Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration–viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22° C) than in non-cooled cells (T = 37° C), but no significant differences are visible between T = 10° C and T = 22° C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided.
Introduction
Chemotherapy-induced hair loss is a feared side effect of cancer treatment (Katsimbri et al. 2000). Scalp cooling during administration of chemotherapy prevents hair loss (e.g., Ridderheim et al. 2003). Cooling can be achieved by means of a cap that is pre-cooled in a freezer or that exchanges coolant with a reservoir. The hair preservative effect of scalp cooling is attributed both to reduced blood flow by vasoconstriction and to reduced reaction rates in the body at the level of the hair follicle. When the scalp is cooled, vasoconstriction is induced, and through this, blood flow to the hair follicle is decreased. This reduced blood flow results in a decrease of the total amount of cytotoxic drug available for uptake in the hair follicle. The effect of reduced perfusion was studied by Janssen et al. (2007), and it was found that cooling the scalp to 20° C reduces local blood flow down to 20% of normal. A further decrease in temperature did not result in a further decrease in local blood flow. Based on this, it might be expected that there is a limit in temperature below which the effectiveness of scalp cooling will not increase anymore.
However, the hair preservative effect of scalp cooling is also attributed to reduced cell metabolism. Because of reduced temperatures during scalp cooling, cellular drug uptake and damage are assumed to be lower, and with this, hair follicles are thought to be less susceptible to cytotoxic drugs. Decorti et al. (2003) showed that in vitro doxorubicin uptake in kidney cells at 37° C was 4.5 times higher than drug uptake at 4° C. In addition, Decorti also showed that doxorubicin uptake is dependent on extracellular concentration. Unfortunately, no studies have been performed to quantify these processes in the human hair follicle.
For a better understanding of the functioning of scalp cooling, it is important to quantify the contribution of reduced drug uptake and drug damage, if any, on the hair preservative effect of scalp cooling. To this end, we investigated experimentally the relationship between doxorubicin exposure and cell damage at different temperatures using normal human epidermal keratinocytes.
Chemotherapy disrupts the rapidly dividing keratinocytes (the cells that actually produce the hair shaft) in the hair follicle (Cotsarelis and Millar 2001). Therefore, the use of keratinocytes as an in vitro model is indicative for chemotherapy-induced damage to the hair follicle. Moreover, human keratinocytes are commercially available and they are easy to cultivate, which means that with this model, a wide range of boundary conditions can quickly be investigated.
The goal of the experiments was to assess the effects of temperature and chemotherapy on keratinocyte survival.
Materials and Methods
Pooled neonatal normal human epidermal keratinocytes (NHEK) were obtained from Cambrex Bio Science Verviers, Belgium (catalog number CC-2507). These cells have a doubling time of approximately 24 h. Cells were cultured in T-75 flasks at a seeding density of 3,500 cells cm2 under an atmosphere of 95% air and 5% CO2 at 37° C using 15 ml of keratinocyte general medium (KGM2; Cambrex Bio Science Verviers, catalog number CC-3107). Medium was refreshed the day after plating, and afterwards every other day until cells reached 70–80% confluence. Then, cells were harvested using 6 ml of trypsin solution and subsequently cryopreserved in liquid nitrogen at a density of 1.2 × 106 cells per milliliter using 80% KGM2, 10% fetal bovine serum, and 10% dimethyl sulfoxide (DMSO).
For the experiments on the effect of temperature and chemotherapy on keratinocyte survival, third passage cells were plated in 24-well plates at a cell density of 6,000 cells per square centimeter. Cells were incubated at 37° C for a period of 24 h to allow the cells to recover from handling. Concentrations of doxorubicin in KGM2 (0.01, 0.04, 0.1, 0.5, 1.0, 3.0, or 10.0 μg ml−1) were prepared. Cells were then exposed to 250 μl of a specific doxorubicin concentration. Plates were incubated for 4 h at either a low temperature (TL = 10° C), a medium temperature (TM = 22° C), or a high temperature (TH = 37° C). For each combination of temperature and doxorubicin concentration, a sample size of eight was used. Cells receiving medium without any doxorubicin were used as a control group for each specific temperature. At the end of the exposure time, doxorubicin was removed, and cells were washed with 500 μl phosphate-buffered saline (PBS). Fresh medium (250 μl per well) was added to each well, and plates were then incubated at 37° C for a post-exposure time of 72 h, after which, a viability assay was performed as described below.
The damage to the NHEK cells exposed to different temperatures and doxorubicin concentrations was determined by a colorimetric MTT (tetrazolium) viability assay. The assay is based on the observation that viable cells have the ability to metabolize a water-soluble tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a product termed purple formazan (Mosmann 1983; Edmondson et al., 1988; Sgouras and Duncan, 1990). The purple formazan can be solubilized, and the optical density of the solute can be determined using a spectrophotometric technique. The resulting absorbance is directly proportional to the number of cells, and this linearity extends over a wide range of cell numbers (Mosmann 1983). A standard MTT solution was prepared by dissolving 5 mg ml−1 of MTT (MTT formazan, Sigma Aldrich, Zwijndrecht, The Netherlands) in PBS. The standard MTT solution was added to complete medium (KGM2) in a ratio of 1 to 10 to obtain a final solution of 10% MTT solution and 90% KGM2. A volume of 220 μl of this solution was added to all test wells, and the plates were left to incubate at 37° C and 5% CO2 for a period of 45 min. After this period, the MTT/medium solution was removed, and 200 μl of a solution of 90% DMSO and 10% Triton X was added to extract the purple formazan salt from the cells. Plates were sealed and kept in dark for 30 min, after which, 100 μl of each well was transferred to a new well of a 96-well plate. The optical density (OD) was measured using an automated spectrophotometric plate reader (Synergy HT, BioTek Instruments Inc., Winooski, VT, USA) set to 570 nm and using a reference wavelength of 650 nm. Blanks (100 μl of DMSO/Triton X solution only) were used as an extra control. For each well, the corrected optical density OD(570–650 nm) was provided by the plate reader. Mean optical density and standard error of each temperature and concentration group [ODT,C ± SE(ODT,C)] and their respective control group [ODT,0 ± SE(ODT,0)] were calculated. Based on these values, the viability (S) for each temperature and concentration group can be calculated as:
An analysis of variance (ANOVA) was performed using statistical software (SPSS version 15.0, SPSS Inc., Chicago, IL). The ANOVA test is used to investigate whether significant differences in mean values exist between two or more groups. In this study, we want to investigate what the effect of temperature on the one hand, and the effect of doxorubicin concentration on the other hand, is on cell survival. Using a general linear model, the viability is statistically modeled as:
This means that we investigated whether temperature, concentration, and an interaction term are significantly relevant for describing our data. Where appropriate, a post hoc analysis was used using Tamhane’s T2 criterium (based on unequal variances) to test for differences between various doxorubicin concentrations and various exposure temperatures. A significance level of p < 0.05 was used in these statistical tests.
Results
Microscopic photographs of cells at selected concentrations are shown in Fig. 1. Here, we can see that the control groups of all temperatures show similar cell counts. With increasing concentration, the number of cells decreases. At 3 μg ml−1 (C4), a clear difference is visible between the high temperature group and both the medium and low temperature group. The difference between these groups at other concentrations is less well defined. It can be seen that there is a difference in cell morphology between 0.04 μg ml−1 (C2) and 0.5 μg ml−1 (C3). Cells in the latter group, and in groups with higher doxorubicin concentrations, show enlarged and flattened cell shape and increased granularity. These are characteristics of senescence or aging, which is known to be caused by doxorubicin (Roninson 2003). The results of the viability determination are shown in Fig. 2. This figure shows the cell viability as a function of doxorubicin concentration for different doxorubicin exposure temperatures. In this figure, we can see that the viability of keratinocytes as a function of doxorubicin concentration shows a decreasing S-curve with increasing concentrations. Viability levels are slightly above 1 for low concentrations (0.01 μg ml−1), and with increasing concentrations, viability gradually drops towards zero for high concentrations (10 μg ml−1). For different temperature groups, cell viability at a specific concentration is always lowest for the high temperature group. In the mid-section of the concentration range (i.e., 0.1–1 μg ml−1), the differences between the high temperature group (TH) and the two lower temperature groups (TL and TM) are more pronounced. Cell viability for these lower temperature groups decreases more slowly than for the high temperature group, until at a concentration of 1 μg ml−1, a rapid drop in cell viability is visible. The difference between the individual temperature groups at low and high concentrations is therefore small.
