Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (IATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the IATP exhibited a strong inward rectification, and the amplitude of the IATP was concentration-dependent. The IATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the IATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at Vhs between −70 and −10 mV when the IATP and ACh-induced current (IACh) were inward, but no occlusion was observed when these currents were outward at a Vh of +40 mV. The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations, by setting Vh close to the equilibrium potential of IACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels.
Introduction
The rat area postrema (AP) is a medullary circum-ventricular organ located in the hindbrain at the level of the obex, with a dense vascular supply devoid of a blood-brain barrier. The lack of a blood-brain barrier makes the AP ideally placed to act as a chemosensitive trigger zone involved in the control of vomiting in response to circulating emetic substances (Borison [1, 2]). Neurons in the AP are also responsive to changes in osmolarity or sodium concentration (Franchini et al. [3]), and can be activated by circulating vasoactive peptides such as angiotensin II (Fink et al. [4]), and arg-vasopressin (Undesser et al. [5]). Anatomical studies have revealed that the AP sends dense efferent projections to the nucleus tractus solitarius, parabrachial nucleus, nucleus ambiguus, and the dorsal motor nucleus of the vagus, and receives afferent inputs from the hypothalamic paraventricular and dorsomedial nucleii, and from the caudal nucleus tractus solitarius (Morest [6]; Kooy and Koda [7]; Shapiro and Miselis [8]). Thus, the AP is not only capable of responding to circulating hormones, but is also anatomically well situated to regulate a range of other central neurons, including those important in cardiovascular control (Sun and Spyer [9]). The low intrinsic firing rates of AP neurons in vivo (Papas et al. [10]) suggests that understanding and modulating excitatory inputs to AP could be particularly important in the functional output of AP neurons. A number of transmitters can evoke excitatory currents in the AP. Inward currents and increases in cytosolic Ca2+ ([Ca]in) via non-NMDA-glutamate receptors have been reported in rabbit and rat AP neurons, respectively (Jahn et al. [11]; Hay and Lindsley [12]). Our preliminary reports indicated that ATP also induces inward currents and [Ca]in increases via the activation of P2X receptor (P2XR) in isolated rat AP neurons (Sorimachi et al. [13, 14]). In addition, pre- and post-synaptic nicotinic ACh receptors (nAChR) have been demonstrated in the AP in rat brain slices (Funahashi et al. [15]), and we have recently reported the presence of nAChRs in dissociated rat AP neurons (Sorimachi and Wakamori [16]). During that study, we also noticed that many of these AP neurons also responded to ATP, which has prompted us to further investigate ATP responses in AP neurons, and potential interactions between nAChR and P2XR responses. In a variety of different peripheral neurons, including sympathetic neurons of bullfrog (Akasu and Koketsu [17]), rat (Nakazawa et al. [18]), and guinea-pig (Searl et al. [19]), cultured guinea-pig enteric and submucosal neurons (Zhou and Galligan [20] Barajas-Lopez et al. [21]), a negative interaction between P2XR- and nAChR responses has been reported. Such an interaction has also been observed for recombinant P2X2 and α3β4 nAChR channels in Xenopus oocytes and HEK cells (Khakh et al. [22, 23]; Boue-Grabot et al. [24]), where it has been recently suggested that this results from direct physical interactions between co-localized receptors (Khakh et al. [23]). In this study we more fully describe P2XR responses in AP neurons, demonstrate cross-inhibition between P2XRs and nAChRs and characterize some of the features of this cross-inhibition.
