectal cancer has provided mechanistic insights that F. nucleatum expresses adhesin protein, FadA, on the bacterial cell surface. FadA can bind to E-cadherin, activates β-catenin signaling, and promotes colorectal cancer cell proliferation (31). In this study, F. nucleatum burden was significantly higher in patients with ESCC with advanced stage. However, T1 ESCC patients with high levels of F. nucleatum exhibited a worse RFS, analogous to patients with advanced disease, suggesting that this bacterium, even in patients with early-stage ESCC promotes aggressive tumor behavior and could impact patient prognosis.

To further investigate the role of F. nucleatum, it was recently demonstrated that mice bearing colorectal cancer and treated with an antibiotic were found to have lower levels of this bacterium and exhibited reduced cell proliferation and tumor growth, suggesting that antibiotics may be helpful in the treatment of F. nucleatum–associated cancers (32). Accumulating evidence suggests that the gut microbiota modulates local immune response, and in turn might alter the efficacy of chemotherapy (18, 33) and immunotherapy (34, 35). In one such study, chemotherapeutic response was modulated by adaptive immunity in ovarian cancer (36). Although these preclinical evidences indicates that microbiota appears to modulate chemotherapeutic response in multiple cancer types (37, 38), to the best of our knowledge, none of the studies have thus far evaluated the clinical significance of F. nucleatum in the context of responsiveness to chemotherapeutic treatment in patients with cancer. Herein, we fill this important gap in knowledge, and evaluated therapeutic response using three commonly used and well-established approaches for drug resistance in ESCC patients. In spite of the use of RECIST as one of the most widely used tumor response metric (25), it has several limitations due to its dependence on morphologic changes (39). RECIST criteria can often select lymph nodes as target lesions in patients with ESCC. In contrast, 18F-FDG PET is considered as a superior method which overcomes the limitations of RECIST. Because metabolic changes are thought to be more closely related to malignant potential of tumors (40), PET/CT is emerging as a more accurate noninvasive imaging modality for initial staging and response assessment in patients with ESCC (41). On the basis of these findings, PET response criteria in solid tumors (PERCIST), which is RECIST using 18F-FDG PET, has recently been proposed as an optimal method for standardized evaluation of the metabolic tumor response rates (39).

In this study, we observed significant differences between response classifications and F. nucleatum levels in ESCC tissues. Interestingly, PET response and tumor regression grade (TRG) were more strongly associated with F. nucleatum levels compared with RECIST, in patients with ESCC receiving NAC treatment. While RECIST in patients with ESCC primarily evaluates shrinkage of lymph nodes, PET/CT and TRG reflect the response of the primary tumor itself. In this study, because F. nucleatum levels in tumor tissues correlate with higher T category, our data imply that this bacterium might be involved in modulating chemotherapeutic response more directly. While specific mechanism(s) underlying chemotherapeutic response of F. nucleatum in cancer remain unclear, several studies have investigated bacteria-induced drug resistance using in vitro and in vivo models. Yu and colleagues reported that F. nucleatum activates autophagy-related pathways in colorectal cancer through modulation of TLR4 and MYD88 innate signaling, along with certain miRNAs that subsequently promote chemoresistance (23). Likewise, Geller and colleagues reported that intratumoral gamma-proteobacteria modulated the chemotherapeutic response by converting gemcitabine into an inactive metabolite through regulation of cytidine deaminase in pancreatic cancer (42). Nonetheless, further studies are required to interrogate and validate the findings of this study, and elucidate the mechanisms by which F. nucleatum modulates the chemotherapeutic response in patients with ESCC.

Although our results indicate that F. nucleatum levels could serve as a potential biomarker for predicting survival and response to NAC in patients with ESCC, there are certain limitations of our study. The detection rates of F. nucleatum were different between the training and validation cohorts. We analyzed frozen tissues in the training cohort of patients, and FFPE tissues in the validation cohort. The qPCR method is currently the most commonly used method for the quantification of F. nucleatum levels; however, most the detection rates of F. nucleatum in frozen tissues are generally higher versus FFPE tissues (43, 44). There is a possibility that tissue fixation during the processing of FFPE tissues might be an important factor for the reduced detection rates of F. nucleatum (45) in clinical specimens.

In conclusion, we demonstrate that high intratumoral F. nucleatum levels associated with tumor recurrence and poor RFS in two large, independent cohorts of patients with ESCC. More importantly, our results indicate that high burden of F. nucleatum in ESCC is predictive of response to neoadjuvant chemotherapy. Collectively, our data highlight that F. nucleatum is not only an important predictive biomarker of chemotherapeutic response, but might be a potential target of antibiotic intervention for improving the therapeutic response rates in patients with ESCC.

No potential conflicts of interest were disclosed.

Conception and design: K. Yamamura, B. Hideo, A. Goel

Development of methodology: K. Yamamura, D. Izumi, R. Kandimalla, A. Goel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Yamamura, D. Izumi, R. Kandimalla, F. Sonohara, Y. Baba, Y. Kodera

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Yamamura, D. Izumi, R. Kandimalla, A. Goel

Writing, review, and/or revision of the manuscript: K. Yamamura, R. Kandimalla, B. Hideo, A. Goel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Yamamura, Y. Kodera, A. Goel

Study supervision: N. Yoshida, B. Hideo, A. Goel

We thank Yuko Ogata, Keisuke Miyake, and Kazuo Okadome for collecting clinical samples and information. We thank Shusuke Toden for editing the manuscript. We thank Lauren J. Patterson, Preethi Ravindranathan, and Divya Pasham for help with performing various experiments. This work was supported by the grants CA72851, CA181572, CA184792, CA202797, and CA187956 from the NCI, a grant (RP140784) from the Cancer Prevention Research Institute of Texas (CPRIT), pilot grants from the Baylor Sammons Cancer Center, as well as funds from the Baylor Scott & White Research Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.