G-CSF-producing renal cell carcinoma characterized by IL-8-induced marked neutrophil infiltration to tumor tissue

Although G-CSF-producing tumor is well-known, co-pr oduction of IL-8 by such tumor and IL-8-induced neutrophil chemotaxis to tumor tis sue have not been recognized. A 65-year-old male was diagnosed with papillary renal cel carcinoma accompanied by marked intratumoral neutrophil infiltration after l eft radical nephrectomy. At presentation, laboratory tests showed a white blood cell count of 47.8×10 /l with 84.1% neutrophils and a high CRP of 18.3 mg/dl. Serum con centrations of G-CSF, IL-6, and IL-8 were elevated to 143 pg/ml, 474 pg/ml, and 1,2 60 pg/ml, respectively. After the operation, the white cell count and serum concentra tions of these cytokines returned to


Introduction
It had been recognized by clinicians that marked leukocytosis without any evidence of infection occasionally occurs in patients with nonhematologic malignancies.Asano et al. first demonstrated the production of human granulopoietic factors by tumor cells from a patient with lung cancer, who was associated with marked leukocytosis, and established the clinical entity of colony-stimulating factor (CSF)-producing tumor, in 1977 (Asano et al. 1977).Thereafter, 3 subtypes of CSF were identified by molecular cloning: granulocyte macrophage colony-stimulating factor (GM-CSF) (Cantrell et al. 1985), granulocyte colony-stimulating factor (G-CSF) (Nagata et al.1986), and macrophage colony-stimulating factor (M-CSF) (Kawasaki et al. 1985).In the first half of the 1990s, Suzuki et al. demonstrated that G-CSF-producing tumors produce not only G-CSF but also interleukin-1 (IL-1) and IL-6, and that IL-1 plays a role as a potent enhancer for the production of G-CSF, IL-6, and IL-1 itself (Suzuki et al. 1991), (Suzuki et al. 1992), (Tsuyuoka et al. 1994).Furthermore, Takahashi et al. showed that G-CSF-producing tumors produce IL-8 in addition to these 3 cytokines and that such tumors are associated with marked intratumoral neutrophil infiltration, presumably due to IL-8 produced by the tumor itself (Takahashi et al. 2007).In addition, co-production of IL-1, IL-6, and IL8 in G-CSF-producing tumors appears to be caused by hyperactivation of NF-κB because the expression of these 4 cytokine genes is up-regulated by this transcription factor (Takahashi et al. 2007).To our knowledge, however, there have been no reports describing IL-8-induced intratumoral neutrophil infiltration in G-CSF-producing tumors other than ours.Recently, we encountered a patient with renal cell carcinoma that produced G-CSF, IL-6, and IL-8.On pathohistological examination, marked neutrophil infiltration in the tumor tissue of surgically resected kidney was observed.We consider this neutrophil infiltration in the tumor tissue to be pathohistologically important, and thus report the serum cytokine profiles, gene expression of G-CSF and IL-8 by the renal tumor, and the histological picture of the tumor in this patient.

Case Report
A 65-year-old male was admitted to a hospital because of fever and lumbago.As the past medical history, he had hypertension and hyperlipidemia.His family history was unremarkable.Laboratory tests showed a white blood cell (WBC) count of 53.7×10 9 /l with 88% neutrophils, a hemoglobin concentration of 7.7 g/dl, and a platelet count of 596×10 9 /l.Serum concentration of C-reactive protein (CRP) was elevated to 15.3 mg/dl (normally below 0.3 mg).Computed tomography(CT)of the abdomen demonstrated a large mass in the upper pole of the left kidney.Therefore, he was referred to the Departments of Hematology and Urology, Shinko Hospital, Kobe, Japan, and admitted to the latter department in October 2012.