Experimental procedures
Preparation of AP neurons
The study was approved by the Committee on Animal Experimentation, Kagoshima University. Wistar rats (13–18 days-old) were anaesthetized with ether and decapitated. The brain was quickly removed from the skull and placed in ice-cold HEPES-buffered saline containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 5.5 mM glucose. The pH of the saline solution was adjusted to 7.4 by adding tris (hydroxymethyl) aminomethane. The brain was sliced at a thickness of 400 µm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan), and the slices were kept in bicarbonate-buffered saline bubbled continuously with 95% O2–5% CO2 at room temperature (21–26 °C). The bicarbonate-buffered saline contained 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM NaHCO3, 2 mM KH2PO4 and 5.5 mM glucose. Following 30–60 min incubation, the slices were treated with pronase (0.2 mg/ml) in bicarbonate-buffered saline for 20 min at 33 °C, followed by incubation for 30 min with bicarbonate-buffered saline containing thermolysin (0.12–0.15 mg/ml). The bilateral AP regions were identified with a binocular microscope (Zeiss, Germany), and were cut out using the tip of an injection needle, and subsequently mechanically triturated with fire-polished glass pipettes of decreasing diameters. Dissociated neurons were placed on to the bottom of 35 mm culture dishes (Falcon, USA) for electrophysiological recordings, or on to glass coverslips (Matsunami, Japan) coated with poly-l-lysine for [Ca]in measurements.
Electrophysiological recordings
Electrical measurements were done using the whole-cell patch-clamp recording configuration under voltage-clamp conditions. Patch pipettes were fabricated from borosilicate glass tubes in five or six stages using a pipette puller (Model P-97, Sutter Instrument, San Rafael, CA, USA). The resistance between the recording electrode filled with the internal solution, and the reference electrode in the external solution, was 3–6 MΩ. Ionic currents were measured, and voltages controlled, using a patch-clamp amplifier (EPC-9, Heka, Darmstadt, Germany). All experiments were carried out at 24–26 °C. Culture dishes were placed on an inverted microscope (TE200, Nikon, Japan), and drugs were rapidly applied to single cells using a Y-tube perfusion device. The internal solution (patch pipette solution) contained 70 mM K-gluconate, 50 mM KCl, 10 mM NaCl, 0.5 mM CaCl2, 3 mM MgCl2, 10 mM HEPES, 10 mM EGTA, and 2 mM ATP. The pH was adjusted to 7.2 with KOH. For measuring current-voltage (I–V) relationships, a Cs-based internal solution was employed, which contained 98 mM CsOH, 40 mM CsCl, 98 mM aspartate, 2 mM MgCl2, 5 mM HEPES, 5 mM EGTA, and 2 mM ATP. pH was adjusted to 7.2 with CsOH. The external solution was the HEPES-buffered saline, pH7.4, described above. When N-methyl-d-glucamine (NMDG+) or sucrose was substituted for external Na+ or NaCl, respectively, the osmolarity of the solution was kept constant as measured using an osmometer (Vogel OM801, Germany). To obtain the Ca2+/Na+ permeability ratio (PCa/PNa), we measured the reversal potential of the ATP-activated current (EATP) in the presence of 1 or 110 mM of external Ca2+ by stepping the holding potential (Vh) between 10 and 30 mV, using increments of 10 mV. The 1 and 110 mM Ca2+ solutions contained 155 or 0 mM NaCl, respectively, in addition to 10 mM HEPES with pH adjusted to 7.4 with NaOH or Ca(OH)2, respectively. The values of PCa/PCs and PNa/PCs were calculated using the constant field equation as described by Lewis ([25]), taking the activity coefficients of Na+, Cs+, and Ca2+ as 0.75, 0.75, and 0.3, respectively.
Measurement of [Ca]in
Dissociated AP neurons on a glass coverslip were incubated with 1–2 µM fura-2 acetoxymethyl ester (fura-2/AM), 0.1% dimethyl sulfoxide, and 1% bovine serum albumin for 45 min at 37 °C. The coverslips were then mounted in a superfusion chamber and placed on the stage of an inverted microscope (Diaphot-TMD, Nikon, Japan). Cells were continuously superfused at a rate of 1 ml/min with HEPES-buffered saline at 24–26 °C via a polyethylene tube placed 1–2 mm away from the cells.
