Serum proteome analysis for profiling protein markers associated with carcinogenesis and lymph node metastasis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), one of the most common cancers in population with Chinese or Asian progeny, poses a serious health problem for southern China. It is unfortunate that most NPC victims have had lymph node metastasis (LNM) when first diagnosed. We believe that the 2D based serum proteome analysis can be useful in discovering new biomarkers that may aid in the diagnosis and therapy of NPC patients. To filter the tumor specific antigen markers of NPC, sera from 42 healthy volunteers, 27 non-LNM NPC patients and 37 LNM NPC patients were selected for screening study using 2D combined with MS. Pretreatment strategy, including sonication, albumin and immunoglobulin G (IgG) depletion, was adopted for screening differentially expressed proteins of low abundance in serum. By 2D image analysis and MALDI-TOF-MS identification, twenty-three protein spots were differentially expressed. Three of them were further validated in the sera using enzyme-linked immunosorbent assay (ELISA). Our research demonstrates that HSP70, sICAM-1 and SAA, confirmed with ELISA at sera and immunohistochemistry, are potential NPC metastasis-specific serum biomarkers which may be of great underlying significance in clinical detection and management of NPC.
Introduction
Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, though it is highly prevalent in Southern Asia, where the incidence is about a 100-fold higher than in other populations. It is one of the most confusing, commonly misdiagnosed and poorly understood diseases. Previous studies show that the cancer is an Epstein-Barr virus-associated malignancy with a remarkable racial and geographical distribution. The etiology of NPC is thought to be associated with a complex interaction of genetic, viral, environmental and dietary factors. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, the etiology, carcinogenesis and progression of the disease is better understood. Research into these components may unravel the pathways in NPC development and potentially decipher the molecular characteristics of the malignancy [1, 2]. NPC is an insidious tumor and is usually at the stage of metastasis involving lymph nodes or other organs before it can be found. Thus early and accurate diagnosis is very important for therapy and prognosis of NPC patients. Unfortunately, no effective method of accurately diagnosing new-onset NPC is available now. We presume if specific serum biomarkers associated with NPC metastasis can be identified on the basis of advancements in genomics, proteomics and bioinformatics, an approach to the early detection and monitoring of NPC may be found.
2D comparative proteome analysis is a new technology for the separation and identification of disease-specific proteins, and it has been applied successfully to screen potential biomarkers for NPC in cell lines and tumor tissues [3–8]. In our previous studies, we employed proteomic techniques to study protein changes of CNE2, a poorly differentiated squamous carcinoma cell line of human NPC cells, induced by 12-O-Tetradecanoyl-phorbol-13-acetate (TPA). It is likely that TPA promotes NPC in necessary cooperation with EBV, or that it may function as an antiproliferative or differentiative revulsant in noninitiated cells [4]. However, if a marker can be detected only in surgical specimens, its clinical significance is limited, especially for early screening or diagnosis. Recently, Saeid R. et al. reported their pioneering work on NPC serum analysis by 2D without any pretreatment, revealing the enhanced expression of relatively abundant proteins as ceruloplasmin (CPL) [9]. In order to find relatively low-abundant serum proteins which may be more valuable in predicting NPC progression, we pretreated serum with sonication, albumin and IgG depletion before 2D analysis. By comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we may identify the differentially expressed proteins so that specific serum biomarkers associated with NPC metastasis can be found.
Materials and methods
Subjects
Serum samples from 42 healthy volunteers and 64 NPC patients were available for comparative proteomic analysis. Samples from NPC patients were obtained before any clinical treatment. All the patients had a nasopharyngeal biopsy and NPC was confirmed by two independent pathologists unaware of the patient’s condition. Careful medical examinations and CT scan determined non-LNM cases and LNM cases in the NPC patients. Clinical data of all the subjects for 2D and ELISA are shown in Table 1. In the screening study, serum samples from the same group were pooled to smooth intrinsic individual differences and enhance common characteristic traits only related to disease status [10], aliquoted, and stored at −80°C before analysis. In the validation study, 26 of the patients received a mean of 17.8 months of follow-up (range, 6–22 months) after radiotherapy and/or chemotherapy. Six of the followed-up patients died of NPC, and the other survived the follow-up period.
Table 1Serum samples in the studySamples for 2-DESamples for ELISAHealthyNPCHealthyNPCLymph node status/NegativePositive/NegativePositiveNumber422737302730Gender (Male/Female)22/2021/623/1415/1521/620/10Age (±SD)42 ± 7.943 ± 10.950.5 ± 11.742.5 ± 8.943 ± 10.947.6 ± 10.2
Depletion of high-abundance proteins
Sigma-Aldrich ProteoPrep® Immunoaffinity Albumin and IgG Depletion Kit was used for depletion of proteins, albumin and IgGs, allowing visualization of co-migrating proteins with albumin and IgG on a 2D gel and also higher sample loads for improved visualization of lower abundance proteins. A total of 30 μl of serum was diluted with 100 μl of equilibration buffer from the depletion kit (Sigma) and sonicated using four ultrasonic bursts of 15 s each. Between sonications, the samples were chilled on ice. Then serum albumin and IgG was depleted according to the manufacturer’s instructions. After collection of the bound proteins, the majority (>95%) of the unbound proteins were left in the depleted serum sample. Modified Bradford assay was used to measure the protein concentrations of all serum samples [11].
2D analysis
The Immobiline Dry strip (pH 4–7, length 17 cm, Bio-Rad) was rehydrated with 600 mg protein in 350 ml rehydration buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 5 mM tributylphosphine, 1% IPG buffer and 0.002% bromophenol blue for 14 h at room temperature. IEF was performed using Protean IEF cell (Bio-Rad, Hercules, CA, USA) with a total of 70 kVh. Then the strip was subjected to two-step equilibration in equilibration buffer containing 6 M urea, 30% glycerol, 2% SDS and 50 mM Tris–HCl (pH 6.8) with 1% w/v DTT for the first step, and 2.5% w/v iodoacetamide for the second step. The second-dimensional SDS-PAGE gel (13% T, 2.7% C, and 20 × 20 × 1 mm3) was carried out using a Protean II xi 2D cell (Bio-Rad) according to the following procedure: 30 min at a constant current of 12 mA followed by 24 mA per gel until the bromophenol blue front reached the bottom of the gel. Subsequently the gels were stained with MS accommodated silver staining [12]. 2D of each sample was run in triplicate to minimize run-to-run variation. The silver-stained 2D gel were scanned using a Power-Look 1100 imaging scanner (Umax, Dallas, TX, USA) and analyzed using PDQuest 7.1 software package (Bio-Rad).
In-gel enzymatic digestion
Protein spots were excised from gel with an operating knife blade, destained twice with 30 mM potassium ferricyanide and 100 mM sodium thiosulfate (1:1 v/v) and then equilibrated in 50 mM NH4HCO3 to pH 8.0. After dehydrating with ACN and drying in N2 at 37°C for 20 min, the gel pieces were rehydrated in 15 ml trypsin solution (10 μg/ml in 25 mM NH4HCO3) at 4°C for 30 min and incubated at 37°C overnight. Peptides were extracted twice using 0.1% TFA in 50% CAN and dried with N2.
MALDI-TOF-MS identification
The peptide mixtures were solubilized with 0.5% TFA, with saturated α-cyano-4-hydroxy-trans-cinnamic (CHCA) solution in 0.1% TFA/50% acetonitrile as the matrix and analyzed using M@LDI R (Micromass, Manchester, UK). Mass spectra were externally calibrated with lock mass 2,465.199 Da and internally calibrated with autodigested peaks of trypsin (MH+: 2,211.105 Da).
Protein identification and database searching
Protein identification using peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (http://www.matrixscience.com/, MatrixSicence Ltd., London, UK) against the MSDB protein database. The errors in peptide masses were in the range of 25 ppm. One missed tryptic cleavage site per peptide was allowed during the search. Proteins matching more than four peptides and with a MASCOT score higher than 64 were considered significant (P < 0.05). Carboamidomethylation of cysteine was selected as the static modification and oxidation of methionine as the differential modification. Protein identification results were filtered with peakErazor software.
Enzyme-linked immunosorbent assay (ELISA) confirmation
ELISAs were conducted to confirm the protein identification and differential expression of sICAM-1, SAA and HSP70 in sera from the three groups. Measurements were done by commercially available ELISA kits (StressXpress Hsp70 ELISA Kit: EKS-700B, Stressgen Biotechnologies, Victoria; High sensitivity sICAM-1 ELISA Kit: BMS241, Bender Medsystems Company, Australian; Human SAA ELISA Kit: EL10015, YES Biotech Laboratories Ltd, Canada), in accordance with the manufacturer’s instructions. All sera were stored at −80°C before they were measured. Both standards and samples were run in duplicate. The main protocols were as follows: monoclonal coating antibody was adsorbed onto microwells; target protein present in the sample or standard bound to antibodies was adsorbed to the microwells; second antibody was added and bound to target protein captured by the first antibody; following incubation unbound enzyme was removed during a wash step; a color reaction was formed and absorbance was measured at 450 nm. The standard curve was used to determine the concentration of target protein in an unknown sample.