Laboratory tests on admission (Table
Contrast CT scanning of the chest and abdomen demonstrated a highly necrotic mass with a major axis of 9 cm arising from the upper pole of the left kidney, which was associated with tumor embolism of the renal vein (Fig. 1A, B).Magnetic resonance imaging (MRI) of the abdomen confirmed the findings of CT scanning and showed direct tumoral invasion to the adrenal gland.These images of the tumor strongly suggested renal cell carcinoma (RCC).CT scanning and MRI also demonstrated no infectious lesion.Bone scintigraphy showed no evidence of bone metastasis.
Because of the marked leukocytosis, the presence of renal tumor, and no evidence of infection, the renal tumor was highly suspected to be CSF-producing.We thus measured serum concentrations of cytokines by enzyme-linked immunosorbent assay (ELISA).The assay was commercially performed at SRL (Hachioji, Japan).Serum concentrations of G-CSF, IL-6, IL-8, and TNF-α were elevated to 474 pg/ml (normally less than 39.0 pg), 143 pg/ml (normally less than 4.0 pg), 1,260 pg/ml (normally less than 2.0 pg), and 12.5 pg/ml (normally 0.6 to 2.8 pg), respectively (Table 2).The high blood levels of G-CSF, IL-6, and TNF-α appeared to have caused the leukocytosis, high CRP, and fever in the present patient.
The patient subsequently underwent radical nephrectomy of the left kidney in November 2011.Intraoperatively, the renal tumor was found to have invaded the left adrenal grand and firmly adhered to the pancreas tail; therefore, resection of the left adrenal gland and pancreas tail, and splenectomy were simultaneously performed.
Histopathological examination of the resected renal tumor revealed it to be stage pT3a, G3, v1, ly1 with sarcomatoid differentiation papillary RCC (Fig. 2A), and the adrenal gland-invaded tumor showed undifferentiated features of RCC, which was accompanied by marked intratumoral neutrophil infiltration (Fig. 2B, C).Interestingly, neutrophil infiltration was not prominent in the differentiated component (Fig. 2A).After the operation, the patient became afebrile, and the WBC counts and CRP levels returned to normal levels (Fig. 3).Serum concentrations of G-CSF, IL-6, IL-8, and TNF-α also returned to normal or nearly normal levels (Table 2).Real-time quantitative PCR (RQ-PCR) of the tumor tissue revealed high gene expressions of G-CSF and IL-8 (described in Materials and Methods, and Results).From these findings, a diagnosis of G-CSF-producing RCC was made.
The patient, however, had to stay in hospital because he developed aspiration pneumonia, adhesive ileus, recurrent esophageal ulcer, and cerebral infarction.In June 2013, serum enzyme levels of the biliary tract and then those of transaminases increased, and he became icteric one month later.He developed cancerous peritonitis and CT scanning showed metastatic lesions in the diaphragm.The WBC count increased to 483×10 9 /l with 93% neutrophils, indicating the recurrence of the G-CSF-producing tumor.The patient died at the end of August, 9 months after the operation.

Materials and methods
Total RNA was extracted from resected tumor tissue and a T-cell tumor cell line, MOLT4, as a negative control, using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA).
One microgram of total RNA was reverse-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, California, USA).For G-CSF and IL-8 gene, RQ-PCR was performed using Sso Fast EvaGreen Supermix (Bio-Rad, Mississauga, ON, Canada).The ABL gene was chosen as a housekeeping gene to evaluate the amount and amplification of cDNA.The gene-specific primers used were 5'-AAGCTGGTGAGTGAGTGTGC-3' and 5'-ATGGAGTTGGCTCAAGCAGC-3' for G-CSF, 5'-GGTGCAGTTTTGCCAAGGAG-3' and 5'-TTCCTTGGGGTCCAGACAGA-3' for IL-8, 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' and 5'-GATGTAGTTGCTTGGGACCCA-3' for ABL.The quantification of each gene expression was performed according to the method by Østergaard et al (Østergaard et al. 2004).To quantify individual expression levels, we measured quantification cycles (Cq) for these genes, in which fluorescence intensity of each PCR product reached the same level (Table 3).Then, we calculated expression levels of each gene per 10,000 copies of abl gene according to the following formula:10,000×2^(Cq ABL -Cq TARGET ), assuming PCR amplification efficiency as 2.