Cells were viewed with a 40× Fluor objective lens (Nikon), and a single cell (10–12 µm diameter) was fixed in a window positioned between the photomultiplier and the microscope. The changes in fluorescence ratios at 340 and 380 nm excitation wavelengths were measured using a CAM-200 spectrometer (Jasco, Japan). The absolute value of [Ca]in was calculated using the formulae as given by Grynkiewicz et al. [26]: . Calibration constants were determined in separate experiments with the same experimental set-up, as described previously (Sorimachi et al. [27]).
Student’s paired t test was used to evaluate differences between mean values obtained from the same cells, and Student’s unpaired t test was used for data obtained from different groups of cells.
Reagents
The following reagents were used: Fura-2/AM [Dojindo, Kumamoto, Japan], ACh [Horai Chem. Co., Japan], PPADS (pyridoxal-phosphate-6-azophenyl-2′, 4′-disulphonic acid) [RBI, USA.], pronase [Calbiochem., USA], thermolysin, ATP, 2-methylthio-ATP, ATPγS, α,β-methylene-ATP, β,γ-methylene-ATP, ADP, nitrendipine, nicardipine, suramin [all from Sigma, Aldrich, Tokyo, Japan], ω-conotoxin-MVIIA, ω-conotoxin-MVIIC and ω-agatoxin-IVA [all from Peptide Institute, Osaka, Japan].
Results
ATP-induced current
Rapid application of ATP (100 µM) to isolated AP neurons voltage clamped at a Vh of −70 mV induced an inward current, which desensitized slowly (Fig. 1A). The ATP-induced current (IATP) was recorded at various Vhs between −70 and +50 mV (Fig. 1: N = 6), and the relative current amplitude was plotted against Vh exhibiting a strong inward rectification. The EATP, estimated from the intersection of the current response and the zero voltage-axis (Fig. 1B), was 22.7 ± 0.9 mV (N = 18).
Fig. 1Current-voltage relationship for ATP-induced currents. A) Representative currents in response to 200 µM ATP recorded at Vhs between −70 and +50 mV. B) Current-voltage relationship for the IATP. All responses are normalized to the peak current amplitude obtained at a Vh of −70 mV, and each point is the mean ± S.E.M. of five neurons. C) Averaged concentration-current relationships for ATP in the presence of 0.1, 2, and 10 mM external Ca2+. All responses were normalized to the mean of two control responses induced by 100 µM ATP in saline containing 2 mM external Ca2+ before and after the test response. Each point is the average ± S.E.M of responses from four to six neurons.
To investigate the ATP concentration-response relationship, care was taken to adjust the pH of the solutions containing higher concentrations of ATP to 7.3, since addition of ATP reduced pH and a small decrease in pH to 7.1 increased the IATP to 189 ± 8% (N = 6) of control (pH7.3), as previously reported for recombinant P2X2 receptors (King et al. [28]). The responses to different ATP concentrations were flanked by responses to the control concentration of 100 µM, and normalized responses were expressed relative to the average of these control responses (Fig. 1C; Ca2mM). We also examined the effects of 0.1 and 10 mM Ca2+ on IATP. As summarized in Fig. 1C, an increase in the external concentration of Ca2+ shifted the concentration-response curve for ATP to the right, with the half-maximum effective concentration (EC50) values at 0.1, 2 and 10 mM Ca2+ being 30, 70, and 190 µM, respectively.
The purinergic agonists, 2-methylthio-ATP (50 µM) and ATPγS (50 µM) induced currents that were 41 ± 3% (N = 4), and 41 ± 4% (N = 5), respectively, of the IATP in response to 100 µM ATP. ADP (0.5 mM) did not produce any response. The IATP in response to 50 µM ATP was inhibited by the P2 antagonists; suramin (10 µM and 20 µM) and PPADS (50 µM), with the response being 35 ± 2% (N = 5), 25 ± 2% (N = 4), and 56 ± 5% (N = 5), of the control IATP, respectively.
