Purpose: To investigate the ferritin distribution in epithelial ovarian cancer patients according to the FIGO stage in the prognosis of epithelial ovarian cancer. Method: All ovarian cancer patients were divided into two groups according their FIGO stage. Benign ovarian tumor patients were analyzed as the control. Serum ferritin, serum iron, and other related medical index were detected by automatic instruments for all patients. In addition, ferritin heavy chain (FHC) and ferritin light chain (FLC) proteins were detected by immunohistochemical staining in 60 epithelial ovarian cancer (EOC) patients and 30 benign ovarian tumor (BOT) patients, which were diagnosed in our department between 2011 and 2016. Results: The serum ferritin concentration was significantly higher in the EOC group than in the BOT group (172.56 ± 99.39 ng/mL vs 78.18 ± 43.06 ng/mL; p < 0.001). The average concentration of serum iron was slightly lower in the EOC group than in the BOT group (11.11 ± 5.68 μmol/L vs 14.92 ± 6.36 μmol/L; p < 0.01). In addition, FHC and FLC proteins were highly expressed in EOC patients and localized at least in the cytoplasm and nucleus. Quantitative data of the FHC or FLC expression showed a significant increase in the average optical density in the benign ovarian tumor group compared with the FIGO I-II stage ovarian cancer group ( p < 0.01 and p < 0.01, respectively) as well as in the FIGO I-II stage ovarian cancer group compared with the FIGO III-IV stage ovarian cancer group ( p < 0.05 and p < 0.05, respectively). In addition, FHC and FLC positive rates of 81.7% and 76.7%, respectively, were found in the ovarian cancer group; the FLC positive rate in the FIGO I-II stage ovarian cancer group was lower than in the FIGO III-IV stage ovarian cancer group (58.3% vs 86.1%; p < 0.05); and the FHC positive rate in the FIGO I-II stage ovarian cancer group was lower than in the FIGO III-IV stage ovarian cancer group (70.8% vs 88.9%), but this difference was not statistically significant ( p > 0.05). Conclusion: Patients showed an overexpression of ferritin and a downregulation of serum iron correlated with the prognosis of epithelial ovarian cancer, which may be a new target for diagnosis and treatment of epithelial ovarian cancer.
Ferritin is a multimeric globular protein of approximately 450 kDa constituted by 24 subunits of the proteins ferritin heavy chain (FHC) and ferritin light chain (FLC), assembled in a nanocage with a central cavity where up to 4500 iron atoms can be stored [
Epithelial ovarian cancer is a serious gynecological with high mortality. More than 50% of the patients relapse and produce chemotherapeutic drug resistance, and the treatment of recurrent resistant ovarian cancer is rather difficult in clinic. It has been reported that patients with ovarian cancer exhibited iron deficiency, anemia, and an increased ferritin concentration [
The present study is a retrospective, observational trial performed after approval by the ethics committee of The First Affiliated Hospital of Yangtze University. Ninety cases were selected from clinically diagnosed cases in The First Affiliated Hospital of Yangtze University between August 2011 and August 2016. The patients were divided into two groups: the first group contained 60 cases with epithelial ovarian cancer (46 serous adenocarcinoma and 14 mucinous adenocarcinoma) and the second group contained 30 cases of epithelial benign tumors as control (24 serous cystadenoma and 6 mucinous cystadenoma).
Inclusion criteria: 1) All cases had protopathic diseases and were pathologically examined to be epithelial ovarian cancer or epithelial ovarian benign tumors; 2) Patients were assessed at diagnosis before receiving any cancer treatment; 3) Operation notes were complete and sufficiently distinct to obtain the clinicopathological stage in accordance with the 2015 International Federation of Gynecology and Obstetrics (FIGO) standards of ovarian cancer. Available tissues of patients should be kept in our pathology department; 4) Essential information and laboratory test results were fully documented. Exclusion criteria: 1) Evidence of infections, chronic inflammatory disease, active bleeding, hemolysis, renal insufficiency, or hypothyroidism; 2) Known history of hematologic disorders, family history of thalassemia or hemocromatosis; 3) Treatment with Erythropoietin; iron or blood transfusion in the preceding 12 weeks; current iron, vitamin B12, or folate supplementation.