Discussion
G-CSF-producing RCC is relatively rare among renal cancers, and being associated with a poor prognosis (Wang et al. 2006).Currently, no standard and effective treatment is available for this type of tumor (Wang et al. 2006).To our knowledge, however radical nephrectomy and/or combination chemotherapy with gemcitabine and nedaplatin (Sugiura et al. 2007) or that with sorafenib and S-1 (Kyono et al. 2011) are potentially effective treatment options.
Regarding IL-8 production by malignant tumors, Takahashi et al. described that there are 2 types: one is IL-8 production in the multi-cytokine production of G-CSF-producing tumors like the present case, and the other is sole IL-8 production by tumors (Takahashi et al. 2007), although the degrees of intratumoral neutrophil infiltration are similar in these two types of tumor (Takahashi et al. 2007).In terms of the important differences between these 2 types, the latter patients are afebrile and exhibit normal CRP levels and mild leukocytosis, presumably caused by IL-8-activated neutrophil mobilization from the marginal pool, but not enhanced granulopoiesis as in G-CSF-producing tumors (Takahashi et al. 2007), (Terashima et al. 1998).
In relation to neutrophil infiltration in tumor tissue, biliary system-dominant liver dysfunction was observed at presentation and during the recurrence of the tumor in the present patient.Furthermore, the patient developed marked jaundice with direct bilirubin dominance.Drug-induced liver dysfunction was ruled out.Suzuki et al.
reported liver damage in patients with G-CSF-producing tumor (Suzuki et al. 1993).In postmortem examination of the liver, neutrophils infiltrated both the portal area and the centrilobular zone even in patients without liver metasatasis, and the authors speculated that the G-CSF-and presumably IL-8-activated neutrophils which passively infiltrated the liver and accumulated themselves in the above mentioned areas, caused the liver damage.We speculate on a similar event caused by infiltrated neutrophils in the liver because the pattern of liver dysfunction was biliary system-dominant, as in patients in the study by Suzuki et al. (Suzuki et al. 1993).
G-CSF-producing tumors are associated with a poor prognosis.For example, there have been no patients who survived for more than 1 year after diagnosis in G-CSF-producing tumors of the urinary tract (Sugiura et al. 2007), (Kanda et al. 2011), (Ito et al. 1990).Regarding the poor prognosis, Gregory et al. discussed  observed that the presence of intratumoral neutrophils is associated with a poor prognosis in bronchioloaveolar carcinoma and localized RCC, respectively (Bellocq et al. 1998), (Jensen et al. 2009).
In conclusion, physicians should be aware of intratumoral neutrophil infiltration, which is caused by IL-8 production by the tumor, and biliary system-dominant liver dysfunction, which may be caused by intrahepatic neutrophil infiltration in G-CSF-producing tumors.association, 105(5)414-417.
Regarding the diagnosis of G-CSF-producing tumor, the present case fulfills the criteria proposed by Asano et al., that is, marked granulocytosis, elevated serum level of 10 G-CSF, normalization of granulocyte count and serum G-CSF level following tumor resection, and demonstration of G-CSF production by the tumor.As for the organ of origin of the G-CSF-producing tumor, it varies and includes the lung the relationship between tumor growth and neutrophil infiltration and proposed the concept of tumor-associated neutrophils (TAN)(Gregory et al. 2011).Although they discussed TAN in non-CSF-producing tumors, the relationship between tumor growth and neutrophils appears also to occur in G-CSF-and IL-8-producing tumors.In terms of the mechanism by which TAN confer an advantage for tumor growth, they proposed several factors: first, infiltrated neutrophils secrete oncostatin M that makes tumor cells produce vascular endothelial growth factor or hepatocyte growth factor, which subsequently enhances tumor invasion or growths.Second, neutrophils produce reactive oxygen species that induce tumor transformation through DNA injury and repair.Third, IL-8-activated neutrophils promote tumor metastasis through their adhesion to vascular endothelium and subsequent expression of integrin by endothelial cells, which leads to the adhesion of the tumor cells and extravascular tumor invasion.Fourth, neutrophil elastase destroys insulin receptor substrate, which leads to phosphoinositide 3-kinase activation and tumor growth.In relation toTAN, Bellocq et al. and Jansen et al.

Figure 1 :
Figure 1: Contrast CT scanning of the abdomen.

Figure 2 :
Figure 2: Histological picture of the tumor taken at nephrectomy.

Figure 3 :
Figure 3: Changes of laboratory data before and after nephrectomy.High