ATP-induced [Ca]in increase
ATP also increased [Ca]in in a dose-dependent manner (Fig. 2A). These responses were all recorded in the presence of 2 mM external Ca2+, and the [Ca]in increases by various concentrations of ATP were normalized to that induced by 100 µM ATP. The normalized and averaged responses to ATP, 2-methylthio-ATP and ATPγS are shown in Fig. 2B. There was no ATP-induced [Ca]in increase in the absence of external Ca2+ (N = 6; data not shown). Furthermore, neither ADP, α,β-methylene-ATP nor β,γ-methylene-ATP increased [Ca]in when tested at concentrations of 200 µM(data not shown). The ATP-induced [Ca]in increases were inhibited by suramin and PPADS (Table 1).
Fig. 2Concentration-response relationships for the [Ca]in increases in response to ATP and ATP analogues. A) Representative [Ca]in increases induced by various concentrations of ATP in the presence of 2 mM external Ca2+. B) Averaged concentration-response relationships for ATP and ATP analogues. All responses were normalized to the mean of two control responses induced by 100 µM ATP before and after the test response. Each point is the mean ± S.E.M. of data from five to nine neurons. MeS-ATP: 2-methylthio-ATP.Table 1Effects of P2 receptor antagonists and Ca2+ channel blockers on the [Ca]in increases induced by ATP and high K+-salineStimulusBlockersPercent of controlATP100 MSuramin 10 M55 ± 7 (N = 5)20 M30 ± 5(N = 6)50 M13 ± 3 (N = 5)PPADS 10 M96 ± 6 (N = 3)20 M70 ± 8 (N = 9)50 M25 ± 5 (N = 6)100 M11 ± 5 (N = 4)110 mMKClNitrendipine 2 M44 ± 4 (N = 16)−CT.M C 2 M61 ± 4 (N = 16)−CT.M A 2 M62 ± 6 (N = 16)−CT.M A 2 M + −CT.M C 2 M58 ± 5 (N = 10)ATP100 MNitrendipine 2 M56 ± 4 (N = 21)Nicardipine 2 M47 ± 9 (N = 10)Cd2+ 50 M27 ± 3 (N = 14)−CT.M A 2 M + −CT.M C 2 M77 ± 9 (N = 4)The mean [Ca]in increase induced by the first and third applications of ATP (100 μM) or 110 μM KCl were averaged and referred to as 100%, and the response in the presence of the P2 antagonist or Ca2+ channel blocker was expressed as a percentage of this control value. The cell was pre-treated for 30 s with the indicated agent before the second stimulation with 100 μM ATP or 110 mM KCl. Number of experiments is shown in parentheses. ω-CT ω-conotoxin.
Effects of Ca2+ channel antagonists on ATP-induced [Ca]in increase
Previous results, using cultured rabbit AP neurons, demonstrated the presence of a ω-conotoxin-M Csensitive Ca2+ response, but that did not involve L- or N-type Ca2+ channels (Hay et al. [29]). We also investigated the effects of various Ca2+ channel antagonists on [Ca]in increases induced by high KCl (110 mM) and ATP (100 µM). Antagonist for the L-type (nitrendipine, nicardipine), N-type (ω-conotoxin-M A), or P/Q-type (ω-conotoxin-M C) Ca2+ channels each substantially inhibited these [Ca]in increases (Table 1). In contrast, the selective P-type Ca2+ channel antagonist, ω-agatoxin IVA (1 µM), did not have any inhibitory effect on the [Ca]in increases (98 ± 5% of control, N = 4).