Figure 1.Microscopic photographs of cells exposed to selected doxorubicin concentrations. Photographs are taken at a magnification of ten times. C1 = 0.01 μg ml−1, C2 = 0.04 μg ml−1, C3 = 0.5 μg ml−1, C4 = 3.0 μg ml−1, C5 = 10 μg ml−1.Figure 2.Cell viability as a function of doxorubicin concentration at different temperatures. Delta: TL (10° C), filled square: TM (22° C), and open circle: TH (37° C). Data points show the mean and standard error of eight replicates. The results of a significance test are shown in Table 1.
From an ANOVA analysis, we found that the effect of doxorubicin concentration on NHEK viability was highly significant [F(6,161) = 165.213, p < 0.001], as was the effect of temperature on NHEK viability [F(6,161) = 37.054, p < 0.001]. The effect size of doxorubicin (; partial eta squared) was higher than the effect size of temperature ( = 0.88 compared to  = 0.36).
For all temperature groups, increased doxorubicin concentration has a significant decreasing effect on cell viability. This effect was highly significant for the high temperature group [F(6,49) = 119.400, p < 0.001], the intermediate temperature group [F(6,49) = 33.166, p < 0.001], and the lowest temperature group [F(6,49) = 68.225, p < 0.001] Based on these results, a post hoc analysis was performed for each temperature group to check where a significant transition in cell viability as function of doxorubicin concentration occurs. This transition point may be viewed as the inflection point of the S-curve. For the low and medium temperature group, we found a transition point between 1.0 and 3.0 μg ml−1. The highest temperature group has a transition point slightly below 0.5 μg ml−1.
The ANOVA analysis also revealed a significant interaction between doxorubicin and temperature on cell viability [F(6,161) = 2.754, p = 0.002], although the effect is smaller than that of temperature and doxorubicin ( = 0.199). This significant interaction term shows that the effect of temperature on cell viability is not the same at the levels of different doxorubicin concentrations. For low and high doxorubicin concentrations, there is hardly any influence of a lower temperature on cell survival. In the mid-section of the graph this influence is far more substantial.
The results of the ANOVA analysis are summarized in Table 1. It can be seen that significant differences exist between TL and TH for concentrations of 0.1 μg ml−1 and up, except for a concentration of 1 μg ml−1. At this concentration, the significance level is not high enough, but still, a trend is visible (p < 0.1). Between TM and TH, significant differences exist for concentrations of 0.5 μg ml−1 and up, except for a concentration of 1 μg ml−1 where no significant difference is visible. The only significant difference between TL and TM exists at a concentration of 10 μg ml−1, although a trend is visible at a concentration of 0.1 μg ml−1.
Table 1.Results of an ANOVA analysis for determining the significance between different temperature groups at a specific doxorubicin concentrationDoxorubicin (μg ml−1)F valuep valueTL vs THTM vs THTL vs TM0.01F(2,21) = 0.3150.733   0.04F(2,21) = 1.9580.166   0.1F(2,21) = 15.187<0.001p < 0.01 p < 0.1*0.5F(2,21) = 31.605<0.001p < 0.001p < 0.001 1F(2,21) = 4.380<0.05p < 0.1*  3F(2,21) = 12.908<0.001p < 0.01p < 0.01 10F(2,21) = 22.587<0.001p < 0.001p < 0.05p < 0.05p Values are shown with their respective significance level. Trends (p < 0.1) are marked with an asterisk. See text for more detail.
Discussion
We examined the role reduced metabolism has on the hair preservative effect of scalp cooling. For this, a controlled in vitro experiment was conducted to assess the damage exerted by doxorubicin on human epidermal keratinocytes for a range of chemotherapy concentrations and exposure temperatures. The effects of temperature and doxorubicin on cell damage were determined from cell viability measurements (MTT assay).
We found that increased doxorubicin concentrations have a significant decreasing effect on keratinocyte cell viability. Viability was close to 1 for low concentrations and close to 0 for high concentrations. In between, the relationship between doxorubicin concentration and cell viability shows an S-curve. We also found that reduced temperature has a significant increasing effect on keratinocyte cell viability. The two cooled temperature groups (10° C and 22° C) show a higher cell viability for each doxorubicin concentration than the 37° C group. At the mid-range of doxorubicin concentrations (0.04–1 μg ml−1), the effect of reduced temperature is more pronounced than in the extreme values of doxorubicin concentration. This observation is confirmed by the fact that an interaction term of doxorubicin concentration and exposure temperature was significant. The increased effect of reduced temperature was significant for doxorubicin concentrations of 0.1 μg ml−1 and higher. We found no significant differences between the low (10° C) and medium (22° C) group.
The results of the experiment are in line with the hypothesis of scalp cooling. In this hypothesis, the reduced temperature caused by scalp cooling will result in lower perfusion and a lower cellular metabolism. With this, the supply, uptake and damage of a chemotherapeutic agent will be diminished.
For low doxorubicin concentrations (0.01 and 0.04 μg ml−1), the MTT assay for each temperature group showed a higher viability than the respective control group. Because of the fact that each specific temperature group has its own control group, it is unlikely that this effect is caused by a measurement error. This means that apparently, cells are metabolically more active when exposed to a small amount of doxorubicin. It is possible that cells in the control group are close to reaching confluence at the time that viability is determined. As cells reach confluence, there is not enough room for new cells. Therefore, the rate at which cells divide will become lower, and hence, their metabolic activity will decrease. Another possibility is that a small amount of doxorubicin will stimulate cells to repair damage exerted to the cells. Hence, it may actually result in larger cell growth compared to a control group receiving no doxorubicin. At this point, however, we have no indication for which explanation may be true.
Another remarkable result is that there is no significant difference between the medium and low temperature group. Thus, it seems that there is a limit in effect of reduced temperature. In a pilot study to determine optimal doxorubicin exposure temperature, we found that an exposure temperature of 26° C also showed increased cell viability (data not shown), and this increase did not differ from other temperature groups. During scalp cooling, a skin temperature of approximately 20° C is reached. Based on the results of our experiment, one might expect that the limit in effect of reduced temperature during scalp cooling may be reached for a temperature as high as 26° C. This means that during scalp cooling, the effect of reduced metabolism is already at its maximum. However, care has to be taken when the results of an in vitro experiment are generalized. Therefore, further studies on the effect of reduced temperature on cell damage are needed to exactly define this limit. Ideally, an in vitro hair follicle model is used in these studies. Our current study confines the boundary conditions in doxorubicin concentration and exposure temperature that can be used in these further studies.
Recently, it was shown that there is a limit in perfusion during scalp cooling (Janssen et al. 2007). Cooling the scalp to approximately 18° C resulted in a decrease in perfusion to 20%. Further cooling did not result in any further decrease in perfusion. The findings of the current study indicate that there is no difference in cell survival between 22° C and 10° C. Based on these findings, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient. Therefore, strong cooling during scalp cooling should be avoided.

doxorubicin
keratinocytes
scalp cooling
viability
chemotherapy induced hair loss


Embolism of the popliteal artery after anterior cruciate ligament reconstruction: a case report and literature review

Arterial complications after anterior cruciate ligament reconstruction (ACLR) are rare. We present a case report of a 44-year-old male patient with a subtotal occlusion of the popliteal artery, with sensory loss in the foot, 17 days after ACLR. Embolectomy and anticoagulant therapy led to full recovery of the peripheral arterial circulation. The sensory loss of the foot also fully recovered. To our knowledge, this is the first case report of an embolus of the popliteal artery after ACLR without relation to graft fixation. A literature review on vascular complications after ACLR is presented.
Introduction
Vascular complications after anterior cruciate ligament reconstruction (ACLR) are rare. Few peer reviewed case reports have been reported with various techniques of reconstruction [1, 3–9]. Allum did not report vascular lesions as a complication after ACLR in a review article on this subject [2]. The origin of vascular lesions after ACLR may be venous or arterial [4]. We present a case of arterial embolism of the popliteal artery after ACLR. A literature review on vascular complications after ACLR is presented.
Case report
A 44-year-old male has a previous medical history of open medial and lateral ligament repair of the right knee 15 years previously (motor accident). Functional instability of the right knee due to ACL deficiency was the reason for referral to our service. There were no signs of posterior nor posterolateral instability. An ACLR was performed with a quadruple hamstring graft. The graft was fixed with a Bone Mulch Screw on the femoral side and a WasherLoc device in the tibia (Arthrotek, Inc. Warsaw, USA). The latter is a spiked washer with bicortical screw fixation. Total tourniquet time was 90 min. No complications were noted during or after surgery. Thromboprophylaxis for deep venous thrombosis was given by means of Low Molecular Weight Heparin (2500 IE daily) during hospital stay. The hospital recovery was uneventful.