Immunohistochemistry (IHC) assay
The previous studies showed that sICAM-1 in serum could be released by membrane-associated ICAM-1 of cancer cell [13, 14]. However, SAA is synthesized and secreted into serum by hepatocytes in the liver during the acute phase [15, 16]. In the present study, therefore, expressions of only HSP70 and ICAM-1 were detected in tissue by IHC assay. Three-micrometer sections of paraffin-embedded tissues were cut for a prior antigen retrieval step in boiling 10 mM citrate buffer pH 6 for 20 min. The fixed tissues were subjected to immunostaining using an ultrasensitive S-P technique (MaiXin, China). Briefly, the slides were incubated overnight at 4°C with goat anti-ICAM and anti-HSP70 polyclonal antibody (1:200) (Wuhan Boster Biological Technology Co., Ltd., China). The concentrations of the primary antibodies used have been optimized previously. After washing with PBS three times, they were treated with biotin-conjugated second antibody before adding streptavidin-peroxidase. For color reaction, diaminobenzidine (DAB) was used. All series included positive controls. For negative controls, the antibody was replaced by PBS. To evaluate the expression, the cellular localization of immunoreactivity was determined and scored for both intensity (negative, weak, moderate and strong) and proportion (5–25, 26–50, 51–75 and 76–100%) of stained cells. To allow comparison of immunostaining with pathological stage and metastasis for each cancer, integer values were assigned to the scores of intensity (0–3) and proportion of tumor cells stained (0–4). If the evaluations did not agree, the specimens were reevaluated and then classified according to the assessments given, most frequently, by the observers. The proportion and intensity scores were then added to obtain a total score, which was labeled as one of the four following categories: (a) high positive (+++), total score of 6–7; (b) moderate positive (++), total score of 4–5; (c) low positive (+), total score of 2–3; (d) negative (−), total score of 0–2.
Statistical analysis
All the analyses performed were descriptive. The serum levels of protein expressions were compared using one-way ANOVA test. Associations between immunohistochemical scores and clinicopathological variables of tissue specimens were evaluated by Fisher’s exact test or χ2 test. PDQuest software was used to establish a master gel representing each group [17]. For differential expression of matched gels, protein spots whose intensities were either increased or decreased two-fold or greater were marked and then confirmed by manual inspection of all relevant 2D gel, not only those included in the matchsets, to ensure consistency. Quantitative analysis was performed using the Student’s t-test among three groups of gels. All statistical analysis was performed with SPSS 10.0 software and P value <0.05 were considered statistically significant.
Results
Quantitative comparison and identification of protein spots on 2D gels
Among the three groups, we found twenty-three spots significantly altered (P < 0.05) or absent/emergent, 13 of which were identified successfully (Fig. 1). The corresponding detailed differential expression patterns are shown in Fig. 2a. Quantitative comparison and MS identification of the 13 differentially expressed proteins are shown in Fig. 2b and summarized in Table 2, respectively. Overall, transferrin (TRF) and transthyretin (TTR) were down-regulated and 12-lipoxygenase (12-LOX), serum amyloid A1 protein precursor (SAA), soluble intercellular adhesion molecule-1 (sICAM-1) and lysine-specific histone demethylase1 (LSD1) were up-regulated in two NPC groups compared with the healthy group. Furthermore, increased expression levels of 12-LOX, sICAM-1 and SAA were observed in LNM NPC as compared with non-LNM NPC. However, heat shock protein 70 (HSP70) and cytochrome P450 (CYP450) were expressed significantly and constantly only in LNM NPC patients. The MALDI-TOF-MS mass spectra are shown in Fig. 3.
Fig. 1Representative 2D patterns of albumin and IgG depleted serum from healthy (a), non-LNM NPC (b) and LNM NPC groups (c). In the 2D images, spots ‘1–10’ indicate up-regulation and ‘11–13’ down-regulation in NPC group. Black arrows indicate protein spots that were identified successfully by MSFig. 2Detailed 2D patterns of the thirteen differentially expressed proteins among healthy, non-LNM and LNM NPC groups (a). Corresponding gel positions are shown in Fig. 1. Magnified 2D maps demonstrating the different expressions of selected proteins are listed in Table 2. The densitometric analysis of each protein was calculated from nine different gels using PDQuest 7.1 software package (b). Each bar represents the mean ± S.D. of intensity, with significant differences between groups found by two-tailed student’s-test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject spot No.6. The break region is from 5000 to 6000. *P < 0.05, compared with healthy group; **P < 0.05, compared with non-LNM NPC groupTable 2Identification results of differentially expressed proteins among healthy volunteers, non-LNM NPC and LNM NPC patientsNo.aProtein descriptionMSDB IDProtein levelbMASCOT scorecSequence coveraged (%)Theoretical Mr/pIe1Ovochymase precursorQ7RTY7↑11652124,947/8.672Arachidonate 12-lipoxygenaseAAA51587↑1936875,694/5.823sICAM-1Q99930↑112544270/6.404Cytochrome P450Q53EX9↑2075755,635/6.835Serum amyloid A1 protein precursorYLHUS↑1708513,524/6.286Hemoglobin beta subunits are s-nitrosylated1BUWB↑2075515,565/6.767E1A 10S proteinQ9YLA2↑1436928,493/4.098Lysine-specific histone demethylase 1O60341↑1725893,358/6.119KIAA1622 proteinQ9HCF0↑7861102,351/8.4210Heat shock 70 kDa protein (HSP70)Q6G1F9↑1575368,161/4.8811TransferrinQ9NQB8↓1676777,050/6.8112UPF0366 protein C11orf67AAD40378↓1045812,798/8.5813TransthyretinAAA61181↓876912,836/5.35aThe numbers indicate the spot positions in 2D gel as shown in Figs. 1 and 2b↑, up-regulation in NPC patients; ↓, down-regulation in NPC patientscBy MALDI-TOF MS analysisdCalculated by amino acid counteCalculated from the database entry without any processingFig. 3PMF of protein spots No. 3, 5 and 10, respectively, representing sICAM-1 (panel A), SAA (panel B), and HSP70 (panel C), which were expressed differentially between the groups of non-LNM and LNM NPC (MASCOT scores are 65, 85 and 78, respectively, P < 0.05)
ELISA confirmation
Figures 4 and 5 show the ELISA results of sICAM-1, SAA and HSP70 performed in the three groups. There were statistically significant differences between health and NPC groups (P < 0.01) as well as between non-LNM and LNM NPC groups (P < 0.05) (Fig. 4). And with the TNM stage advanced, the serum levels of sICAM-1 and HSP70, were detected significantly elevated, but that of SAA not (Fig. 5). Furthermore, bivariate correlation analysis showed a significant relationship between the serum levels of the three proteins (P < 0.01). The follow-up showed that the serum levels of sICAM-1 and HSP70, but not of SAA, were strongly associated with NPC mortality (Student’s t-test, P = 0.001, 0.001 and 0.132, respectively). Our ELISA findings were consistent with results of 2D and silver-staining.
Fig. 4Quantitative comparison of sICAM-1, HSP70 and SAA in sera among healthy, non-LNM NPC and LNM NPC groups. Each bar represents the mean ± S.D. of the concentration, with significant differences between groups found by one-way ANOVA test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject SAA. The break region is from 500 to 4500 μg/l. *P < 0.05, compared with healthy group; **P < 0.05, compared with non-LNM NPC groupFig. 5Quantitative comparison of sICAM-1, HSP70 and SAA in sera among NPC patients at different TNM stage. Each bar represents the mean ± S.D. of the concentration, with significant differences between groups found by one-way ANOVA test. Note that this panel has a different vertical (y) axis and a break in the axis to accommodate the poor performance of the subject SAA. The break region is from 600 to 2000 μg/l. *P < 0.05, compared with NPC patients at TNM stage I; **P < 0.05, compared with NPC patients at TNM stage II; ***P < 0.05, compared with NPC patients at TNM stage III
IHC assay
In NPC patients, HSP70 and ICAM-1 were observed in tumor cells but not in stromal, endothelial or inflammatory cells. The immunoreactivity was located in both membrane and cytoplasm for ICAM-1 and only in cytoplasm for HSP70 (Fig. 6). The immunoreactivities of HSP70 and ICAM-1 were observed in 37 (64.9%) and 43 (75.4 %) of the 57 cases of NPC, respectively. The correlation of positive rates showing HSP70 and ICAM-1 immunoreactivity with clinicpathological characteristics of the cases is summarized in Table 3. Statistically, positive expression rates of HSP70 and ICAM-1 were significantly correlated with lymph node metastasis (LNM) of NPC. Furthermore, we found that positive expression rates of ICAM-1 (6/6, 100%) and HSP70 (5/6, 83.3%) were high in the cases that died of NPC during the follow-up period (P > 0.05). The results manifested that ICAM-1 and HSP70 expressions were strongly associated with NPC metastasis and mortality, indicating poor prognosis.
Fig. 6Immunohistochemical detection of HSP70 and ICAM-1 in NPC (Magnification ×200). The immunoreactivity was located in both membrane and cytoplasmic stainings for ICAM-1 but only in cytoplasmic staining for HSP70. The black arrows indicate the target proteins stained in brown-yellow in the NPC nest. The red arrow in E shows negative immunoreactivity of normal nasopharyngeal epithelia. a, negative expression of HSP70; b and c, positive expressions of HSP70; d, negative expression of ICAM-1; e and f, positive expressions of ICAM-1; a, b, d and e, non-LNM NPC; C and F, LNM NPCTable 3Characteristics of NPC patients dependent on ICAM-1 and HSP70 expressionsCharacteristicsNumberICAM-1 expressionHSP70 expressionNegativePositive (%)P valueNegativePositive (%)P valueGender    Male41932 (78.0) 0.4641526 (63.4) 0.704    Female16511 (68.8) 511 (68.8) AgeMean (45.4)     <45 years26719 (73.1) 0.704917 (65.4) 0.945    ≥45 years31724 (77.4) 1120 (64.5) Lymph node status    Positive30327 (90.0) 0.012525 (83.3) 0.002    Negative271116 (59.3) 1512 (44.4) TNM stage    I + II241113 (54.2) 0.0021212 (50.0) 0.044    III + IV33330 (90.9) 825 (75.8) Survival rate    Survival20614 (70)0.280614 (70)1.000    Mortality606 (100)15 (83.3)Note: Values in parentheses are percentages
Discussion
Serum is a complex body fluid, containing a large diversity of proteins. More than 10,000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [18]. Consequently, proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [19]. Serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact with, all tissues. During this contact it is likely to pick up proteins secreted or shed by tissues, which has recently been tested and confirmed [20]. However, serum has been termed as the most complex human proteome [21] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [22]. Another analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [23]. Despite these limitations, human serum holds immense diagnostic potential. In the last decade, several large-scale projects have been initiated, aimed at characterizing the human plasma/serum proteome.