Parameters directly collected from previous medical records include red blood cell (RBC), hemoglobin (HB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum iron concentration, the liver enzymes alanine aminotransferase (ALT) and aspartate transaminase (AST), the kidney health indicator blood urea nitrogen (BUN), and albumin (ALB). Serum ferritin and cancer antigen 125 (CA125) were additionally examined using the saved blood samples collected at the time of the preoperative examinations and kept at −80˚C until sampling. Serum ferritin and CA125 were detected by the Cobas 6000 analyzer (Roche Diagnostics GmbH, Germany) and the corresponding kit (Roche Diagnostics GmbH, Germany).
Four-μm thick paraffin-embedded, formalin-fixed slides of de-identified human tissues were deparaffinized, and high-temperature heat pressure antigen retrieval (120˚C, 120 kPa) was performed by tissue incubation in 0.01 mol/L citrate buffer (pH = 6.0) for 3 min. The sections were then incubated with 5 µg/mL ferritin heavy chain (FHC) antibodies (ab65080, Abcam, Cambridge, United Kingdom) and 5 µg/mL ferritin light chain (FLC) antibodies (ab69090, Abcam, Cambridge, United Kingdom) at 4˚C overnight and analyzed with an horseradish peroxidase-conjugated compact polymer system using DAB as the chromogen. The sections were then counterstained with hematoxylin and mounted with DPX.
Imaging of immunohistochemically (IHC) stained tissue was performed using the imaging system HMIAS-2000W (Wuhan Champion Image Technology Co., Ltd., Wuhan, China) and the Olympus BX41 microscope (Olympus Corporation, Japan), and the positive rate and average optical density were calculated from the images.
Positive criteria: protein expression was assessed by scoring the percentage of the labelled cells. FHC and FLC expressions were quantified using a visual grading system based on the proportion of staining (percentage of positive tumor cells graded on a scale from 0 to 3: 0, none; 1, 1% - 30%; 2, 31% - 60%; 3, >60%) and the intensity of staining (graded on a scale of 0 to 3: 0, none; 1, weak staining; 2, moderate staining; 3, strong staining). The product PI = P × I of proportion (P) and intensity (I) of the staining ranged between 0 and 9 and was obtained as a combined parameter for each spot. For statistical analysis, a PI score of 0 - 3 was considered as negative expression and a PI score > 3 was considered as positive expression [
Continuous data are expressed as mean ± standard deviation (S.D.), and differences between two groups were compared by Student’s t-test. Differences in the positive rates were analyzed using chi-square tests. Continuity correction chi-square tests were performed for contingency values between 1 and 5, while Pearson’s chi-square test was performed for contingency values larger than 5. Values of p < 0.05 were considered statistically significant. Computations were performed using SPSS version 19.0 (International Business Machines Corporation, New York City, United States of America).
All 90 ovarian tumor patients were treated in our hospital. Among these patients, 30 patients with an age range of 20 - 71 years (average 45.10 ± 15.05) had benign ovarian tumors (BOT), and 60 patients with an age range of 25 - 75 years (average 52.18 ± 9.79) had epithelial ovarian cancer (EOC). From the blood samples of ovarian cancer patients, ferritin blood levels and other clinical characteristics were analyzed in parallel. These other clinical characteristics involved the patients’ age, red blood cell counts, the liver enzymes alanine aminotransferase (ALT) and aspartate transaminase (AST), the kidney health indicator blood urea nitrogen (BUN), albumin (ALB), the ovarian cancer marker cancer antigen 125 (CA125), and the serum iron level.