On the 17th day of post surgery, he experienced pain and swelling in the popliteal fossa of the right knee. The complaints partially resolved with physiotherapy. Two days later, the fossa pain returned with alterations of skin color, sensory loss and an increasing cold right foot. He was referred to a vascular surgeon. Adequate dorsal pedal and posterior tibial pulses were noted. Duplex ultrasound examination showed no sign of venous thrombosis. Angiography revealed a subtotal occlusion of the popliteal artery at the level of the superior genicular artery (Fig. 1). An embolectomy was performed using a Fogarty catheter inserted in the femoral artery. The pedal pulses were diminished after embolectomy and a second angiography was performed. The occlusion at the level of the popliteal artery was no longer detected. No further emboli were noted, however, the peripheral flow qualified as too slow and suspect of small distal occlusions. Anticoagulant therapy with intravenous heparin as well as epidural analgesia was administered until complete recovery of peripheral circulation. The patient developed a superficial infection of the groin wound, treated by antibiotics. He was mobilized and discharged after 8 days. Sensory loss of the foot slowly recovered after 4 months. Vascular analysis in rest and strenuous activity was performed at 4 months. He had no more complaints, symmetrical ankle-brachial index in both legs and intact pulses at the foot and ankle. Vascular analysis did not reveal any other possible cause for arterial emboli. The patient has full range of motion of the right knee with a Lachman and anterior drawer test of 0–2 mm (International Knee Documentation Committee) and absent pivot shift test.
Fig. 1Angiography of the right knee showing subtotal occlusion of the popliteal artery at the level of the superior genicular artery (reproduced with permission from [4])
Discussion
Vascular complications after ACLR are rare. The origin of vascular lesions may be venous or arterial [4]. A case of fatal pulmonary embolism after ACLR with venous origin has been published recently [5]. Hypothesis of the cause was a hereditary coagulopathy. Arterial lesions of the popliteal artery after ACLR have been presented in few peer reviewed case reports even with an all-inside technique of arthroscopic ACL reconstruction and fixation as well as any type of graft [4].
Roth et al. [8] described an occlusion of the proximal popliteal artery. A composite graft consisting of a polypropylene ligament augmentation and the middle third of the quadriceps-patellar tendon was fixed to the lateral femur with a staple. The artery was trapped between the graft and the femur. A saphenous bypass was performed 6 weeks post surgery.
Spalding et al. [9] reported a case of unilateral claudication 8 years after ACL reconstruction with use of a Gore-Tex ligament. A cyst had formed around the femoral insertion of the ruptured synthetic ligament and was excised without vascular repair.
Evans et al. [3] reported a pseudoaneurysm of the medial inferior genicular artery following ACL reconstruction using a central third patellar tendon graft fixed with interference screws. At 5 weeks, ligation of the artery and removal of the thrombus led to full recovery. The cause of the lesion was elevation of the periosteum on the medial side of the tibia for tibial tunnel preparation.
Aldridge et al. [1] described an avulsion of the middle genicular artery after a bone-patellar tendon-bone autograft fixed with interference screws. Surgical exploration at 4 weeks revealed a tear in the popliteal artery. There was no rupture of the posterior capsule. Probable cause for the avulsion of the middle genicular artery was the debridement of the femoral ACL remnant tissue.
We have previously published two cases of popliteal artery lesions caused by a drill for bicortical tibia fixation after quadruple hamstring ACLR [6, 7]. In the first case, the drill had caused an intimal lesion at the level of the infragenicular popliteal artery which led to the pseudoaneurysm. Vascular repair was performed 12 days after ACL reconstruction but sensory loss of the saphenous and medial plantar nerves was still present at 4 months follow-up [7]. The second case was a simultaneous traumatic pseudoaneurysm and thrombosis of the popliteal artery after ACLR. At surgical exploration, the thrombosis was in line with the drill hole for bicortical tibial fixation. There was no apparent relation of the femoral fixation device to the pseudoaneurysm of the supragenicular popliteal artery. This pseudoaneurysm was thought to be pre-existent. The pseudoaneurysm was ligated and a venous jump graft was performed to bypass the thrombosis located more distally in the popliteal artery [6].
In this review of the literature on arterial complications after ACLR, all cases are associated with direct damage to the popliteal artery at time of ACLR [1, 3, 6–9]. There was no apparent direct damage to the popliteal artery in the 44-year-old male patient presented in this case report. The popliteal artery occlusion was not in line with either the femoral nor tibial fixation device. Vascular analysis did not reveal any pre-existent vascular causes for arterial embolus formation proximal to the popliteal artery. Our hypothesis of the cause was the traumatic knee dislocation 15 years previously. Precursors could have been pre-existent intimal vascular damage or adhesions of the artery at the level of the superior genicular artery in combination with the use of the tourniquet and ACLR.
Conclusion
Awareness of possible arterial complications after ACL reconstruction is essential for early diagnosis. Clinical symptoms of pain in the popliteal fossa and sensory deficits in lower leg and foot should prompt the physician to analyze possible injuries of the popliteal artery. The differential diagnosis should include compartment syndrome and deep venous thrombosis. Doppler examination as well as intact pedal arterial pulses are unreliable in diagnosing arterial lesions after ACL reconstruction. Contrast-, CT- or MRI-angiography are the diagnostic tools of choice. Immediate surgical exploration is indicated to limit limb ischemia and neurological damage [4].


Editorial to Echocardiographic assessment of left atrial ejection force and kinetic energy in chronic congestive heart failure

It is well known, that the left atrium plays an important role in filling of the left ventricle: In first instance by being a reservoir of expansion during systole, than by being a conduit phase during diastole and finally by an active phase (if sinus rhythm is present) during late diastole [1]. This active phase has an important role in compensation of decreased left ventricular compliance in patients with heart failure. It is well known, that cardiac output decreases with 15–20% in patients who are in atrial fibrillation or are only paced in the ventricle [2]. This is not only of importance from pathophysiological point of view, but left atrial function is also an independent determinant of prognosis in patients with heart failure [3]. Stretch of the left atrium is generally controlled by intra-atrial pressure. The Frank–Starling mechanism of the left atrium can be described by a biphasic increase of contraction force after increase of the stretch level.
Left atrial function has been investigated with echocardiography for a long time [4].
One can make simple measurements of left atrial size or left atrial volume but also try to quantify left atrial function with Doppler and tissue Doppler. During heart failure, atrial function is characterised by augmented reservoir and pump function. This can be measured with pulsed wave Doppler across the mitral valve, which is generally expressed by a decreased E/A ratio. However, there is a marked heterogeneity among heart failure patients with pseudo normal patterns and restrictive filling patterns. This makes it difficult to use pulsed wave Doppler as a determinant of prognosis and guide therapy. The current paper of Tripokiadis et al. [5], describes the measurement of left atrial ejection volume and left atrial ejection force in patients with heart failure. The method is not new and has been described in normal volunteers and patients with atrial fibrillation [6]. Although there were some limitations of the study in sample size, matching of age groups and gender, the authors were able to demonstrate, that left atrial function increases during heart failure and so compensated the decrease of left ventricular function. The model, described by Tripokiadis et al. can be used to estimate atrial function in patients with systolic and diastolic left ventricular dysfunction but also to evaluate the influence of atrial contribution to ventricular filling after medical treatment. Doing so, individual treatment can be optimised not only taking into account the clinical status, renal status, ventricular remodelling and neurohormonal status (BNP), but also atrial contribution to left ventricular filling.

heart failure
left atrial function
echocardiography


Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation

The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation.
Introduction
In eukaryotes, genomic DNA is first packaged into nucleosomes and then organised into higher-order chromatin structures. Chromatin organisation is locally or globally changed in response to external and internal signals. The changes are required for executing critical biological functions, most notably in regulated gene expression and chromosome segregation. Various post-translational modifications take place on histones, mostly in their tail domains, and play critical roles in the regulation of chromatin structure and function, either directly or indirectly through the recruitment of specific chromatin binding proteins [1]. The importance of histone modifications in gene expression is well appreciated and has led to the hypothesis of ‘the histone code’, which proposes that the combination of various histone modifications defines the pattern of gene expression [2].
Upon entry into mitosis, chromatin undergoes dramatic morphological changes to form mitotic chromosomes. On mitotic chromosomes, centromeres form unique chromosomal domains which are crucial for chromosome segregation in two respects [3]. First, centromeres are sites which connect two sister chromatids through cohesins until anaphase. Second, they serve as the foundation for kinetochores which provide the sites for microtubule attachment. To execute these functions, centromeres need to adopt a specialised chromatin structure which also changes during the cell cycle, particularly at the entry into mitosis, at the metaphase–anaphase transition and during exit from mitosis. Moreover, different regulation is also required for meiotic divisions to achieve a correct meiotic chromosome segregation pattern [4].