In the mean time, several serum proteomic studies on NPC have been reported. Cho et al. performed protein chip profiling analysis with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology on sera from NPC patients and demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. However, the technology for discovery of new cancer biomarkers has recently been questioned for its flaws, such as its qualitative nature, high identification error rate, poor reproducibility and nonspecific absorption matrices [25–27]. 2D-based comparative proteome analysis, though a new technology for the separation and identification of disease-specific proteins, has been applied successfully to screen potential biomarkers for NPC in cell lines and tumor tissues [3–8].
Recently, Saeid et al. reported their pioneering work on NPC serum analysis by 2D, revealing the enhanced expression of such relatively abundant proteins as ceruloplasmin (CPL) [9]. However, in 2D study, abundant proteins, such as albumin and IgG, that account for approximately 60–97% of the total serum proteins [28], mask other proteins that migrate to the surrounding areas and limit the loading amount of serum. As albumin and IgG are known to function as carriers and transporters of important proteins such as hormones, cytokines, and lipoproteins within the blood [29–31], the depletion of these two highly abundant proteins may result in the loss of potentially important proteins bound to them at the same time. In order to release those adsorbed or bound proteins, we sonicated the diluted sera before the depletion and desalting steps as suggested by Quero et al. [32, 33]. The improvements we made in sample preparation enabled us to find some valuable low abundant proteins.
Of the 13 successfully identified protein spots, we focused on 10 up-regulated proteins in NPC for further validation. We also accumulated certain knowledge about three proteins by literature profiling. Finally, we decided to further investigate sICAM-1, HSP70 and SAA, which seemed more associated with our research interest, using both ELISA and IHC to validate their differential expressions. Intriguingly, most of these identified proteins have been reported to be associated with carcinogenesis and tumor metastasis. Our research is herein chiefly concerned with the functional implications of the three proteins to NPC at the serum level.
Although the source of sICAM-1 has not been fully elucidated, researches show it can be released by cancer cells [13, 14] as well as by peripheral blood mononuclear, endothelial, and fibroblastic cells [34]. Proteolytic cleavage of membrane-bound ICAM-1 may be the most likely mechanism for the generation of sICAM-1 [35]. In patients with certain malignancies, the serum sICAM-1 titers have been found elevated in association with tumor growth and distant metastasis of malignant melanoma [36], lung [37], breast [38], gastric [39], hepatocellular [40], and colorectal cancers [41]. Poor survival of cancer patients correlated with a high level of serum sICAM-1 has also been demonstrated [36, 38, 39]. In our study, the positive expression of tissue ICAM-1 and levels of serum sICAM-1 were significantly correlated to the presence, progression, metastasis and mortality of NPC. sICAM-1 possesses most of the necessary extracellular structures to retain the functional activities of ICAM-1 [13, 35]. It has been reported that ICAM-1 on the surface of cancer cells or antigen presenting cells (i.e., macrophages) is a costimulatory factor that stabilizes T-cell receptor-mediated binding between these cells and T lymphocytes [42]. sICAM-1 would work as an immunosuppressive agent by blocking LFA-1 on T lymphocytes, thus rendering it less available for binding with ICAM-1 on the surface of cancer cells [43]. In this manner, the shedding of sICAM-1 may speed up the metastatic process by escaping host immune surveillance. This, probably, presents an additional potential mechanism accounting for high serum levels of sICAM-1 in NPC patients who have metastasized via hematogenous and lymphatic routes. As serum sICAM-1 may be useful for monitoring hematogenous metastasis, measuring the serum sICAM-1 level might be potentially significant in clinic.
SAA is an acute-phase protein with various isoforms in a molecular mass range of 11–14. In normal individuals, SAA is produced by hepatocytes in the liver [16]. After secreted into serum, it rapidly binds to high-density lipoprotein, with 90% of the protein particles bound [15]. A review of the literature shows that only a low level of SAA can be found in the sera of healthy individuals, despite the ubiquitous nature of SAA [44]. This is in sharp contrast to the patients with neoplastic diseases, such as those with renal [45] and colorectal [46] cancers, who showed dramatic elevation of serum SAA. The prognostic significance of SAA for other cancers has also been found by conventional radioimmunoassay, in line with the findings of this study [47]. Cho et al. demonstrated that SAA may be a potentially usefully biomarker for NPC [24]. The report fully confirmed relatively huge concentrations of SAA in serum (0.2–2 g/l) that are thousands of times higher than classical cancer biomarkers (such as CEA, PSA, CA125, etc.) originating from tumor cells. In a similar way, we further confirmed that serum SAA was much elevated in NPC patients, particularly at the process of lymph node metastases when compared with non-LNM patients. However, such biomarkers (acute-phase reactants) are not commonly considered as cancer-specific ones and expected to be elevated in other malignant diseases or inflammatory diseases as well [26, 27]. In previous reports, SAA was found elevated in different malignancies, such as cancers of kidney, colon and prostate, as well as in leukemias and lymphomas [24, 48]. Therefore, SAA may represent a cancer epiphenomenon, unlikely to be of much clinical use in diagnosing and monitoring cancer [49], but it will be interesting to explore whether the rapid production of SAA in the liver or the epithelia of different organs [50] may be related to the stimulation by cytokines abundantly present in the NPC cells. It is also meaningful to investigate how SAA is produced at different stages of clinical manifestation in NPC patients.
HSP70 is the main protein produced during cellular response to varied stresses, such as heat shock, ischemia/reperfusion, and oxidative changes [51, 52]. Because HSP70 confers cell protection against different stresses, it has been hypothesized that it plays a protective role in tumor growth in vivo [53, 54]. Indeed, HSP70 is overexpressed in human tumors of varied origins [55], such as colorectal [56], breast [57, 58], prostate [59], liver [60] cancers and melanoma [61]. LNM and poor survival of cancer patients correlated with HSP70 overexpression has also been documented [57, 58, 61–63]. It is suggested that HSP70 is needed for in vivo tumor progression. In addition, HSP70 may be released from tumor cells involving involves both active secretion and passive release from necrotic cells [64]. Physiological mechanisms include co-secretion in exosomes of HSC70 with TRF and the active HSP70 secretion by the nonclassical pathway employed by cytokines [65]. Therefore, HSP70 is detectable in serum so that it could potentially be used as a biomarker for diagnosis or disease classification. Abe et al. reported that HSP70 is a marker of prostate cancer, and may be used in conjunction with PSA to identify patients with early-stage prostate cancer [59]. However, to the best of our knowledge, the association between serum level of HSP70 and NPC status has not been reported. Since our findings demonstrated that there was a modest association between serum HSP70 level and NPC staging, the serum HSP70 level should not be considered as an independent prognostic factor in NPC patients, although it might be a prognostic predictor by univariate analysis. In short, serum HSP70 may have an adjunctive clinical value in monitoring tumor progression and evaluating prognosis in NPC patients, though its clinical application as a major tumor marker is limited.
In conclusion, our serum proteomic analysis, using a comprehensive pretreatment strategy, provides a practical and exemplary tool of screening progression-associated serum proteins in NPC research. After comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we successfully identified 13 differentially expressed protein spots. We further explored the differential expressions of three of the proteins, namely, sICAM-1, HSP70 and SAA, by ELISA at serum and IHC at tissue, though more work should be completed to pinpoint the direct correlation between serum level of sICAM and HSP70 and their expression level in the tissues. We suggest that the three proteins may be potential serum biomarkers which can serve as effective target points for early diagnosis and therapy of NPC patients, though further clinical research should be done before the potential come true.

serum proteome
carcinogenesis
lymph node metastasis
nasopharyngeal carcinoma


Trochanteric fractures in the elderly: the influence of primary hip arthroplasty on 1-year mortality

Introduction The aim of the study was to compare the mortality risk and complication rate after operative treatment of pertrochanteric fractures with primary arthroplasty, dynamic hip screw (DHS) or proximal femoral nail (PFN).
Introduction
While relative consensus exists about the treatment of femoral neck fractures for elderly patients, the optimal treatment for per- and intertrochanteric fractures is still under debate [1, 7]. In a previous study of 1,173 patients with proximal femoral fractures it appeared that trochanteric fractures are still a challenge, as 1-year mortality risk and complication rate were considerably higher compared with femoral neck fractures [7]. As restoration of the preoperative ambulatory level correlated with survival rate after 1 year [7] and elderly patients are often unable to cooperate with partial weight bearing, the primary stability of the device is crucial to allow early mobilization to prevent cardio-pulmonal complications and thrombosis. Excessive collapse of the fracture site and varus displacement is a common problem of sliding hip screws combined with plates or femoral nails especially in elderly women who often suffer from osteoporosis and poor bone quality [16]. Therefore, primary arthroplasty was proposed by some authors [4, 10, 28] but their patient collectives ranging from 37 to 54 were to small to allow general recommendations. Because we used hip arthroplasty even for trochanteric fractures for a long time as a standard procedure [7, 26], we are now able to report on our experiences in a large number of patients.
This study compares the mortality risks and 1-year postoperative complications between arthroplasty and osteosynthetic fixation (DHS or PFN) for intertrochanteric fractures. A 1-year mortality was chosen as a main indicator as it depends on the surgical trauma as well as the rapid return to preinjury activity and further complications. A 1-year period was chosen as the mortality risk approaches that of an age-matched reference population after this interval [32, 35].