Benign ovarian tumor patients and ovarian cancer patients exhibited differences in various parameters, except for blood urea nitrogen (BUN). For example, the red blood cell (RBC) counts among ovarian cancer groups were 3.93 ± 0.47 × 1012/L, which was significantly smaller than the counts of 4.26 ± 0.61 × 1012/L in the benign ovarian tumor groups (p < 0.01;
Evidently, the average concentration of the ovarian cancer marker CA125 was
Variable | Benign Ovarian Tumor (n = 30) | Ovarian Cancer (n = 60) | T | p |
---|---|---|---|---|
Number | 30 | 60 | ||
Age, years (range) | 45.10 ± 15.05 (20 - 71) | 52.18 ± 9.79 (25 - 75) | 2.342 | 0.024* |
HB, g/L | 122.68 ± 15.13 | 114.12 ± 15.68 | 2.470 | 0.015* |
RBC,×1012/L | 4.26 ± 0.61 | 3.93 ± 0.47 | 2.779 | 0.007** |
MCV, f L | 88.35 ± 7.41 | 88.49 ± 4.93 | 0.100 | 0.921 |
MCH, Pg | 29.03 ± 2.80 | 29.06 ± 1.97 | 0.052 | 0.958 |
MCHC, g/L | 328.47 ± 10.70 | 328.40 ± 11.33 | 0.027 | 0.979 |
ALT, U/L | 10.30 ± 7.86 | 14.50 ± 9.19 | 2.140 | 0.035* |
AST, U/L | 18.13 ± 4.17 | 23.8 ± 10.04 | 2.961 | 0.004** |
BUN, mmol/L | 5.01 ± 1.78 | 4.41 ± 1.29 | 1.806 | 0.074 |
ALB, g/L | 44.83 ± 9.70 | 41.80 ± 4.38 | 2.046 | 0.044* |
CA125, U/mL | 17.91 ± 14.97 | 255.64 ± 246.45 | 5.264 | 0.000** |
Serum ferritin, ng/mL | 78.18 ± 43.06 | 172.56 ± 99.39 | 6.272 | 0.000** |
Serum iron, μmol/L | 14.92 ± 6.36 | 11.11 ± 5.68 | 2.883 | 0.005** |
Results are considered significant for values of p < 0.05* and highly significant for p < 0.01**. Abbreviations: HB: hemoglobin; RBC: red blood cell; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; ALT: alanine aminotransferase; AST: aspartate transaminase; BUN: blood urea nitrogen; ALB: albumin; CA125: cancer antigen 125.
much higher in the EOC group than in the BOT group (255.64 ± 246.45 U/mL vs 17.91 ± 14.97 U/mL; p < 0.01). Similarly, the level of serum ferritin, one of the storage forms of iron, was significantly higher in the EOC group than in the BOT group (172.56 ± 99.39 ng/mL vs 78.18 ± 43.06 ng/mL; p < 0.001). In contrast, the average concentration of serum iron was slightly lower in the EOC group than in the BOT group (11.11 ± 5.68 μmol/L vs 14.92 ± 6.36 μmol/L; p < 0.01), and this difference was statistically significant (
As mentioned above, the serum ferritin level was increased, and the serum iron level was decreased in the EOC group. To further ascertain the levels of secreted serum ferritin and serum iron in EOC patients with different FIGO clinical stages, the patients were divided according their FIGO stage into two groups with either FIGO stage I-II or III-IV (
Variable | FIGO stage | T | p | ||
---|---|---|---|---|---|
I-II (n = 24) | III-IV (n = 36) | ||||
Serum ferritin, ng/mL | 148.27 ± 91.92 | 188.76 ± 102.11 | 1.565 | 0.123 | |
Serum iron, μmol/L | 12.39 ± 5.58 | 10.25 ± 5.66 | 1.438 | 0.156 | |
HB, g/L | 116.80 ± 15.96 | 112.34 ± 15.46 | 1.078 | 0.285 | |
RBC,×1012/L | 4.00 ± 0.45 | 3.89 ± 0.49 | 0.865 | 0.391 | |
MCV, f L | 88.20 ± 2.94 | 88.68 ± 5.93 | 0.368 | 0.714 | |
MCH, Pg | 29.05 ± 1.60 | 29.06 ± 2.21 | 0.021 | 0.983 | |
MCHC, g/L | 329.50 ± 14.86 | 327.67 ± 8.35 | 0.549 | 0.587 |
Abbreviations: FIGO: International Federation of Gynecology and Obstetrics; HB: hemoglobin; RBC: red blood cell; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration.