Recently a novel phosphorylation site was identified at threonine 119 (T119) in the C-terminal tail of Drosophila H2A [5]. The site is conserved in H2A amongst eukaryotes (serine in yeasts), but not in H2A variants, such as H2Av and H2AX. Here we show H2A T119 phosphorylation is enriched at centromeres during Drosophila mitosis. The Aurora B complex is required for this phosphorylation in centromeric regions, while Polo kinase suppresses phosphorylation by NHK-1 on chromosome arms. Inactivation of Cdc2 kinase is required for loss of centromeric phosphorylation at the metaphase-anaphase transition. Therefore, these mitotic kinases together control the temporal and spatial pattern of H2A phosphorylation at centromeres.
Materials and methods
Molecular and immunological techniques
Standard immunological, DNA manipulation and protein techniques were followed throughout [6,7]. Mouse α-tubulin antibody DM1A (Sigma) was used as a loading control in western blots. For immunoblotting, peroxidase-conjugated secondary antibodies (Jackson Lab) were used and detected using an ECL kit (Amersham). Primary antibodies used in this study include antibodies against Histone H2A (Upstate), dH2A-pT119 [5], phospho-H3 (Ser10; Upstate), CID [8], α-tubulin (DM1A; Sigma), GFP (3E6; Molecular Probes) and Aurora B [9].
Immunofluorescence microscopy
Culture and RNAi of S2 cells were carried out as described [10,11]. Effective depletion of target proteins was monitored by immunoblots or appearance of predicted phenotypes. S2 cells were immunostained as described with the exception that cells were fixed with 4% paraformaldehyde in PBS for 5 min [10]. Larval central nervous systems were dissected from late third instar larvae and fixed with 11% formaldehyde in 0.7% NaCl as described [12]. Secondary antibodies conjugated with Cy3 or Alexa488 (Jackson Lab or Molecular Probes) were used at 1/250–1/1000 dilution. S2 cells were transfected using Effectene Transfection Reagent (Qiagen). Non-degradable cyclin B fused to GFP (pUASp-CBTPM-GFP [13]) was co-transfected with ubiquitin-GAL4 to induce expression. Transfected cells were identified by the presence of GFP. The presence of dH2A-pT119 on centromeres of segregated chromosomes (> 50 cells) was scored.
Cultured cells were examined using a Plan-Apochromat objective lense (100×, 1.4NA; Zeiss) attached to an Axioplan2 (Zeiss). Images were captured by a CCD camera (Orca; Hamamatsu) using OpenLab2 (Improvision). Larval central nervous systems were taken using a Plan-Apochromat lense (63×, 1.4NA; Zeiss) attached to an Axiovert 200 M (Zeiss) with a confocal scan head (LSM510meta; Zeiss). Confocal images were presented as a maximum intensity projection of the Z-stacks. All digital images were imported to Photoshop (Adobe) and adjusted for brightness and contrast.
Phosphatase treatment
For western blotting of phosphatase treated cell extract, cell extracts were obtained by resuspending S2 cells in lysis buffer (150 mM NaCl, 20 mM Tris, 5 mM EDTA, 1% NP-40) with or without phosphatase inhibitors (100 mM NaF, 2 μM okadaic acid, 100 mM β-glycero-phosphate, 15 mM p-nitrophenylphosphate) and incubating on ice for 10 min. Lambda phosphatase (NEB) was added to the cell extract without phosphatase inhibitors and both samples incubated for 30 min at 37 °C. 2× SDS sample buffer was then added to the extracts and boiled for 3 min. Samples were then western blotted with anti-dH2A-pT119 to compare phospho-protein levels. In addition, cells immediately resuspended in 1× SDS sample buffer were included for comparison.
For phosphatase treatment of fixed cells for immunofluorescence with the anti-dH2A-pT119 antibody, cells were fixed with 4% paraformaldehyde in PBS followed by incubation with lambda phosphatase for 1 h at 37 °C. Cells were then washed and immunostained as described above. Microscope images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment. Average pixel intensity of dH2A-pT119 staining on the DNA was measured in interphase and mitotic cells (16 cells in 2 separate experiments).
Fly stocks
Standard techniques for fly manipulation were followed [14]. All stocks were grown at 25 °C in standard cornmeal media. A null nhk-1 mutant (nhk-1E107) used in this study was previously described [15].
Results
H2A T119 phosphorylation is specific to centromeres in mitosis
To examine the spatial and temporal control of H2A T119 phosphorylation in cells, we immunostained Drosophila S2 cells using an antibody which specifically recognises this phosphorylated form of H2A (anti-dH2A-pT119 [5]). We found a dynamic change in the phosphorylation pattern of H2A during the cell cycle. In interphase, phosphorylation was present throughout the chromatin in the nucleus (Fig. 1A). Interestingly, in mitosis, as the chromosomes begin to condense, phosphorylation was no longer spread throughout the chromatin but produced a more punctate pattern (Fig. 1B). Co-staining with a centromeric marker CID (the CENP-A homologue; [8,16]) revealed that in prometaphase and metaphase, phosphorylation was enriched in regions between and surrounding CENP-A positive regions, which we refer to as the centromeric regions (Figs. 1C–E). This phosphorylation became dramatically reduced at the onset of anaphase (Fig. 1F). Phosphorylation only returned on decondensed chromatin at the end of mitosis.
Specificity of the signal obtained by this phospho-H2A antibody was confirmed by treatment with lambda protein phosphatase. Lambda phosphatase treatment of S2 cell extracts eliminated a single band (which comigrates with H2A) recognised by the antibody on immunoblots (Supplementary Fig. 1). Furthermore, the immunofluorescent signals obtained by the phospho-H2A antibody were greatly reduced by lambda phosphatase treatment of fixed S2 cells (Supplementary Fig. 1).
In syncytial embryos and oocytes, entire mitotic/meiotic chromosomes are stained with the anti-dH2ApT119 antibody [5,17]. To confirm that the phosphorylation pattern found in S2 cells is not specific to this cell line, we examined H2A phosphorylation in somatic cells of developing flies. The larval central nervous system (CNS) is the tissue most commonly used for the study of standard mitotic cell cycles, which have two gap phases and checkpoint regulation [18]. Immunostaining of larval CNSs revealed a similar temporal and spatial pattern of H2A T119 phosphorylation as found in S2 cells (Supplementary Fig. 2A).
Centromeric H2A T119 phosphorylation depends on Aurora B kinase
Previously, the conserved protein kinase NHK-1 was identified as phosphorylating H2A T119 in vitro [5]. A female sterile mutation in NHK-1 greatly reduced phosphorylation at this site in oocytes, but not in follicle or nurse cells [17]. This indicated that NHK-1 is the major kinase responsible for this phosphorylation at least in the oocyte nucleus. To test whether NHK-1 is responsible for this phosphorylation in S2 cells, we examined whether depletion of this kinase by RNA interference (RNAi) affects the phosphorylation. Down-regulation of NHK-1 in S2 cells did not eliminate the signal of the phospho-H2A antibody in immunostaining (Supplementary Figs. 3 and 5). This result was further confirmed by immunostaining of larval CNSs from a null mutant of NHK-1 (nhk1E107
[15]) (Supplementary Fig. 2B). These results indicated that either a residual amount of NHK-1 kinase is sufficient to phosphorylate this site or kinases other than NHK-1 can phosphorylate this site in the absence of NHK-1.
To identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation, we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation [19]. After Aurora B was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. In Aurora B-depleted cells, the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms (Fig. 2A). However, nuclear staining in interphase cells remained high (Supplementary Fig. 5), suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms.
Aurora B kinase is part of at least two functionally distinct complexes [19], a core complex (containing INCENP) and a larger complex (additionally containing Survivin and Borealin/DasraB). To understand which complex is required for the H2A phosphorylation, we tested the requirement of other subunits for the phosphorylation. Depletion of any one of INCENP, Survivin and Borealin by RNAi greatly reduced H2A phosphorylation in centromeric regions in mitosis (Supplementary Fig. 4). Interphase phosphorylation was not affected in any of the cases. These results indicated that the large AuroraB complex is required for centromeric phosphorylation of H2A at T119 in mitosis.
Polo kinase down-regulates the H2A phosphorylation on chromosome arms
To further study the regulatory mechanism of the phosphorylation, we examined the role of the crucial mitotic regulator Polo kinase [20]. After Polo kinase was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. Surprisingly, in Polo-depleted cells, H2A T119 phosphorylation was not restricted to centromeric regions in mitosis but remained at a high level on the entire chromosome arms (Fig. 2A). Quantitative analysis indicated that the fluorescent signal from the phospho-H2A antibody on chromosome arms was dramatically increased in the absence of Polo kinase (Fig. 2B). This result suggests that Polo kinase is directly or indirectly required for down-regulating H2A T119 phosphorylation on chromosome arms to enrich the phosphorylation at centromeric regions.