Patients and methods
A total of 308 patients who were treated for trochanteric femoral fractures between 1992 and 2005 with a minimum age of 60 years were enrolled in this study. Seventeen patients with pathologic fractures due to metastases were excluded. Information about survival after 1 year could be achieved from 283 of the 291 patients.
Two hundred and seventeen patients (76.7%) were female. The mean age at the time of surgery was 80.5 (±9) years ranging from 60 to 98 years. Women (82 ± 8 years) of our cohort were significantly older than men (75.5 ± 11 years; P < 0.01).
The fracture type was classified according to the System of the Orthopedic Trauma Association (AO/OTA) [24]. Pertrochanteric two part fractures are classified as A1-type, multi-part fractures as A2-type and reversed oblique intertrochanteric fractures as A3-type fractures. While all A1-type fractures are considered to be stable, most of the A2 and all A3 type fractures are unstable [19]. The distribution of the fracture types within the treatment groups is displayed in Fig. 1.
Fig. 1Distribution of the surgical treatment depending on the fracture type. The dynamic hip screw (DHS) was mostly used in more stable fractures, while the proximal femoral nail (PFN) was mainly used in A3-type fractures (X² = 22.4, P < 0.001)
A total of 109 patients (38.5%) were treated with a “dynamic hip screw” (DHS by Synthes®). Within this group there were mainly stable fractures type A1 (52%) or A2 (44%; Table 1); 132 patients (46.6%) were treated with primary arthroplasty, 117 with a total hip replacement (THR) and 15 with a bipolar hemiarthroplasty (HA). Since 2000 PFN (Synthes®) was used in 42 of the 100 cases, mainly for unstable fractures in patients without advanced osteoarthritis. As demonstrated in Table 1, these patients were younger and more frequently male (P < 0.05).
Table 1Description of the baseline variables within the treatment groupsArthroplastyDHSPFNAge (years)83 ± 779 ± 975 ± 12Number of patients13210942Gender Female 115 (87%)80 (73%)26 (62%) Male17 (13%)29 (27%)16 (38%)≥4 comorbidities63 (48%)56 (51%)17 (41%)Fracture type A137 (28%)57 (52%)7 (17%) A282 (63%)48 (44%)29 (69%) A312 (9%)5 (4%)6 (14%)Year of injury 1992–1999111 (84%)72 (66%)0 (0%) 2000–200521 (16%)37 (34%)42 (100%)
Outcome variables
Primary outcome variable was the 1-year mortality risk (Table 2). Furthermore, the occurrence of complications which led to further interventions was analyzed in multiple regression analysis. These were dislocation of the hip, non-union of the fracture, cutting out of the hip screw, infections, thrombosis and hematoma.
The Merle d'Aubigne test (0 to 18 points) was used to assess the patients [10]. We further questioned whether the patients returned to their preoperative status (e.g. living at home).Table 2The distribution of fracture type and treatment and its specific mortality1992–19992000–20051992–2005nDeathMortality (%)nDeathMortality (%)nDeathMortality (%)A1711926.829413.81002323.0 THR34926.50034926.5 HA003133.33133.3 DHS371027.019315.8561323.2 PFN000.0700.0700.0A2952829.5661319.71614125.5 THR652436.96233.3712636.6 HA001218.31218.3 DHS30413.318211.148612.5 PFN0030826.730826.7A317635.35120.022731.8 THR12541.60012541.6 HA000000 DHS5120.0005120.0 PFN005120.05120.0All fractures1835329.01001818.02837125.1 THR1113834.26233.31174034.2 HA0015213.315213.3 DHS721520.837513.51092018.4 PFN0042921.442921.4THR total hip replacement, HA hemiarthroplasty, PFN proximal femoral nail, DHS dynamic hip screw
Surgical technique
The proximal femoral nail (PFN by Synthes®-Switzerland) or the dynamic hip screw (DHS by Synthes®-Switzerland) was used according to the manufacturer’s instructions. For closed reduction the patients were positioned supine on a fracture table. Only 135° four hole-DHS plates without additional trochanteric stabilizing plates or tension band wires were used in this series.
For hemiarthroplasty a standard cemented stem (Weller, Aesculap, Germany) and a bipolar head (DePuy, Leeds, England) were used. All femoral stems were cemented using the modern 3D generation cementing technique [2]. In cases of total hip replacement, a polyethylene cup (Aesculap®, Germany) was cemented in the same manner (Fig. 3).
Statistics
First we performed univariate analysis of independent variables (age, sex, treatment, preoperative risk factors, etc.) and mortality using Fischer’s exact test for qualitative variables and ANOVA for quantitative variables. The homogeneity of variance was checked by Levene test. All tests were two-sided and a P ≤ 0.05 was considered to be significant.
The influence of treatment on mortality was checked by a multiple logistic regression model adjusted for the presence of preoperative comorbidities (four or more vs. up to three), fracture classification, age, gender, complications requiring revision surgery and year of surgery.
We used RR additional to OR because the more frequent the outcomes becomes, the more the odds ratio will overestimate the risk ratio when it is more than 1 or underestimate the risk ratio when it is less than 1 [34]. Odds ratios (OR) were transferred to relative risks (RR) according to the method of Zhang et al. [34] with the formula: RR = OR/[(1 − P0) + (P0 × OR)], where P0 is the incidence of the outcome in the nonexposed group (reference category), and OR the odds ratio given by the multiple analysis. A RR < 1 refers to a factor category that is mitigating (decreasing) the risk of the outcome (e.g. dying) when present, an RR > 1 refers to a factor category that is increasing that risk when present compared with the reference category.
All analyses were conducted with the SPSS statistic software for Windows 12.0 (SPSS, Chicago, IL, USA).
Results
Surgery
Eighty-five percent of our patients were operated within 1 day after admission. In the other cases, operation was delayed to improve the preoperative condition of the patient, e.g. by transfusion or change of medication. This was independent of the treatment groups (P = 0.307). Mean blood loss and operating time differed significantly depending on the type of operation. The mean blood loss for arthroplasty (1,050 ± 700 ml) was significantly higher than for fixation with a DHS (409 ± 360 ml) or PFN (332 ± 277 ml; P < 0.001). Implantation of a hip replacement took a mean of 115 ± 36 min, that of a DHS 73 ± 38 min and of a PFN 84 ± 32 min (P < 0.001) including time for closed reduction on the extension rag. Fracture classification significantly influenced operating time if an arthroplasty was performed. It was 100 ± 24 min for A.1, 121 ± 39 min for A.2 and 127 ± 26 min for A.3-fractures (P = 0.012). The blood loss was similarly influenced (800 ± 460 ml for A.1, 1,200 ± 800 ml for A.2, 900 ± 500 ml for A.3 fractures; P = 0.019)
Mortality
Six patients (2.1%) died during hospitalization, 25 (8.9%) within 90 days. These were patients with considerable preoperative risk factors; 71 patients (25.2%) died within 1 year (Table 2).
Influence of gender and age
As demonstrated in Fig. 2, survival was correlated to age and gender; 34.8% of the male and 24.9% of our female patients dies within the first year (relative risk 0.34, P = 0.004; Table 4). Not considering the different treatment groups patients over the age of 80 years had an increased mortality risk (P = 0.004).
Fig. 2Influence of age and gender. Independent of the treatment and fracture type the mortality risk was significantly influenced by age for both genders (P = 0.018 for men, P = 0.002 for women). After multivariate analysis, the risk to die was significantly lower for woman (relative risk 0.34, P = 0.004) and higher for patients over the age of 90 years (relative risk 3.61; P = 0.009)
Influence of comorbidities
The comorbidities and their prevalence are shown in Table 3. Of the single comorbidities only cardiac arrhythmia and cerebral diseases (P = 0.04) showed a significant influence on mortality. Logistic regression analysis demonstrated that four or more comorbidities increased the risk to die by 78% (relative risk 1.78, P = 0.009; Table 4). Forty-eight percent of our patients had four or more comorbidities and showed a mortality risk of 33.3% compared with 17.4% if they had less than four (P = 0.032 in χ2-test).
Table 3The prevalence and relative risks of the most common comorbiditiesComorbiditynPoP (Fisher’s exact)RRORCI 95%P (log. reg.)Heart attack1240.3400.431.121.190.56–2.140.69Cardiac arrhythmia470.3540.01**1.101.161.02–2.430.04*Cardiac insufficiency1110.3040.821.441.790.67–3.180.55Hypertonus1540.2681.000.980.970.55–1.730.92Pulmonary diseases660.2880.731.071.10.57–2.160.77Anemia470.3190.351.261.430.69–2.960.33Renal insufficiency470.2611.000.850.810.38–1.750.60Cerebral diseases1270.3360.301.421.811.02–3.220.04*Diabetes mellitus920.3260.281.241.410.79–2.520.25Metabolic diseases300.2330.820.750.70.27–1.800.46Alcohol/nicotine/other drugs290.3210.351.511.980.79–4.990.14Gastrointestinal disease420.2440.310.760.710.32–1.640.43Others570.3040.851.011.010.51–2.030.96Relative risks (RR) are computed from odds ratios (OR) by the following formula: RR = OR/[(1 – P0) + (P0 × OR)]P0 is the proportion of patients with the respective disease dying within 1-year after proximal femoral fractureRR < 1.0 represents a minor risk of mortality; RR > 1.0 represents a major risk of mortality compared with patients without this criteria; * P ≤ 0.05; ** P ≤ 0.01Table 4Multiple logistic regressions analysis of a 1-year mortality after proximal femoral fractures of elderly patientsDescription of the analysisInfluence of interaction of kind of treatment and fracture classification after adjusting age, gender, comorbidities, revision surgery and date of surgery Odds ratio95% CIPRelative riskInfluence: interaction of kind of treatment and fracture classificatione A1*PFN0.0000.0000.9990.00 A1*DHS0.6380.228–1.7870.3920.71 A2*TEP1.3910.566–3.4200.4721.25 A2*PFN1.1240.272–4.6470.8721.08 A2*DHS0.3720.112–1.2370.1070.46 A3*TEP2.1200.502–8.9540.3071.59 A3*PFN1.0170.087–11.8330.9891.01 A3*DHS1.0930.096–12.3870.9431.06Influence: age categoryc 70–79 years1.2310.334–4.5320.7551.19 80–89 years2.7980.785–9.9700.1122.23 >90 years6.3811.603–25.4030.0093.61Influence: genderd Female gender0.3380.161–0.7080.0040.44Influence: comorbidities complications and year of surgery  ≥4 comorbidities2.2381.219–4.1090.0091.78 Revision surgery1.1950.448–3.1840.7221.13 Surgery after 20000.7750.343–1.7520.5410.83Relative risks (RR) are computed from OR (odds ratio) by the following formula:RR = OR/[(1 − P0) + (P0 × OR)]P0 is the proportion of patients in the respective reference category dying within 1-year after proximal femoral fractureRR < 1.0 represents a minor risk of mortality; RR > 1.0 represents a major risk of mortality compared with patients without this criteriaNumber of cases in all models: 282aReference: TEPbReference: A1cReference: <70 yearsdReference: male gendereReference: A1*TEP
Influence of fracture-classification and treatment
Only pertrochanteric fractures were enrolled in this study and sorted according to the AO/OTA classification (Fig. 1). While the mortality did not differ between A1- (23.0%) and A2-type fractures (25.5%), it was higher in A3-type fractures (31.8%; Table 2). As this fracture type affected only 7% of our patients, it did not significantly influence the mortality risk (P = 0.42).