ATP-induced current in the absence of external Na+
When external Na+ was completely replaced by NMDG+, and in the absence of external Ca2+ (0 Ca2+ plus 0.5 mM EGTA), the IATP was markedly reduced to 8 ± 1% of the control IATP recorded in the presence of external Ca2+ and 150 mM Na+ (N = 9, Fig. 3A). This current was further reduced to 4 ± 1% (N = 3) in the presence of 20 µM suramin. The small suramin-sensitive, Na+-independent current supports the previous suggestion that the P2XR channel is permeable to glucosamine (Nakazawa [30]). To confirm this, ATP did not induce a current at all when sucrose (0.25 M) was substituted for NaCl (again in the absence of external Ca2+, N = 5). Addition of Ca2+ to the NMDG+-substituted saline further increased IATP. The currents induced by 500 µM ATP in the presence of 2 and 10 mM Ca2+, but in the absence of external Na+ (replaced by NMDG+) were 18 ± 2% (N = 11) and 29 ± 2% (N = 14, Fig. 3B) of the control IATP, respectively. Similarly, the currents induced by 500 µM ATP in the presence of 2 and 10 mM Ca2+ added to the sucrose-substituted saline were 6 ± 1% (N = 6), and 13 ± 2% (N = 16; Fig. 3C) of the control IATP, respectively.
Fig. 3ATP-induced current in Na+- and NaCl-free saline containing 0 and 10 mM Ca2+. A) A representative IATP recorded in Na+-free (Na+ replaced by NMDG+) and Ca2+-free saline (containing 0.5 mM EGTA) at a Vh of −70 mV. B) A representative IATP in Na+-free (NMDG+) saline containing 10 mM Ca2+. C) A representative IATP in NaCl-free (NaCl replaced by sucrose) saline containing 10 mM Ca2+.
We also measured the EATP of IATP in the presence of 1 mM external Ca2+ and 150 mM NaCl or 110 mM external Ca2+ and 0 mM NaCl. The EATP at 110 mM Ca2+ was 21.1 ± 1.0 mV (N = 18), from which we calculated a PCa/PCs of 6.3. The EATP at 1 mM Ca2+ was 20.2 ± 1.0 mV (N = 13), from which we calculated a PNa/PCs ratio of 2.1. From these, we obtained a PCa/PNa ratio of 3.0, confirming the substantial Ca2+ permeability of P2XRs.
Negative interaction between P2XR and nAChR channels
Requirement of actual current flow through receptor channels for cross-inhibition
It has previously been reported that there is mutual occlusion between P2XR and nAChR in some neurons (Nakazawa et al. [18]; Nakazawa [30]; Searl et al. [19]; Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). To examine whether there were negative interactions between the ACh-activated current (IACh) and IATP, one receptor agonist was added in the presence of the other. As shown in Fig. 4A, the IACh (100 µM) was markedly reduced when activated in the presence of ATP (50 µM). We next examined IACh (100 µM) in the presence of various concentrations of ATP. With 2, 10, 20, and 100 µM ATP, IACh was reduced to 95 ± 1% (N = 20), 84 ± 2% (N = 24), 60 ± 4% (N = 15), and 46 ± 6% (N = 7), respectively, of control (P < 0.01 except at 2 µM ATP). Thus, the IACh inhibition became stronger as the concentration of ATP was increased and with a higher agonist-receptor occupancy. In fact, when IATP at 100 µM ATP was markedly inhibited in the presence of 200 mM PPADS (8 ± 2% of control, N = 6), IACh was not occluded (97 ± 1% of control; N = 6). When the peak amplitude of IATP, evoked by various concentrations of ATP, was plotted against the ratio of IACh in the presence and absence of ATP, there was an inverse correlation between the amplitudes of these responses (Fig. 4B). Conversely, when ATP (50 µM) was applied in the presence of ACh (100 µM), IATP was also occluded (Fig. 4C). Again, when IACh at 200 mM ACh was nullified in the presence of 1 mM hexamethonium, a nAChR antagonist (0.2 ± 0.2% of control, N = 6), IATP was 96 ± 2% (N = 6) of control. IACh at 10, 20, 50, and 200 µM ACh were 6 ± 2% (N = 5), 20 ± 4% (N = 5), 61 ± 8% (N = 6), and 139 ± 8% (N = 6) of IACh at 100 µM. There was also an inverse correlation between the peak amplitude of IACh, evoked by various concentrations of ACh, and the ratio of IATP in the presence and absence of ACh (Fig. 4D). However, the inhibition of IATP by nAChR activation was weaker than that of IACh by P2XR activation. As the IACh desensitized faster than the IATP, the weaker inhibition of IATP by ACh could be due to the reduced amplitude of the IACh at the time of ATP application.