Polo suppresses phosphorylation by the H2A kinase NHK-1
To identify the relationship between Aurora B and Polo actions, both of the kinases were depleted simultaneously. If a loss of Polo kinase misregulates Aurora B kinase, simultaneous depletion would suppress H2A T119 phosphorylation on chromosome arms. Immunostaining of cells depleted of both Aurora B and Polo showed a high level of phosphorylation on the entire chromosome arms (Fig. 2). This indicated that H2A T119 phosphorylation on chromosome arms induced by loss of Polo kinase was independent of Aurora B activity.
Next we tested the relationship between Polo and the H2A kinase NHK-1 by co-depletion. We found that NHK-1 depletion suppresses H2A T119 phosphorylation on arms induced by a loss of Polo (Fig. 3). Quantitative analysis confirmed that the phospho-H2A signal on chromosome arms in Polo NHK-1 double depletions was decreased to a level comparable to that of the control or NHK-1 depletion.
Finally we tested the phenotype of double depletion of Aurora B and NHK-1. Like Aurora B single depletion, H2A T119 phosphorylation was greatly reduced from centromeric regions of mitotic chromosomes (Supplementary Fig. 5).
These epistasis studies suggested that Polo functions upstream of NHK-1 to suppress H2A T119 phosphorylation, but is independent of Aurora B.
Cyclin B degradation triggers a loss of H2A phosphorylation at initiation of anaphase
Centromeric H2A T119 phosphorylation becomes greatly reduced at the onset of anaphase indicating a change in its regulation at this time. After alignment of all chromosomes, APC/Cdc20 triggers degradation of Cyclin B and securin, leading to inactivation of Cdc2 kinase and activation of separase which cleaves cohesin to initiate anaphase [21]. To separate Cyclin B degradation from securin degradation, we expressed non-degradable Cyclin B in S2 cells and examined H2A phosphorylation by immunostaining. As previously reported [22,23,13], expression of non-degradable Cyclin B did not inhibit the onset of anaphase but prevented exit from mitosis, resulting in an accumulation of anaphase cells with overcondensed chromosomes. In cells expressing non-degradable Cyclin B, H2A phosphorylation was still retained at centromeric regions in most anaphase cells (Figs. 4A, B). Therefore, we concluded that cyclin B degradation, not anaphase onset, is required for triggering loss of phosphorylation at the metaphase-anaphase transition.
Discussion
In this study, we found dynamic changes in H2A T119 phosphorylation during the Drosophila cell cycle. This phosphorylation is enriched at centromeric regions early in mitosis and lost at the onset of anaphase. In interphase, H2A T119 phosphorylation was found throughout chromatin. Furthermore, our evidence showed that the combined action of at least four conserved mitotic kinases is required for precise spatial and temporal regulation of H2A T119 phosphorylation (Fig. 4C). Aurora B kinase is required for the enrichment of phosphorylation at centromeric regions in mitosis. Polo kinase is required for suppressing H2A phosphorylation by NHK-1 on chromosome arms. Furthermore, inactivation of Cdc2 kinase induced by Cyclin B degradation is required for the loss of centromeric phosphorylation at the onset of anaphase.
Currently we do not know what the function of this H2A phosphorylation is in cells. In higher eukaryotes which have many copies of histone genes, the function of histone modifications has been studied only indirectly by down-regulating responsible modifying enzymes. Unfortunately this approach is not suitable for kinases as they are likely to have multiple substrates (for example, Cdc2 and Aurora B mediating H1 and H3 phosphorylation).
Centromeric distribution and regulation by conserved mitotic kinases may tempt us to speculate possible involvement of H2A T119 phosphorylation in chromosome segregation in mitosis. The phosphorylation might be important for generating or sensing tension between sister chromatids, or modes of microtubule attachment to kinetochores through the formation of centromere-specific chromatin or recruitment of centromere proteins during mitosis. A loss or misregulation of the H2A phosphorylation may be responsible for a subset of the highly pleiotropic phenotypes observed after down-regulation of Aurora B or Polo [19,20]. It would be a future challenge to define the precise roles of this H2A phosphorylation.

aurora polo nhk-1 kinase centromere histone drosophila cyclin


Treatment of Gastric Adenocarcinoma May Differ Among Hospital Types in the United States, a Report from the National Cancer Data Base

The concept that complex surgical procedures should be performed at high-volume centers to improve surgical morbidity and mortality is becoming widely accepted. We wanted to determine if there were differences in the treatment of patients with gastric cancer between community cancer centers and teaching hospitals in the United States. Data from the 2001 Gastric Cancer Patient Care Evaluation Study of the National Cancer Data Base comprising 6,047 patients with gastric adenocarcinoma treated at 691 hospitals were assessed. The mean number of patients treated was larger at teaching hospitals (14/year) when compared to community centers (5–9/year) (p < 0.05). The utilization of laparoscopy and endoscopic ultrasonography were significantly more common at teaching centers (p < 0.01). Pathologic assessment of greater than 15 nodes was documented in 31% of specimen at community hospitals and 38% at teaching hospitals (p < 0.01). Adjusted for cancer stage, chemotherapy and radiation therapy were utilized with equal frequency at all types of treatment centers. The 30-day postoperative mortality was lowest at teaching hospitals (5.5%) and highest at community hospitals (9.9%) (p < 0.01). These data support previous publications demonstrating that patients with diseases requiring specialized treatment have lower operative mortality when treated at high-volume centers.
Introduction
Approximately 22,000 patients in the United States (US) will be diagnosed with gastric carcinoma in 2006, a number that pales in comparison to more common malignancies such as colorectal (172,000 new cases) and breast cancer (211,000 new cases).1 Because of the relative infrequency of gastric cancer, most individual hospitals evaluate and treat a limited number of patients with stomach cancer, impairing the ability to develop expertise at many institutions. A lack of expertise may contribute to the dismal survival of gastric cancer in the US. In an earlier report from the National Cancer Data Base (NCDB), Hundahl et al.2 observed a 5-year survival rate of 78% for stage IA, 58% for stage IB, 34% for stage II, 20% for stage IIIA, 8% for stage IIIB, and 7% for stage IV disease.
A recent report from the World Health Organization3 shows a decline in the mortality of gastric cancer worldwide. However, the observed rate of decline in mortality is less in the US than what is observed for Japan. Factors that might contribute to the improved Japanese survival includes greater operative experience leading to more skilled surgeons, earlier diagnosis, different biologies of gastric cancer between countries, improved pathologic staging with stage migration (Will Rogers effect),4 and the frequency of neoadjuvant or adjuvant chemoradiation therapy use.
These data highlight the need for improved treatment of gastric cancer to increase patient survival. A controversial way to accomplish this might be to limit gastric cancer care to high-volume centers, if the results were better at such sites. Evidence-based hospital referral has been adopted by some insurance companies and consortiums of large health care purchasers, such as the Leapfrog group, based on studies showing better outcomes for surgical services at high-volume centers.5–7 Birkmeyer et al.8 reported that Medicare patients had a lower operative mortality if they had cancer-related procedures (gastrectomy, esophagectomy, colectomy, pancreatectomy, cystectomy, nephrectomy, and pulmonary resection) or cardiovascular disease at a high-volume hospital. The Japanese have also found a similar pattern of improved survival after the treatment of gastric carcinoma at high-volume centers. Nomura et al.9 reported 5-year survival rates to be significantly higher at centers with high surgical volumes (96–205/year), when compared to very-low-volume hospitals (1–28 cases/year).
To determine if patients treated for gastric cancer at high-volume and specialized centers in the US had better postoperative outcomes, we analyzed the preoperative evaluation and surgical treatment of gastric adenocarcinoma at three categories of hospitals, defined by the Commission on Cancer (CoC), namely, the Community Hospital Cancer Program (CHCP), Community Hospital Comprehensive Cancer Program (COMP), and Teaching Hospital Cancer Program (THCP). Data collected for the 2001 Gastric Cancer Patient Care Evaluation (PCE) by the NCDB were utilized.
Material and Methods
NCDB, Data Source, Case Selection, and Data Handling
The NCDB is a project of the American College of Surgeons (ACS) CoC. The NCDB was established in 1989 to serve as a comprehensive clinical surveillance resource for all forms of cancer diagnosed in the US and its operations have been supported in part by the American Cancer Society. In 2001, the database captured 73% of all newly diagnosed cancer cases in the US.
Data were submitted electronically in accordance with specified North American Association of Central Cancer Registries data transmission standards.10 Hospital cancer registrars abstracted each case according to a standardized set of data elements and definitions as described in the CoC’s Registry Operations and Data Standards, volume II.11 The NCDB elements include patient characteristics: sex, age or date of birth, and race/ethnicity; tumor characteristics: primary site, histology, behavior, grade, and American Joint Committee on Cancer (AJCC), fifth edition stage groups; and first course of treatment: surgery, radiation, chemotherapy, and others. In addition to the annual call for data, a call for participation in a 2001 PCE was issued and 711 of the 1,423 CoC-approved institutions in the US responded. Cancer registrars were also asked to provide information describing additional diagnostic and treatment information, 30-day mortality, and patient comorbidities. Data quality checks were conducted at the local and the depository level.