About 18.4% of the patients who received a dynamic hip screw, 21.4% of those who received a proximal femoral nail and 33% in the arthroplasty groups (THR and HA) died within 1 year (P = 0.011). As the indication for surgery was not randomized but decided on fracture classification and clinical status we attempted to reduce these influences in a multiple logistic regression analysis (Table 4). All potential influencing factors as gender, age, fracture type and amount of comorbidities were taken into account. If categorical variables were used to evaluate the interaction between fracture classification and treatment, we could not find a statistical influence of surgical treatment on mortality (Table 4). The relative risk to die was decreased for A.1- and A.2-type fractures, treated with a DHS, but this did not reach significance levels. As only seven patients with a A.1-type fracture were treated with a PFN (and all of them survived), no sensible statistical analysis can be done for this category. As all devices showed good results for this kind of fracture, the decision should be based on other parameters for A.1-type fractures.
Because our treatment regime was changed in the beginning of 2000 (introduction of the PFN, new bipolar hemiarthroplasty instead of total hip arthroplasty; Table 2), we implicated the time of surgery in our analysis. Since then the 1-year mortality after arthroplasty decreased from 34.2 to 19% (P = 0.26, Fisher’s exact test). Although over all mortality decreased from 29 to 18%, no influence of time of surgery on the mortality risk could be found using multivariate regression analysis (P = 0.54; Table 4).
Influence of complications
The only postoperative complication which influenced the survival rate was postoperative pneumonia. It occurred in 13 cases independent of the treatment groups (P = 0.26). Six of the patients died within 1 year (P = 0.048).
The DHS had a revision rate of 8.1%, while the PFN needed reoperations in nine cases (22.5%). The main reason was cutting out of the screw in the osteoporotic bone and postoperative hematoma. Due to cutting out and fixation failure secondary hip arthroplasty was performed after failed internal fixation ten times. It happened six times (5.4%) after a DHS and four times (9.8%) after a PFN.
The main complication after arthroplasty was dislocation, which occurred in 16 patients (12%). It did not occur with bipolar hemiarthroplasty. While dislocation was only a minor problem in A1-type fractures (7%), it occurred in ten A2-type (12%) and two of the nine A3-type fractures (22%). Patients were usually treated by closed reduction and physiotherapy and it did not influence mortality (P = 0.78).
Rehabilitation
The mean hospital stay was 18 days and did not differ significantly between the three treatment groups (P = 0.7). It prolonged to a mean of 30 days if a reoperation became necessary (P = 0.003). Patients who were ambulatory before the injury could walk with a walker or sticks at time of discharge; 80% of the surviving patients who were independent before the injury regained their preoperative status after 1 year. Since the year 2000 patients were encountered to a prospective registry. The mean Merle d’Aubigné score (0–18 points) for the surviving 78 patients was 13.8 ± 2.5. No significant differences could be found between the three treatment groups (P = 0.122).
Discussion
While primary arthroplasty is a standard procedure for femoral neck fractures, little experience exists for trochanteric fractures [4, 5, 9, 28]. They were primarily used as a salvage procedure after failed internal fixation [12, 15, 21, 23, 31]. Most authors implanted special calcar replacement devices, which are expensive and require the removal of large portions of the proximal femur [3, 8, 13, 28]. We used a standard cemented stem and either a standard cemented cup or a bipolar head since the 1970s in our hospital. As it proved superior to former methods of internal fixation even for pertrochanteric fractures it was the treatment of choice since 1992 [26]. From the year 2000 on hemiarthroplasty was routinely used instead of total hip arthroplasty and the proximal femoral nail was introduced for patients without advanced osteoarthritis.
The ideal treatment for intertrochanteric fractures is still under debate as none of the existing osteosynthetic devices could prove its superiority in former studies [16, 25, 29, 30]. The most frequent problem in elderly patients who usually are not able to walk without weight bearing was a cutting out of the hip screw which occurred in 4 to 20% of the reported cases [4, 6]. In our series this complication was only seen in comminuted fractures (A2 and A3 type). Thus, 10 times (6.2% of the internal fixation devices) a secondary hemiarthroplasty was performed. Immediate full weight bearing regardless of bone quality is an advantage of primary cemented arthroplasty. In our hospital, all patients are encouraged to stand up the day after surgery. Dislocation was the major complication in patients treated with a total hip replacement after comminuted pertrochanteric fractures. This high dislocation rate is rarely seen after primary hip arthroplasty but well known even for femoral neck fractures [22] or revision surgery [33] and can be attributed to insufficient muscular stability. Fractures of the greater or lesser trochanter might exaggerate this problem. The use of bipolar arthroplasty instead of total hip replacements can reduce this complication to an acceptable rate [11, 28]. Since we use total hip arthroplasty only in patients of good physical shape with severe osteoarthritis we did not see any dislocations.
Ambulatory levels and clinical scores are considerable affected by the preoperative status. Since clinical examination usually cannot be obtained for all patients, reliable comparison is difficult. Some authors use telephone interviews [4] or report about less than 50% of their patients for a 1-year follow-up [10]. In both cases it can be assumed that patients with bad functional results are not regularly included. We could also examine only 78% of the last 100 patients clinically and therefore do not want to overemphasize these findings. Instead we chose the 1-year mortality risk as the primary outcome parameter as it could be obtained for all patients and is related to the postoperative ambulatory status as well as to the operative trauma [14]. In a previous study with more than 500 fractures, we could demonstrate that patients who regained their independency and were able to attend a rehabilitation institution had a significantly lower mortality risk [7], which was further supported in this study.
Mortality was significantly influenced by patient related factors such as gender, age and comorbidities but not by the fracture type. In accordance with our former studies and other authors [7, 17, 20, 26] few single comorbidity influenced the mortality risk whereas the sum of four or more comorbidities increased the risk to die by approximately 78% (Tables 3 and 4). Patients who received a PFN or DHS were significantly younger and healthier than those in the arthroplasty group. These influences were reduced by multivariate regression analysis (Table 4). Furthermore, the interaction of fracture type and treatment was taken into account. Considering these influences, no significant influence of one of the three treatment groups on the mortality risk could be found. In studies that only use internal fixation devices mortality risks comparable with our over all mortality were found [27], thus—in accordance with other authors [3, 13]—we think that cemented hemiarthroplasty is an alternative to internal fixation in elder patients especially as our revision rate was significantly lower for arthroplasty compared with internal fixation.
Only Chan et al. [4] published a study about the use of a standard hemiarthoplasty for the treatment of displaced intertrochanteric fractures. In their small group of 54 patients they found a mortality rate of 31.5% within 1 year which did not differ from comparable age-matched studies. The walking ability of their patients was favorable compared with other studies with internal fixation. We agree that immediate full weight bearing, which can be achieved after cemented arthroplasty, is crucial for these patients.
Haentjens et al. compared 37 patients who received a calcar replacement device with 42 patients of a retrospective group, who were treated by internal fixation and found an advantage in functional outcome [10]. Although the mortality rate could not be decreased, early walking with full weight bearing reduced the incidence of pressure sores, pulmonary infection and atelectasis. Kim et al. [18] who compared a calcar replacement prosthesis with intramedullary fixation in a prospective study in two small groups of 29 patients could not find a significant difference concerning the functional outcomes, but the cut-out rate of the hip screw was 7% in their patients.
Up to now a clear indication of arthroplasty in the treatment of pertrochanteric fractures does not exist. Although operation time and blood loss are higher no significant difference concerning the mortality rate between primary arthroplasty and internal fixation could be found, but the revision rate in the arthroplasty group was significantly smaller especially since we use bipolar hemiarthroplasty instead of a total hip replacement. If the cemented arthroplasty has been performed accurately, there is very little concern about weight bearing. Furthermore, primary arthroplasty eliminates the possibility for malunion, cut-out of the hip screw and avascular necrosis of the femoral head. But primary arthroplasty is a technically challenging procedure. All lose fragments including the greater and the lesser trochanter have to be attached with cerclage wires before cementing the stem to prevent extrusion (Fig. 3) and especially in utterly comminuted fractures orientation of the rotation and leg length can be demanding.