To investigate the expression of ferritin and its subtype positive rate, the two ferritin subtypes ferritin heavy chain (FHC) and ferritin light chain (FLC) were detected by immunohistochemical methods in benign ovarian tumor and epithelial ovarian cancer patients. As shown in
Furthermore, the positive rates of FHC and FLC were calculated according to the ovarian cancer FIGO clinical stage. As shown in
Ferritin can be expressed in its intracellular iron cargo protein form or its extracellular form of secreted ferritin [
The present study evaluates the prevalence of cancer-related anemia in a large cohort of oncology patients with cancers of the ovaries. For example, our data showed that both the red blood cell (RBC) count and the average concentration of hemoglobin (HB) were significantly decreased among ovarian cancer groups compared with benign ovarian tumor groups. MCV, MCH, and MCHC showed
no significant differences between the two groups. In addition, the average concentration of the liver enzymes ALT and AST in the two groups were normal while the EOC group showed a slightly higher concentration than the BOT group. In contrast, the concentration of albumin (ALB) was lower in the EOC group than in the BOT group. Anemia and nutrition deficiency in oncology patients is often considered a side effect of cancer therapy [
We found that the serum iron concentration decreased with the tumor growth. We hypothesized that the tumor has an intrinsic need for iron, and some evidence supports this hypothesis. Some studies showed that cancer tissue usually has an increased iron uptake and a decreased iron release and usually exhibits an iron deficiency in the serum and an iron overload within the tumor
Variable | FHC | x2 | p | FLC | x2 | p | ||
---|---|---|---|---|---|---|---|---|
Positive, n (%) | Negative, n (%) | Positive, n (%) | Negative, n (%) | |||||
Ovarian cancer | 49 (81.7) | 11 (18.3) | 46 (76.7) | 14 (23.3) | ||||
Ovarian cancer (I-II) | 17 (70.8) | 7 (29.2) | 2.045 | 0.153 | 14 (58.3) | 10 (41.7) | 5.926 | 0.015 |
Ovarian cancer (III-IV) | 32 (88.9) | 4 (11.1) | 31 (86.1) | 5 (13.9) |
FIGO: International federation of gynecology and obstetrics. FHC: ferritin heavy chain; FLC: ferritin light chain.
[
Overall, we conclude that an abnormal distribution of ferritin was found in epithelial ovarian cancer patients, and ferritin overexpression and serum iron downregulation in epithelial ovarian cancer were consistent with the cancer prognosis, indicating that ferritin may be a new target for diagnosis and treatment of epithelial ovarian cancer.
We acknowledge the excellent laboratory work performed by the technicians of the pathology department. This study was supported by the Iron-deficiency & Iron-deficiency Anemia Research Grant project and Jingzhou science and technology development plan (key projects, 2015AC45 and 2016AE51-2), as well as the Hubei Province health and family planning scientific research project (key project, WJ2017Z024).
Jiang, J.P., Wang, S.F., Zhang, L., Lu, J.Z. and Yi, C.J. (2018) Characteristics of the Distribution of Ferritin in Epithelial Ovarian Tumor Patients: Results of a Retrospective, Observational Study. Yangtze Medicine, 2, 51-61. https://doi.org/10.4236/ym.2018.22006