Patient Population
The 2001 Gastric PCE included data submitted from 711 CoC-approved institutions in the US. Participating institutions submitted data for consecutive hospital admissions and clinic visits between January 1, 2001 and December 31, 2001. Patients eligible for participation had a “microscopically confirmed neoplasm of the stomach that was either diagnosed or initially treated at the reporting facility.” Of 7,084 total patients, 6,099 (86%) were diagnosed with gastric adenocarcinoma. For this study, 52 patients were excluded because they were treated at nine institutions without a specialized hospital type, leaving 6,047 patients at 691 cancer program types for evaluation. This group comprises the study population.
Cancer Program Categories
Cancer programs were characterized as CHCP, COMP, or THCP. Community Hospital Cancer Programs diagnose and/or treat 100–650 cancer patients every year and will commonly refer patients to other institutions for diagnostic evaluation or treatment. A CHCP has neither a medical school affiliation nor residency programs but does posses a medical oncology unit or functional equivalent and infrequently participates in cancer research. Community Hospital Comprehensive Cancer Programs diagnose and/or treat more than 650 cancer cases per year, but are not associated with a medical school. A COMP may make outside patient referrals, has a medical oncology unit, and participates in cancer research. Teaching Hospital Cancer Programs are defined as facilities associated with a medical school that participates in the training of residents in at least four fields, two of which are medicine and surgery. A THCP offers a full range of diagnostic and therapeutic services on site and has an in-patient medical oncology unit. A THCP hospital also participates in cancer-related clinical research and has board-certified medical oncologists.
Statistical Analysis
All analyses were performed using the SPSS statistical software (SPSS for Windows, version 14.0; SPSS Inc, Chicago, IL, USA). Frequency distributions were calculated to get the mean number of cases by hospital category type. The chi-square (χ2) test was used for comparisons of proportions across levels of categorical variables. When the overall test was significant, pairwise comparisons among the three hospital categories were also calculated to asses which hospitals differed in rates. The p values reported were adjusted for multiple comparisons using the Bonferroni adjustments.12 Results were based on two-sided tests with a p = 0.01 significance level, except where indicated. Where specific values were unknown, these cases were excluded from the analysis when appropriate.
A forward stepwise binary logistic regression model was used to evaluate the impact of age, stage, and comorbid burden on determination of type of treatment, i.e., the odds of “surgery, with or without other treatment” compared to the odds of nonsurgical treatment (radiation and/or chemotherapy). The Wald statistic was used to test significance. Exponentiated estimates of the beta coefficients were interpreted as the estimates for the effect (odds ratio) of a particular variable, controlling for the other variables in the equation. A receiver operating characteristic (ROC) curve was created to examine the prediction results. The true-positive probability was calculated to define the sensitivity of the classification rule and the true-negative probability was calculated to determine the specificity to summarize how well the model performed.
Confidentiality
Data reported to the NCDB are retrospective in nature. No patient or physician identifiers were collected as part of the study. Case identification information (facility identification number and local registry accession number) was collected for administrative purposes only. Analyses were reported only at the aggregate level to assist hospital cancer programs with quality assurance, rather than used to make decisions about individuals and their care.
The ACS has executed a business associate agreement that includes a data-use agreement, with each of its CoC-approved hospitals. Results reported in this study were in compliance with the privacy requirements of the Health Insurance Portability and Accountability Act of 1996 as reported in the Standards for Privacy of Individually Identifiable Health Information; Final Rule (45 CFR Parts 160 and 164).
Results
Treatment Volumes
A total of 691 cancer programs were included in the study: 258 CHCP sites, 267 COMP sites, and 166 THCP sites. Although 37.3% of the programs were CHCPs, only 22% (n = 1329) of the cases were treated in this setting; 40.8% (n = 2,468) of the cases came from COMPs; and 37.2% (n = 2,250) of the patients came from THCPs. Community Hospital Cancer Programs saw on average of 5.2 cases/year; COMPs, 9.2; and THCPs 13.6. The THCPs treated more surgical patient on average (7.6 cases) than either the COMPs (5.3 cases) or the CHCPs (2.9 cases) (Table 1).
Table 1Number and Percent of Cancer Programs, Number and Percent of Patients by Cancer Program, Mean Number and Range of All Cases by Cancer Program Type, Number and Percent of Surgically Treated Cases, and Mean Number of Surgical Cases and Range by Cancer Program Type Community Cancer CentersComprehensive Community Cancer CentersTeaching/Research HospitalsTotalNumber (%) of cancer programs258 (37.3)267 (38.6)166 (24.0)691Number (%) of patients in study1,329 (22.0)2,468 (40.8)2,250 (37.2)6,047Mean number and range of all cases5.2 (1–39)9.2 (1–49)13.6 (1–55)Number (%) of surgical cases673 (20.5)1,369 (41.8)1,235 (37.7)3,277Mean number and range of surgical cases2.9 (1–16)5.3 (1–37)7.6 (1–40)
Patient Demographics
Men, 3,751(62%), and women, 2,296 (38%), were equally distributed across hospital types. The mean age in years was 69.3 and the median age 71 (18–103 years). Significantly more 70 and older patients were seen in CHCPs when compared to COMPs or THCPs (p < 0.01). Significantly more 50 and younger patients were seen at THCPs when compared with CHCPs and COMPs (p < 0.01) (Table 2).
Table 2Patient Characteristics by Type Cancer Program Community Cancer CenterComprehensive Cancer CenterTeaching/ResearchTotaln (%)n (%)n (%)n (%)GenderMale805 (60.6)1,502 (60.9)1,444 (64.2)3,751 (62.0)Female524 (39.4)966 (39.1)806 (35.8)2,296 (38.0)Total1,3292,4682,2506,047Age<50106 (8.0)207 (8.4)264 (11.7)577 (9.5)50–69402 (30.2)865 (35.0)836 (37.2)2,103 (34.8)70 and older821 (61.8)1,396 (56.6)1,150 (51.1)3,367 (55.7)Total1,3292,4682,2506,047Race/EthnicityCaucasian966 (72.7)1,723 (69.8)1,387 (61.6)4,076 (67.4)African American143 (10.8)278 (11.3)406 (18.0)827 (13.7)Hispanic120 (9.0)206 (8.3)207 (9.2)533 (8.8)Asian78 (5.9)218 (8.8)176 (7.8)472 (7.8)Other22 (1.7)43 (1.7)74 (3.3)139 (2.3)Total1,3292,4682,2506,047AJCC stageO2 (0.2)8 (0.3)2 (0.1)12 (0.2)I13 (1.0)28 (1.1)26 (1.2)67 (1.1)IA105 (7.9)210 (8.5)182 (8.1)497 (8.2)IB118 (8.9)218 (8.8)178 (7.9)514 (8.5)II160 (12.0)286 (11.6)242 (10.8)688 (11.4)III6 (0.3)12 (0.5)12 (0.5)30 (0.5)IIIA156 (11.7)347 (14.1)337 (15.0)840 (13.9)IIIB47 (3.5)96 (3.9)97 (4.3)240 (4.0)IV427 (32.1)856 (34.7)835 (37.1)2,118 (35.0)Unknown295 (22.2)407 (16.5)339 (15.1)1,041 (17.2)Total1,3292,4682,2506,047
The study population included 4,076 (67.4%) Caucasians, 827 (13.7%) African Americans, 533 (8.8%) Hispanic, 472 (7.8%) Asians, and 139 (2.3%) patients of other or unknown racial or ethnic background. Significantly fewer Caucasians were seen in THCPs when compared to CHCPs and COMPs (p < 0.01). THCPs saw significantly more African Americans than the other two types of cancer programs (p < 0.01). Significantly more Asians were seen at COMP hospitals when compared to CHCP hospitals (p < 0.01), but there was no significant difference when comparing the proportion of Asians in COMPs to the proportion found in THCPs.
A large percentage of this patient population did not have a documented AJCC Stage reported (n = 1,041, 17.2%). Of those patients with a documented stage, more than 64.5% had advanced stage (stage III or IV) at presentation. The largest subgroup had stage IV disease (n = 2,118, 35.0%). There were no significant differences in the stage at presentation between the different types of medical institutions. When specified, the most common location of the primary cancer was the cardia 27.4% (n = 1,656), followed by the antrum 18.3% (n = 1,107) and stomach, not otherwise specified (NOS) 17.9% (1,080) (Fig. 1). The distribution of tumor location was similar among all these categories of treatment institution.