Fig. 3Examples of treatment of A2-type fractures with a hemiendoprosthesis and tension band wires. Depending on the fracture type different ways of fixating the trochanteric region with wires were used. In the case of a calcar fragment reaching distal to the lesser trochanter a temporary reduction with a cerclage wire or reduction bone clamp was used before reaming. Final fixation of the greater trochanter was performed after reduction of the hip with cerclages or a tension band
The DHS is not suitable for reverse oblique and comminuted fractures [19, 30] but for stable fractures it seems to be advantageous to short femoral nails regarding reoperation rate and fracture fixation failure in the literature [16, 29]. We further support the recommendation that the DHS should be used for stable A1- and A2.1-type fractures while an intramedullary device is advantageous for unstable fractures. Primary cemented arthroplasty is a viable option for the treatment of trochanteric fractures in a selected group of previously independent mobile patients especially if osteoporosis would prevent full weight bearing or osteoarthritis would make further operations necessary. The routine use of total hip arthroplasty was abandoned in our hospital as the luxation rate was higher than after hemiarthroplasty. It is still used as salvage procedure after failed internal fixation in elder patients with bad bone quality.

mortality risk
trochanteric femoral fractures
total hip replacement


Differential effects of tamoxifen and anastrozole on optic cup size in breast cancer survivors

Introduction The main purpose of this study was to determine whether the optic cups of tamoxifen users and anastrozole users differ in size, with the cups of the tamoxifen users being smaller.
Introduction
Selective estrogen receptor modulators (SERMS) and aromatase inhibitors are the two major classes of medications used in the United States as adjuvant endocrine therapy for early-stage, hormone-receptor-positive breast cancer. Of the SERMS that are FDA-approved for this purpose [1], tamoxifen has been the most widely used, and for several decades it has been prescribed extensively for women of all ages [2]. The first aromatase inhibitor to be FDA-approved for early-stage breast cancer was anastrozole (Arimidex®), in 2002 [2]. However, because aromatase inhibitors do not block estrogen production adequately for pre-menopausal women [3], they are indicated for use by post-menopausal women only [4], for whom estrogen production is entirely non-ovarian [5]. Initial reports indicate that anastrozole is more effective than tamoxifen at preventing cancer recurrence [6, 7]. Thus, anastrozole now is being recommended for many women who would have been prescribed tamoxifen several years ago [8]. Because the use of aromatase inhibitors is likely to continue to increase and because the use of tamoxifen may become progressively more restricted to younger women, the potential side effects of anastrozole need to be evaluated, and the influence of age on tamoxifen side-effects needs to be closely examined.
Of all the documented or putative effects of tamoxifen on the visual system [9], tamoxifen retinopathy may be the most widely known. However, tamoxifen retinopathy is uncommon [10] and possibly cumulative-dose-dependent [11]. In contrast, Eisner et al. [12] found that short-term tamoxifen users (i.e., ≤2 years of use) often have smaller optic cups than age-matched female control subjects, in a study of women who had been selected for the absence of any overt eye disease. This result was consistent with the possibility that tamoxifen often causes a subclinical degree of swelling at the optic cup, particularly since tamoxifen is a potent blocker (at least in culture) of swelling-activated chloride channels [13, 14], such as those in astrocytes [15, 16], and astrocytes are the predominant glial cell in the cup [17, 18]. If tamoxifen indeed causes optic-cup swelling via its ability to block swelling-activated chloride channels, the cup sizes of anastrozole users would be expected to more closely resemble those of female control subjects than those of tamoxifen users. The present study tested this prediction.
Because of changing trends in adjuvant breast cancer treatment, the present study also assessed the effect of age on optic nerve head (ONH) parameters. The results support the inference that tamoxifen usage can lead to small cups, although apparently mainly for women older than about 50 years. All measurements for this study were obtained using confocal scanning laser ophthalmoscopy with the Heidelberg Retina Tomograph II (HRT). This commercial instrument was introduced in 1999 for routine clinical use, but until now, it has been used mainly to assess glaucoma and related conditions.
Methods
Subjects
Three groups of amenorrheic women ages 40–69 years old were recruited for this study: (1) women using the standard dose of 20 mg tamoxifen daily as adjuvant therapy for breast cancer for at least 4 months but no longer than 2 years, (2) women using the standard dose of 1 mg anastrozole daily as adjuvant therapy for breast cancer for at least 4 months but no longer than 2 years, and (3) women not using any hormonally acting medications. This third group served as an approximately age-matched control group. The 2-year duration-of-use requirement was based on results from several studies of vision [19, 20] and the eye [12] indicating that short- versus long-term tamoxifen use can be distinguished operationally using a 2-year cutoff. The 4-month duration-of-use requirement was included to help ensure that the medications had time to act and that the body’s response had a chance to stabilize [21, 22].
All tamoxifen and anastrozole users had completed primary treatment for breast cancer, and all were fully active and able to perform their daily pre-cancer activities without restriction. None of the control subjects had positive breast cancer histories or had previously used breast cancer medication for any purpose. Similarly, none of the tamoxifen users had previously used a hormonally acting breast cancer medication other than tamoxifen, and none of the anastrozole users had previously used a hormonally acting breast cancer medication other than anastrozole. In addition, none of the subjects in any group had ever used the SERM raloxifene.
With one exception, all analyses for this report are based on data from subjects who had been amenorrheic for at least 6 months at the time of testing. The one exception is an analysis comparing data from control subjects with data from an additional group of women (the fourth subject group overall) who met the same eligibility criteria as the control group, except for the absence of menses.
All subjects, regardless of group, met a rigorous set of eligibility criteria for excellent ocular health in order to reduce the roles of confounding factors that could complicate the data. These are the same criteria we have used previously for several types of studies [12, 19, 23]. These criteria are: (1) 20/20 or better visual acuity in one eye and 20/25 or better visual acuity in the other eye, (2) no evidence or suspicion of eye disease on undilated direct ophthalmoscopic examination and on subsequent evaluation of individuals’ stereoscopic color fundus and ONH photographs by a glaucoma specialist (author JRS) who was masked from identifying subject information, (3) no history of eye disease or ocular hypertension, (4) no diabetes, (5) intraocular pressure (IOP) ≤22 mmHg on Goldmann applanation and no between-eye IOP difference >2 mm Hg, (6) no myopia >5 diopters, (7) no use of any medication (other than tamoxifen) known to affect vision, (8) no history of ocular surgery, and (9) normal color vision (i.e., no worse than a single minor transposition error on the D-15 test conducted under Macbeth illumination). There was no suggestion of papilledema for any subject (as for criterion #2).
Demographic data for eligible subjects with usable HRT data (see second paragraph of Procedures) are given in Table 1 for each of the three amenorrheic subject groups plus the additional group comprised of women without breast cancer histories and still experiencing menses. The mean ages of the tamoxifen users were slightly lower than the mean ages of the anastrozole users and amenorrheic control subjects possibly because some tamoxifen users were not menopausal despite being amenorrheic [24]. For reference, the median age of natural menopause in American society is 51 years [25].
Table 1Demographic data for each of the subject groupsAmenorrheic subjects 40–69 years oldAmenorrheic subjects 51–69 years oldNot amenorrheic, no breast cancer history n = 19Anastrozole n = 20Tamoxifen n = 34Control, no breast cancer history n = 32Anastrozole n = 18Tamoxifen n = 25Control, no breast cancer history n = 29Age (years)58.2 (SD = 6.8)54.5 (SD = 5.2)57.2 (SD = 6.0)59.6 (SD = 5.4)56.5 (SD = 4.7)58.2 (SD = 5.1)48.0 (SD = 3.3)IOP (mmHg)15.7 (SD = 2.9)15.5 (SD = 2.3)15.6 (SD = 2.9)15.7 (SD = 3.0)15.5 (SD = 2.5)15.7 (SD = 3.0)15.5 (SD = 2.3)Duration of medication use (years)1.07 (SD = 0.45)1.31 (SD = 0.49)01.09 (SD = 0.471.36 (SD = 0.48)00IOPs are from subjects’ test eyes only
All subjects were unpaid volunteers. Recruitment methods have been described previously [19]. All subjects were Caucasian, except for one Asian subject still experiencing menses. After being informed of the nature and possible consequences of the study, all subjects gave written informed consent to participate in this study. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the OHSU Institutional Review Board and the OHSU Cancer Institute. For each subject, HRT data and IOP measurements were obtained at the same testing session. Subject recruitment and assessment lasted several years, until usable HRT data had been collected for 20 eligible anastrozole users.
Procedures
ONH scans were obtained using the HRT II according to standard techniques, after which a contour line is drawn on the average of three rapid-succession scans to define the margin of the ONH (i.e., of the optic disk) [26]. Stereo color photographs of the ONH were used to assist with contour-line placement, and extensive use was made of the ability of the HRT II software to rotate images in three dimensions. Contour lines were drawn with the grader masked from all other subject information, including subject group and age. Additional details have been presented previously [12]. However, we had previously placed the contour line as close as possible to the dark-to-light color change in the HRT reflectance image in order to define the margin of the ONH [12], but for the present study we ensured that the contour line was placed on stable rim tissue on top of the ring of Elschnig [27], which is often most discernable from the underside of the 3D image, as a ridge circumscribing the ONH. With these 3D-based contour lines, the mean measured disk areas for each of our subject groups were very nearly centered within the norms provided by Heidelberg Engineering on the HRT II printout. Thus, with one exception, all analyses of HRT values are based on the contour lines drawn according to the more recent set of criteria, which are intended to place the contour line exactly at the disk margin, rather than slightly within it. The one exception was made to assess the importance (or lack of importance) of the precise contour line placement for documenting between-group differences. Thus, we conclude the Results section by briefly reporting the results of analyses based on data obtained using the earlier contour-line placement criteria.