Fig. 4Inhibition of IACh and IATP, respectively, in the presence of ATP and ACh. A) Sample currents in response to ACh (100 µM) in the presence of ATP (50 µM). The responses to ACh applied in the presence of ATP were reduced. The extent of occlusion was normalized to the control ACh responses recorded before and after ATP application. This sequence was routinely repeated twice or three times, and the averaged amount of occlusion was plotted in B. B) Relative amplitude of the ACh current amplitude in the presence of ATP (ordinate) plotted against the peak amplitude of IATP obtained in the same neuron (abscissae). Note the negative correlation between these two variables. The line was drawn using KaleidaGraph (Synergy Software). Vh: −70 mV. C) The IATP was recorded in the presence of 100 µM ACh and this response was flanked by control ATP alone responses. This sequence was routinely obtained twice or three times in the same cell, and the averaged amount of occlusion was plotted in D. D) The ratio of the ATP current amplitude in the presence and absence of ACh (ordinate) is negatively correlated with the peak amplitude of IACh (abscissae). Vh : −70 mV.
To examine this possibility, IATP was measured in the presence of both ACh and d-tubocurarine (dTc, 10 µM) or serotonin (5HT, 50 µM), two experimental conditions which change the time course of the IACh response. The former agent, dTc, has been shown to be both an open channel blocker and a competitive antagonist of nAChR (Manalis [31]), and, as expected, a low concentration (10 µM) slightly reduced the peak amplitude of IACh (79 ± 4% of control, N = 11) and caused the IACh to be terminated within 0.5 s (Fig. 5A, insert). Once the IACh response had returned to baseline (in the presence of ACh and 10 µM dTc) and ATP was applied, there was no inhibition of IATP (95 ± 1% of control, N = 5; Fig. 5A). The IATP in the presence of ACh alone was 77 ± 4% of control (N = 5, P < 0.01; Fig. 5A). When we measured the current (IACh+ATP+dTc) induced by a combination of ACh, ATP and dTc, the peak amplitude of IACh+ATP+dTc was occluded under these conditions, being 74 ± 2% (N = 10) of the predicted sum of IATP and IACh+ dTc (Fig. 5B). The level of IACh+ATP+dTc at 0.5 s following ligand application, which corresponds to a time when the nAChR channels are completely blocked (Fig. 5A, insert), was 96 ± 3% (N = 8) of the control IATP, measured immediately before and after (Fig. 5C). These results suggest that the inhibition of IATP by IACh disappears immediately after the IACh is abolished, although ACh is still present and bound to the nAChRs. 5HT has been shown to also accelerate the decay of IACh (Grassi et al. [32]; Garcia-Colunga and Miledi [33]; Sorimachi and Wakamori [34]), but not to the same extent as dTc. 5HT at 50 µM decreased the peak amplitude of IACh slightly, to 88 ± 4% (N = 6) of control, and reduced the time constant (tau) of IACh decay from 2.02 ± 0.18 to 1.06 ± 0.19 s (N = 6). As shown in Fig. 5D, the extent of inhibition of IATP by ACh was smaller (to 92 ± 2% of control, N = 6) in the presence of ACh and 5HT compared to that in the presence of ACh alone (to 73 ± 3% of control, N = 6; P < 0.01).