Figure 1Location of cancer at presentation by cancer program types: A Community Hospital Cancer Program (CHCP), B Community Hospital Comprehensive Cancer Program (COMP), and C Teaching Hospital Cancer Program (THCP). Location of lesions: cardia, funds, body, antrum, pyloric, lesser curve, and greater curve by percent and others indicated by asterisk (C168, overlapping lesions and C169, stomach, NOS).
Diagnostic Testing
Diagnostic evaluation included computed tomography (CT) of the abdomen (n = 4,417, 73%) and pelvis (46.9%) over all hospital types. The abdominal CT suggested a diagnosis of cancer in 63.9% of patients at CHCP, 63.0% at COMP, and 68.2% at THCP. The CT of the abdomen was more likely to suggest cancer at the THCP when compared to the COMP (p < 0.01), but no significant difference was noted between the THCP and the CHCP. Computed tomography of the pelvis was suggestive for cancer in 36.9, 38.1, and 42.6% at CHCP, COMP, and THCP, respectively. The differences across cancer programs were not significant (p > 0.01). Only 15.6% (n = 946) of these cases were evaluated with endoscopic ultrasonography. That procedure was used more often at THCPs (33.5%) when compared to both CHCP (13.8%) and COMP (17.6%) (p < 0.01). 2-18F-Fluoro-2-deoxy-d-glucose positron emission tomography (F18-FDG-PET) was rarely used at any type of hospital (4.6%). When done, THCPs were more likely to use the F18-FDG-PET than CHCP for diagnostic testing (p < 0.01). No significant differences were noted between THCP and COMP use.
Intraoperative Assessment
Staging laparoscopy was performed significantly more often at THCPs (18.9%) than at COMPs (13.6%) or at CHCPs (10.5%) (p < 0.01). No significant differences were seen between COMPs and CHCPs. Peritoneal lavage with cytology for the assessment of occult peritoneal disease was rarely used at THCPs, COMPs, and CHCPs with rates of 5.7, 4.3, and 2.7%, respectively. Significantly fewer peritoneal lavages occurred at CHCPs when compared to THCPs (p < 0.01). No significant differences were found between THCPs and COMPs.
Surgical Treatment
Surgical intervention was undertaken for 54.2% (n = 3,277) of the all patients in the study. At CHCP hospitals, 673 out of 1,329 (50.6%) patients were surgically treated, 1,369 out of 2,468 (55.5%) were surgical patients at COMP, and 1,235 out of 2,250 (54.9%) patients underwent surgery at THCP. When surgeries were grouped as less extensive or more extensive, significantly more extensive surgeries were performed at THCPs than at CHCPs (p < 0.01); however, no significant difference was observed between COMPs and THCPs (p > 0.01). More specifically, the most frequently recorded type of surgical resection was gastrectomy, NOS (overall, 21.7%; CHCP, 26.0%; COMP, 20.5%; and THCP, 20.6%) followed by distal gastrectomy (overall, 16.2%; CHCP, 15.6%; COMP, 17.3%; and THCP, 15.2%), and partial or subtotal gastrectomy (overall, 13.3%; CHCP, 12.2%; COMP, 12.7%; and THCP, 14.6%). A near total or total gastrectomy was uncommonly performed (overall, 5.7%; CHCP, 4.3%; COMP 5.7%; and THCP, 6.4%). After pathologic analysis, 83.5% of the entire surgical patient population had a resection with curative intent (R0 resection), whereas 16.5% had a palliative resection (R1 resection). The frequencies of R0 and R1 resections were similar (CHCP, 81.2/18.8%, COMP, 84.6/15.4%, and THCP, 83.6/16.4%) among all institutions and not statistically different between different types of institution (p = 0.25).
Lymph node staging, a highly significant predictor of outcome, was evaluated using the number of lymph nodes examined after surgical resection. Analysis of at least 15 lymph nodes is required by the AJCC for accurate staging and exclusion of nodal metastases. Only 31.1% of patients at CHCPs, 31.0% at COMPs, and 38.4% at THCPs had more than 15 nodes examined (p < 0.01). A large number of patients from each hospital type had an unknown number of lymph nodes pathologically evaluated (26.6% CHCP, 22.6% COMP, and 15.4% THCP), but THCPs had significantly better documentation of lymph node evaluation (p < 0.05) (Fig. 2). A D1 lymphadenectomy with removal of only perigastric nodes was noted in 56.7% of surgically managed patients. Some patients had removal of some lymph nodes that are included in D2 or D3 nodes but most across all hospital types did not have a formal extended lymphadenectomy (Table 3).
Figure 2Percent of lymph nodes sampled at each hospital. CHCP = Community Cancer Center Program, COMP = Community Hospital Comprehensive Cancer Program, THCP = Teaching Hospital Cancer Program. p < 0.05 (asterisk). Error bars: 95% CI.Table 3Lymph Nodes Sampled During Surgical ResectionResectedCommunity Cancer CenterComprehensive Cancer CenterTeaching/Researchn (%)n (%)n (%)PerigastricaYes367 (71.1)775 (68.9)715 (70.4)No149 (28.9)350 (31.1)301 (29.6)HepaticbYes21 (4.2)68 (6.5)70 (7.5)No478 (95.8)979 (93.5)867 (92.5)CeliaccYes40 (7.9)100 (9.5)96 (10.1)No465 (92.1)952 (90.5)854 (89.9)SplenicdYes14 (2.8)49 (4.7)52 (5.5)No481 (97.2)988 (95.3)887 (94.5)ParaaorticeYes94 (18.8)170 (16.8)189 (20.2)No405 (81.2)840 (83.2)745 (79.8)Comparison of column proportions did not include the unknown values in calculations.aPerigastric unknown, n = 620bHepatic unknown, n = 794cCeliac unknown, n = 770dSplenic unknown, n = 806eParaaortic unknown, n = 834
Morbidity and Mortality
The prevalence of operative morbidity, specifically for postoperative hemorrhage, wound infection sepsis, and an anastomotic leak, was similar across all hospital categories (Table 4). Operative mortality, defined by 30-day mortality, was different among the institution types. Teaching Hospital Cancer Programs had the lowest 30-day mortality at 5.5%, compared to 7.9% at COMPs and 9.9% at CHCPs (Table 4). The proportional difference between THCPs and COMPs was not significant (p > 0.01), whereas, when compared to CHCPs, THCPs had significantly fewer deaths within 30 days of surgery (p < 0.01). For those that died within that 30-day period, no significant differences were found across all hospital types for stage (p > 0.01) or age (p > 0.01). In the logistic regression model stage was the most significant predictor of 30-day postoperative death (p = 0.0001), followed by age (p = 0.0001), and category of hospital (p = 0.004). Stage IV patients were 2.6 times (99% confidence interval [CI] = 1.6–4.3) more likely to die within 30 days of surgery; no other stage was significant (p > 0.01). Patients in CHCPs were almost twice as likely to die within 30 days when compared to THCPs (p = 0.001). Race and extent of surgery were not significant factors. The area under the ROC curve defined by the logistic model was 0.69 (99% CI = 0.64–0.74), where 0.5 represents a nondiscriminatory result. Long-term 5-year survival data are unavailable for this patient population and will not be until the fall of 2007.
Table 4Postoperative Mortality and Complications Community Cancer CenterComprehensive Cancer CenterTeaching/Researchn (%)n (%)n (%)30-Day mortalityaYes55 (9.9)93 (7.9)59 (5.5)No501 (90.1)1,080 (92.1)1,020 (94.5)BleedingbYes20 (4.2)40 (3.9)47 (5.1)No451 (95.8)993 (96.1)881 (94.9)Wound infectioncYes35 (7.4)54 (5.2)61 (6.6)No440 (92.6)981 (94.8)868 (93.4)SepsisdYes34 (7.1)62 (6.0)43 (4.7)No443 (92.9)973 (94.0)881 (95.3)Anastomotic leakeYes32 (6.7)68 (6.5)64 (6.6)No447 (93.3)979 (93.5)884 (93.2)Comparison of column proportions did not include the unknown values in calculations.a30-Day mortality unknown, n = 469bBleeding unknown, n = 845cWound infection unknown, n = 840dSepsis unknown, n = 841eAnastomotic Leak unknown, n = 803
Neoadjuvant and Adjuvant Therapy
Approximately 38.9% of the surgical patients received chemotherapy; 30.5% received radiation therapy. Neither neoadjuvant radiation therapy nor chemotherapy was frequently provided at all hospital types, but highest, although not significant (p > 0.01) at the THCPs when compared to CHCPs and COMPs (Table 5). Patients more frequently received adjuvant chemotherapy and radiation than neoadjuvant chemotherapy or radiation therapy at all the categories of hospitals. There were no statistically significant differences in the frequency of treatment with chemotherapy or radiation therapy by tumor stage between the hospital categories.