Eyes were considered to have usable HRT data only if the average standard deviation for the height of each pixel in the 3 HRT scans did not exceed 50 μm, since average topographic standard deviation values above 50 μm are considered by Heidelberg Engineering to signify “low image quality” [28]. Image quality was equally good for each subject group. The mean average-topographic-standard-deviation for each of the three amenorrheic subject groups ranged from 20 to 22 (SD = 7–8) μm for all eligible subjects and also for subjects older than 50 years. Among women still experiencing menses, the mean was 23 (SD = 9) μm.
Although HRT scans were obtained for each subject’s 2 eyes, only one eye per subject was used for data analysis for this paper. In this way, analyses could be limited to eyes with 20/20 or better acuity for a maximal number of subjects. Thus, test eyes were defined initially using the following steps applied in order as necessary: (a) the eye with the better acuity, even by 1 letter, than the fellow eye, (b) the eye with a lesser degree of spherical equivalent refractive error than the fellow eye, and (c) subject preference. In cases where the initially designated test eye had an HRT average topographic standard deviation value exceeding 50 μm, the fellow eye became the test eye for analysis purposes, but only if the fellow eye had 20/20 or better acuity and an HRT average topographic standard deviation value ≤50 μm. These are the same methods we used previously to designate test eyes [12].
Data analyses
Comparisons of central tendency across more than two subject groups were made using Kruskal–Wallis non-parametric analyses of variance (ANOVAS) because the distributions of some of the HRT indices were significantly non-Gaussian (1-sample Kolmogorov-Smirnov test) in ways consistent with the literature [29]. Similarly, post hoc comparisons between pairs of groups were made using Mann–Whitney U tests. Relations of HRT indices to age were evaluated for significance using Spearman rank-order coefficients because the strongest age trends were not linear. None of the reported P-values have been adjusted for multiple comparisons, but the P-values required for statistical significance of post-hoc tests as determined using a step-down Bonferroni procedure [30] are provided in the legend to Table 2. All analyses were conducted using SYSTAT 10.2 (Richmond, CA). All P-values are for 2-sided tests.
Table 2ONH indices: between-group comparisons (subjects 40–69 years old)ANOVA (Kruskal–Wallis)Anastrozole versus tamoxifen Tamoxifen versus control Anastrozole versus control Cup volumeP = 0.021P = 0.007P = 0.052P = 0.560 Maximum cup depthP = 0.016 P = 0.008P = 0.036P = 0.430Mean cup depthP = 0.028P = 0.011P = 0.050P = 0.645Cup areaP = 0.074P = 0.028P = 0.106P = 0.612Cup/disk area ratioP = 0.030P = 0.013P = 0.045P = 0.763Cup shapeP = 0.593P = 0.325P = 0.797P = 0.429Rim areaP = 0.115P = 0.173P = 0.053P = 0.492Rim volumeP = 0.084P = 0.059P = 0.061P = 0.940Disk areaP = 0.510P = 0.474P = 0.653P = 0.232Mean RNFL thicknessP = 0.718P = 0.654P = 0.438P = 0.735RNFL cross sectional areaP = 0.562P = 0.573P = 0.287P = 0.721Height variation contourP = 0.617P = 0.375P = 0.893P = 0.387Left-most data column––P-values for the non-parametric ANOVAS (Kruskal–Wallis) across all three amenorrheic subject-groups simultaneously. Comparisons based on the results for the larger contour circles (see Methods). Right 3 columns––unadjusted P-values for the post hoc comparisons (Mann–Whitney U tests) between pairs of subject-groups. The top 6 rows of variables are for cup data, the next 2 rows are for rim data, and the bottom 4 rows are for disk-margin data. The bold entries signify P-values that are considered statistically significant. For the ANOVAS, statistical significance was considered to be p ≤ 0.05. For the post-hoc comparisons, a step-down Bonferroni approach was used, so that the first (i.e., the most significant) comparison required a significant ANOVA plus P ≤ 0.05/3, the second most significant comparison required a significant first comparison plus P ≤ 0.05/2, and the third most significant comparison required a significant second comparison plus P ≤ 0.05. “RNFL” signifies retinal nerve fiber layer thickness. The height variation contour signifies the maximal minus the minimal retinal nerve fiber layer thickness, as defined in the text
Results
The optic cup volumes, maximum optic cup depths, and cup/disk area ratios were each significantly smaller for the tamoxifen users than for the anastrozole users. In contrast, none of the comparisons between anastrozole users and control subjects approached significance for any cup dimension. Table 2 provides P-values for the between-group comparisons for every summary ONH index. The cup-volume results are represented graphically as a function of age in Fig. 1, which presents the data from the three groups of amenorrheic subjects: tamoxifen users (filled symbols), anastrozole users, (shaded symbols), and control subjects (unfilled symbols).
Fig. 1Graph of cup volume versus age for all amenorrheic subjects. Filled circles represent tamoxifen users, shaded circles represent anastrozole users, and unfilled circles represent control subjects
The data in Fig. 1 indicate that among tamoxifen users, cup volumes tended to be relatively small mainly for those subjects older than about 50 years. Indeed, cup volumes decreased significantly with age (Spearman r = −0.41, P = 0.018) among tamoxifen users. However, a correlational analysis would necessarily reduce or obscure the significance of any age-related change if such change occurred predominantly within a narrow age range. Thus, although the corresponding results for cup area (Spearman r = −0.33), maximum cup depth (Spearman r = −0.30), and cup/disk area ratio (Spearman r = −0.34) were each non-significant when assessed using correlations, the cup volumes, cup areas, maximum cup depths and cup/disk area ratios were each significantly lower (P < .05) for subjects older than 50 years compared to subjects 50 years and younger when based on between-group comparisons of central tendencies. An age cutoff at 51 rather than 50 years yielded the same result. For each of these 2 age cutoffs, the difference in cup volumes between the older and younger tamoxifen users was highly significant (p ≤ .005).
Among control subjects and anastrozole users, there were not enough younger subjects to make meaningful comparisons between these same 2 age classes. However, we also tested an additional group of women without breast cancer histories, comprised of subjects who met all the same eligibility criteria as the control group, except for the absence of menses. None of the ONH indices differed significantly between the amenorrheic control group (32 women with an average age of 57.2 years) and this additional group (19 women with an average age of 48.0 years), and there was no suggestion of any differences in the cup indices specifically. For example, the median cup volume equaled 0.077 mm3 for the 32 control subjects and 0.076 mm3 for the 19 subjects still experiencing menses. Similarly, the median cup/disk area ratio equaled 0.20 for the 32 control subjects and 0.21 for these 19 additional subjects. Among the 32 control subjects themselves, cup volume was not correlated with age (Spearman r = −0.08). Among anastrozole users, Spearman r = 0.27 (P = 0.255), but the regression line relating cup volume to age accounted for only 5% of the total variance, and for only 2% when the calculation was restricted to subjects older than 50 years.
Because cup dimensions varied appreciably with age only for tamoxifen users, and because this variation appeared to be quite marked at about age 50 years, we recomputed the between-group comparisons of all the ONH indices for subjects older than 50 years. (None of the 19 women still experiencing menses were included in any of these comparisons). The results are shown in Table 3. As expected, all of the between-group differences that were significant when the data were compared for all subjects remained significant when the data were compared for only the older subjects, and many of the comparisons between tamoxifen users and control subjects became significant (e.g., for cup volume). In addition, rim volumes (i.e., the volume above and outside the cup but within the disk) now differed significantly between groups when compared for all three groups simultaneously, but not when compared for any two groups on subsequent post hoc analysis. Had we used a less conservative means of comparison (a parametric ANOVA followed by Tukey’s HSD test), the rim volumes of the tamoxifen users would have been considered to be significantly larger than the rim volumes of the anastrozole users and also than the rim volumes of the control users. This is probably the appropriate interpretation, since rim volumes, unlike cup volumes, were fairly normally distributed, allowing the use of parametric statistics.
Table 3ONH indices: between-group comparisons (subjects 51–69 years old)ANOVA (Kruskal–Wallis)Anastrozole versus tamoxifen Tamoxifen versus control Anastrozole versus control Cup volumeP = 0.006P = 0.003P = 0.019P = 0.330Maximum cup depthP = 0.007P = 0.005P = 0.014P = 0.309Mean cup depthP = 0.016P = 0.007P = 0.038P = 0.463Cup areaP = 0.025P = 0.009P = 0.051P = 0.431Cup/disk area ratioP = 0.011P = 0.005P = 0.020P = 0.540Cup shapeP = 0.340P = 0.146P = 0.400P = 0.463Rim areaP = 0.113P = 0.161P = 0.049P = 0.511Rim volumeP = 0.030P = 0.018P = 0.035P = 0.678Disk areaP = 0.435P = 0.301P = 0.883P = 0.217Mean RNFL thicknessP = 0.542P = 0.350P = 0.336P = 1.000RNFL cross sectional areaP = 0.482P = 0.369P = 0.259P = 0.878Height variation contourP = 0.341P = 0.160P = 0.775P = 0.242Same as Table 2, except based on data from subjects older than 50 years)
Table 4 presents the means and standard errors of the mean, along with the corresponding medians, for every ONH index for each of the three groups of subjects restricted to women older than 50 years. Note that the means and medians for many ONH indices (e.g., rim volume) were similar within groups, but that for some ONH indices (e.g., cup volume), means and medians were dissimilar, signifying a skewed distribution. Thus, Figs. 2–6 present between-group comparisons of medians with interquartile intervals plus overall ranges for cup volume (Fig. 2), cup area (Fig. 3), maximum cup depth (Fig. 4), cup/disk area ratio (Fig. 5), and rim volume (Fig. 6), in all cases for subjects older than 50 years. Although the cup areas of the tamoxifen users were not significantly smaller than those of the control subjects, their cup/disk ratios were significantly lower, probably because a major source of anatomic variability unrelated to tamoxifen use now was factored out.