Table 5Radiation and Chemotherapy/Surgery Sequence Community Cancer CenterComprehensive Cancer CenterTeaching/Research# (%)# (%)# (%)RadiationaNeoadjuvant23 (12.1)53 (13.0)68 (19.1)Adjuvant167 (87.9)356 (87.0)288 (80.9)ChemotherapybNeoadjuvant36 (14.6)71 (15.0)87 (20.3)Adjuvant210 (85.4)401 (85)342 (79.7)Comparison of column proportions did not include the unknown values in calculations.aRadiation sequence unknown, n = 43bChemotherapy sequence unknown, n = 128
Discussion
When examining the current status of gastric cancer patient care in the US and seeking ways to improve survival, looking to the East for guidance is a reasonable strategy. The standards of gastric cancer therapy have largely been set by the practices of Japanese physicians and surgeons, in a large part, because of their large experience with this disease. In Japan, there are 104,000 new cases annually in a population of 128 × 106,13 compared to the 22,000 cases seen annually in the US in a population of 296 × 106,1 an incidence that is more than 10 times greater than that of the US.
The high incidence seen in Japan has allowed the Japanese to develop surgical and medical strategies to improve mortality. However, several of the surgical principles practiced in Japan are difficult to incorporate into Western practices. Early detection programs in Japan have led to a significant decrease in mortality but this has not been replicated in the US because of the high cost to benefit ratio associated with the much lower incidence of gastric cancer in the US. A more controversial standard Japanese practice is the extended D2, D3, or even D4 lymphadenectomy performed for gastric cancer. Whereas extended lymphadenectomy has been associated with improved survival in Japan, with retrospective analysis, the value of this technique has not been proven in the West. Wanebo et al.,14 in a retrospective study from the US, as well as Bonenkamp et al.15 and Cuschieri et al.16, in randomized controlled trials from The Netherlands and Great Britain, respectively, have reported a lack of survival benefit with D2 lymphadenectomy. Both Bokenkamp et al.15 and Cuschieri et al.16 observed significantly higher mortality rates among those patients that had a D2 resection of 10 vs 4% and 13 vs 6.5%, respectively. The operative mortality after a D2 resection is much lower in Japan with reported rates below 2%.17,18 Kodera et al. from Japan reported an operative mortality rate of 0.8% from 523 patients in a D2 vs D3 study.19
Studies examining large national databases have found improved surgical mortality after gastrectomy for gastric cancer at high-volume centers. A study examined using the National Inpatient Sample reported on 23,690 hospitalized patients with a hospital discharge code of gastric cancer and any gastrectomy noted significant differences in mortality among hospital types. Very-low-volume hospitals (<4 cases/year) had a mortality rate of 8.9% compared with a rate of 6.4% seen at high-volume hospitals (>9 cases/year).20 In a study by Birkmeyer et al.21 of the Medicare population, the observed mortality rate was 13% at very-low-volume centers (<5 cases/year) compared to 8.7% at very-high-volume centers (>21 cases/year). Improved survival among high-volume hospitals was also reported in Swedish22 hospitals that treated >20 surgical patients a year and German23 hospitals that treated >50 patients/year.
The Japanese have also noted an association of lower patient survival rates among gastric cancer patients treated at low-volume centers when compared to high-volume centers. Nomura et al.9 analyzed a database of more than 55,000 patients and grouped them into the following time periods: 1975–1979, 1980–1984, 1985–1989, and 1990–1994. He found “positive relationships between hospital volume and 5-year survival” but over time the survival benefit seen at high-volume centers decreased and persisted in comparison with the very-low-volume centers.
Not all authors, however, have observed improved survival at high-volume centers. A Dutch study evaluated the impact of patient volume on operative mortality and found no differences. This study analyzed 1,987 gastric cancer patients treated at 22 hospitals between 1987 and 1997.24 A limitation of this study is the fact that only 1 of the 22 hospitals was a university hospital and the others were “general hospitals.” Many of the participating hospitals were considered to be low-volume centers.
Interpretation of the Results
Our study had similar results to the aforementioned American, Japanese, and European studies. In this study, there was a marked improvement (>50%) in operative mortality at higher volume centers (≥14 cases/year) when compared to lowest volume institutions (≤5 cases/year). The average 30-day postoperative mortality at the low-volume community centers was almost 10%. The annual volume of gastric surgeries performed seems to be inversely related to 30-day postoperative mortality. On average 2.9, 5.5, and 7.6 gastric surgeries were reported from CHCPs, COMPs, and THCPs, respectively. Corresponding 30-day mortality was 9.9, 7.9, and 5.5%, respectively. There were no significant differences seen in postoperative morbidities such as wound infection and hemorrhage by hospital category. The logistical model revealed three predictors of perioperative mortality: stage IV disease, advanced age, and institution type. Patients that had surgery at a CHCP were twice as likely to die postoperatively compared to patients treated surgically at THCPs. Among those that died, there were no significant differences of stage or age at the different institutions.
Because the absolute differences in surgical case number among the hospital types were not vastly different, this observed difference in outcomes may be a reflection of the infrastructure of the institution rather than individualized surgeon skill. Centers affiliated with a medical school might have more experience with caring for the critically ill in the form of larger and better equipped intensive care units, resident and fellow coverage, newer technology, and more subspecialized physicians to help manage patient care. Unfortunately, we could not analyze the infrastructure for each hospital type nor look at surgeon-specific experience in this study as this was not a part of the study.
Clinical staging is affected by the sensitivity and specificity of the diagnostic studies performed during the preoperative evaluation. An assessment of staging at the different institutions revealed some major differences. Teaching hospitals were more likely to detect malignancy on a CT of the abdomen and pelvis than the other two hospital types. This might be a reflection on the quality of CT scans obtained and the experience of the radiologists. Preoperative utilization of endoscopic ultrasound was higher at teaching hospitals, a predictable finding given the recent adaptation of this technology and the expertise required to interpret these images.
In terms of pathologic staging, Karpeh et al.25 previously reported that evaluation of more than 15 lymph nodes allows a better estimate of patient survival. In fact, a study by Smith et al.26 found that overall survival was largely dependent on the number of nodes examined and found an increase in survival when up to 40 lymph nodes was assessed. We found that teaching hospitals did a significantly better job meeting this recommendation; however, even at THCPs only 38.4% of patients had greater than 15 nodes assessed. The D1 lymphadenectomy was the most common operation for of nodal dissection, probably as a result of controlled Western surgical trials showing no survival benefit from a D2 dissection. Only a limited number of patients had any D2–D4 nodes resected and usually without a standardized extended lymphadenectomy. A notable observation was that the percentage of D2–D4 lymphadenectomy performed at THCPs was not higher than what was observed at COMPs or CHCPs.
To improve and obtain accurate surgical staging of gastric cancer patients, current practice will have to improve and will be dependent upon both the excision of nodes by the surgeon and their retrieval from the specimen by the pathologist. Given so few patients had 15 or more nodes removed at the time of surgery, regardless of hospital type, this practice could be improved by surgeons and pathologists working together to achieve the goal of identifying at least 15 nodes followed by the proper documentation of the microscopic evaluation of these nodes in the pathology report. If intraoperative pathologic evaluation of the surgical specimen is possible and the lymph node sampling is inadequate, the surgeon should excise additional lymph nodes.
A strength of this study is the large number of patients and hospitals included in the study. This broad sampling of hospitals leads to a close approximation of the current practice of gastric cancer treatment in the US. The main shortcoming is that many of the critical data categories had at least 20% “unknown” responses, and may have biased the reported results of this study. In addition, analysis of hospital infrastructure or surgeon volume was limited by lack data availability in the database. Another limitation is that the 5-year survival information has not yet been documented; these data will provide long-term outcome by type of treatment center.
Conclusion
Data from the Gastric PCE project suggests that there is significant room for improvement in the surgical management of gastric cancer in the US. Most hospitals, regardless of category, do not document the evaluation at least 15 lymph nodes necessary to meet AJCC standards. With a little over one third of all patients having more than 15 lymph nodes examined, pathologic staging is less accurate and results of surgical and adjuvant therapy are likely to be worse. Significantly more patients had appropriate staging with the recommended number of nodes included in the lymphadenectomy at teaching centers than at nonacademic centers. Thirty-day mortality rates after gastric cancer resection were significantly lower at teaching centers, further establishing the recommendation that complex oncologic operations should be performed at high-volume centers to obtain better patient outcomes, corroborating the results of several previous studies. Long-term survival data will provide more information on effectiveness of treatment at each of the different institution types.

gastric cancer
operative mortality
survival
ncdb
hospital volume