Table 4ONH indices: means and standard errors of the mean, and medians (subjects 51–69 years old)Anastrozole n = 18Tamoxifen n = 25Control n = 29Cup volume (mm3)0.107 ± 0.0170.043 ± 0.0150.090 ± 0.0170.1040.0120.073Maximum cup depth (mm)0.611 ± 0.0470.423 ± 0.0400.559 ± 0.0400.5910.3780.594Mean cup depth (mm)0.207 ± 0.0170.145 ± 0.0140.189 ± 0.0140.2060.1300.198Cup area (mm2)0.476 ± 0.0590.275 ± 0.0500.423 ± 0.0570.5230.1390.385Cup/disk area ratio0.207 ± 0.0220.123 ± 0.0210.193 ± 0.0220.2290.0790.192Cup shape−0.229 ± 0.015−0.199 ± 0.012−0.219 ± 0.013−0.229−0.205−0.210Rim area (mm2)1.724 ± 0.0661.895 ± 0.0711.697 ± 0.0611.7971.8671.732Rim volume (mm3)0.453 ± 0.0250.593 ± 0.0420.475 ± 0.0280.4610.5800.506Disk area (mm2)2.200 ± 0.0942.169 ± 0.0742.120 ± 0.0672.3162.1562.154Mean RNFL thickness (mm)0.245 ± 0.0100.264 ± 0.0140.246 ± 0.0080.2460.2620.242RNFL cross sectional area (mm2)1.281 ± 0.0591.366 ± 0.0141.266 ± 0.0491.2821.3350.242Height variation contour (mm)0.359 ± 0.0190.401 ± 0.0190.383 ± 0.0150.3750.3880.391Fig. 2Box plot of cup volume for women older than 50 years for each of the three amenorrheic subject groups. For each group, the horizontal line inside the box represents that group’s median value. The bottom and top of the box represent the upper and lower hinges, respectively, i.e., the first and third quartiles between which lie 50% of all the values. The whiskers represent the range of values that fall within a distance D from the hinges, where D equals 1.5 times the inter-quartile range. Asterisks represent individual points that fall between a distance of D and 2D from the hinges. Open circles represent individual points that are more distant outliersFig. 3Same as Fig. 2, except that box plots are for cup areasFig. 4Same as Fig. 2, except that box plots are for maximum cup depthsFig. 5Same as Fig. 2, except that box plots are for cup/disk area ratiosFig. 6Same as Fig. 2, except that box plots are for rim volumes
When the between-group comparisons of the ONH indices were further restricted by excluding tamoxifen and anastrozole users using these medications for less than 1 year, every comparison that was significant (bold numerical entries in Table 3) remained significant. In addition, the duration of medication use was observed to matter little or not at all for the anastrozole users. For instance, the regression line relating cup volume to the duration of anastrozole use accounted for only 5% of the total cup-volume variance among all 20 anastrozole users, and for only 4% when the calculation was restricted to the 18 anastrozole users older than 50 years. These several sets of results indicate that the cup differences between the tamoxifen and anastrozole groups did not occur because the anastrozole users had experienced too short a period of medication use.
The bottom 4 indices listed in Tables 2–4 represent biomorphometric assessments at the disk margin alone. These are: (1) disk area, (2) mean retinal nerve fiber layer (RNFL) thickness, (3) RNFL cross-sectional area, and (4) the height variation contour, calculated as the maximal minus the minimal RNFL thickness along the contour line. None of these indices differed significantly between groups, although there was some suggestion of greater RNFL thickness for the tamoxifen users (see Table 4).
As stated in the Methods section, we also conducted analyses using the ONH indices derived from contour lines placed slightly interior to the anatomic disk margin rather than exactly at the disk margin. Every statistically significant result concerning the cup remained significant when based on the smaller contours. Conversely, whereas some of the cup-area comparisons involving tamoxifen users were not statistically significant when based on the larger contours (see Tables 2, 3), every corresponding comparison became significant when based on the smaller contours. In particular, the cup-area values for the older tamoxifen users (>50 years) became significantly lower than the cup-area values for the older control subjects (>50 years), presumably because using the smaller contours enhanced the importance of effects occurring predominantly within the interior of the cup.
Discussion
Because tamoxifen users and anastrozole users share similar medical histories, the results indicate that tamoxifen often leads to reductions of optic-cup size among middle-aged women older than about 50 years. Any effects of anastrozole at the ONH remain unproven, and if they exist at all, they are probably minor and opposite to the effects of tamoxifen. In contrast to tamoxifen [9, 10], there seem to be no reports in the peer-reviewed literature concerning any visual or ocular side effects of anastrozole. One study has found that anastrozole can cause regression of breast cancer metastases within the choroid [31].
Because all of the women in this study met a rigorous set of criteria for excellent ocular health, any cup-size changes presumably would be designated as subclinical. Data from several reports suggest that the color vision of tamoxifen users is often altered [10, 19, 20], although such alterations either are subtle or can be revealed only under specialized laboratory conditions, and there is no evidence that color vision changes are caused by effects occurring at the ONH. A recent survey found that about 13% of tamoxifen users reported experiencing vision changes that they attributed to tamoxifen [32], but the nature of these purported changes was not specified, nor were the subjects’ reports related to the results of any eye examination. Nevertheless, the subjects reporting vision changes had significantly higher serum levels of tamoxifen and a potent tamoxifen metabolite (N-DMT) than did the women not reporting these changes [32]. In contrast, reports of well-known side effects associated with tamoxifen use, such as hot flashes and vaginal dryness, were not related significantly to these same serum levels [32]. Cases of tamoxifen-induced optic neuropathy have been described in the literature [12, 33–38], but such cases usually are considered rare [9, 10, 38]. Perhaps tamoxifen users sometimes experience a degree of optic neuropathy that would not be detected or confirmed on a presenting eye examination owing to the wide range of normal cup appearances. If so, the results of this study point to a potential new application for intraocular imaging devices such as the HRT, which provides spatial resolution at the ONH on the order of tens of microns and is designed to detect longitudinal change [39]. Several types of high-resolution intraocular imaging device are now available clinically [39], and they allow high-resolution biomorphometric data to be collected in ways that are non-invasive and not intimidating to patients. The ease of data collection with these devices may allow patients who report tamoxifen-related vision change to be evaluated before and after cessation of tamoxifen use, and also after any rechallenge. Until this is done, and then only if changes in vision are shown to correspond to anatomical changes, might it be justified for the type of results described in this study to direct therapy. Presently, the only assured clinical application is to alert ophthalmologists and neurologists that assessment of the ONH for conditions such as glaucoma or optic neuritis may be complicated by the use of tamoxifen.
A human-subjects study such as ours cannot definitively identify the mechanism(s) by which tamoxifen may reduce optic-cup size in vivo. Nevertheless, there is evidence to suggest that tamoxifen might reduce cup size via astrocytic swelling. First, tamoxifen is a potent blocker of swelling- (or “volume-”) activated chloride channels for astrocytes assessed in culture [16, 40], and astrocytes are abundant in the optic cup [18, 41]. Second, although astrocytes are present in the RNFL [17, 41], they comprise proportionally more of the neural volume as the cup is descended [41], and astrocytes along with ganglion cell axons are abundant at the base of the cup and also within the sides [42]. However, if the ganglion cell axons increased in volume, one would expect more visual dysfunction than is observed. In addition, there is evidence that tamoxifen-induced development of posterior subcapsular cataracts [10] may involve the blockage of volume-activated chloride channels [43], and that the utility of high-dose tamoxifen as salvage therapy for astrocytomas [44] may depend partly on the ability of tamoxifen to cause astrocytic swelling [45]. Based on photographic assessment of the ONH and retina by a neuro-ophthalmologist (author JF), there did not appear to be any difference in the vasculature of the three subject groups that would have led to the between-group cup differences quantified in this study and also observed in the ONH photographs when examined for groups of subjects rather than for isolated subjects.
The presence of a steep reduction of cup size with age at about 50–51 years among the tamoxifen users suggests that any ability of tamoxifen to reduce cup size, by whatever means, depends on the absence or near absence of natural female hormones, given that the median age of natural menopause is 51 years [25] and given that tamoxifen elevates the estrogen levels of pre-menopausal women [46]. Hormone replacement therapy might, in principle, prevent or alleviate tamoxifen-induced reductions in cup size, but hormone replacement is generally contraindicated for breast cancer survivors, especially for women who have had hormone-receptor-positive tumors [47] (although diverse opinions exist [48]). Hormonal reduction by itself, whether occurring naturally or induced by anastrozole, does not appear to result in cup-size reductions of nearly the magnitude or the frequency inferred for the older tamoxifen users, at least not for the age range that we investigated. The presence of small cups among the tamoxifen users probably did not result from selectively low IOP levels, since the measured IOPs for each subject group were quite similar, differing by no more than 0.2 mmHg on average. Because the presence of small cups among tamoxifen users appeared to be unaccompanied by corresponding significant effects at the disk margin, the effects of tamoxifen on the ONH may be regarded as predominantly localized to the cup.
For this study, we found no evidence of any age-related change of ONH indices for healthy women not using hormonally acting medications. However, a recent study of hundreds of subjects has documented the presence of ONH differences between healthy men and women older than 65 years [49]. In that study, several ONH indices were found to differ significantly between men and women, but the greatest percentage difference was for cup area, which averaged about 14% smaller for women than for men [49]. There is separate evidence that cup areas can depend slightly on the phase of the natural menstrual cycle [50]. These 2 sets of results along with our own results suggest that further studies be conducted to elucidate the effects of hormonally acting medications on the ONH. Such studies should include elderly women using tamoxifen, if possible, since elderly women have higher circulating levels of tamoxifen than do younger women [51].

tamoxifen
anastrozole
optic cup
breast cancer
adjuvant endocrine therapy
eye
optic nerve head