Fig. 5Loss of the inhibition of IATP by ACh in the presence of dTc and 5HT. A) Representative IATP in control conditions (applied alone), in the presence of ACh and in the presence of ACh and dTc. This sequence was routinely repeated twice, and the averaged response was obtained. Note the very transient nature of IACh in the presence of dTc, with the current returning to baseline levels within 0.5 s (insert). Note also the lack of occlusion under these conditions. Vh: −70 mV. B) Effect of dTc on IACh+ATP. The sequence of applications was repeated multiple times in each neuron. The peak amplitude of IACh+ATP+dTc was smaller than the predicted sum of IATP and IACh+dTc. C) Rapid recovery of IATP from inhibition by ACh in the presence of dTc. The amplitude of IACh+ATP+dTc 0.5 s following application was equal to the mean of two control IATP at an equivalent time. Inset shows the rapid decline of IACh+ATP+dTc on an expanded time scale. D) Representative IATP in control conditions, in the presence of ACh, and in the presence of both ACh and 5HT. In this example, the tau of IACh in response to the first application was 2.7 s, while the tau of IACh+5HT was 0.9 s, showing the acceleration of the IACh decay by 5HT. Note the reduced occlusion under these conditions. Vh : −70 mV.
Next, we measured the current induced by the concomitant applications of ACh and ATP (IACh+ATP) at a Vh of −70 mV. As shown in Fig. 6A, IACh+ATP was 75 ± 1% (N = 44; P < 0.01) of the predicted sum of IACh and IATP. The decay phase of IACh, IATP and IACh+ATP were fit to a single exponential function, giving tau of 3.0 ± 0.2, 7.5 ± 0.6 and 3.7 ± 0.2 s, respectively. When the decay of IACh was accelerated by the presence of 100 µM 5HT, the tau of IACh, IATP, and IACh+ATP were 0.8 ± 0.3 s (N = 7), 7.4 ± 1.4 s (N = 7) and 2.6 ± 0.8 s (N = 7), respectively (the amplitude of IACh+ATP under these conditions was 76 ± 2% of the predicted sum of IACh and IATP). Thus, IACh+ATP desensitization occurs with kinetics that cannot be explained by the desensitization kinetics of IACh or IATP alone, suggesting that IACh+ATP is mediated via both receptors.
Fig. 6Voltage dependence of IACh and of IATP occlusion. A) At a Vh of −70 mV, co-application of ACh (200 µM) and ATP (100 µM) induced an inward current that was smaller in amplitude than the predicted sum of the individual IACh and IATP. B) At a Vh of +40 mV, the amplitude of the outward current induced by the co-application of ACh (500 µM) and ATP (200 µM) was equal to the predicted sum of the individual IACh and IATP. C) Co-application of ATP and ACh does not markedly change EACh or EATP. Representative currents induced by ACh, ATP, and by co-application of ACh and ATP at a Vh of +10 mV. Note that IACh+ATP is composed of early outward and delayed inward current, corresponding to the ACh component and the ATP component, respectively.
When Ca2+ was omitted from extracellular solution, the negative interaction between the two receptor channels was still obtained; with IACh+ATP being 77 ± 1% (N = 5; P < 0.01) of the predicted sum of IACh and IATP. Hence negative interaction is not mediated by a Ca2+ influx-dependent mechanism, although both nAChR and P2XR are Ca2+-permeable cation channels (Fieber and Adams [35]; Rogers and Dani [36]). This result, however, does not necessarily rule out the possible involvement of [Ca]in in negative interaction.
Occlusion was not only observed at a Vh of −70 mV; IACh+ATP at Vhs of −20, and −10 mV were 80 ± 2% (N = 12; P < 0.01), and 76 ± 2% (N = 8; P < 0.01), respectively, of the predicted sum of IACh and IATP. In sharp contrast, such occlusion was not observed at a positive potential; the outward current caused by coapplication of ACh and ATP at a Vh of +40 mV was 100 ± 1% (N = 10) of the predicted sum of individual current (Fig. 6B).
We next investigated the possibility that the combined addition of ACh and ATP alters the driving force for Na+, by attempting to measure EACh+ATP. In these studies, currents in response to ACh, ATP and both ligands were measured at various fixed potentials close to the reversal potential for IACh and IATP. The EACh and EATP were 8.7 ± 1.5 mV (N = 5) and 21.0 ± 1.6 mV (N = 7), respectively, and IACh+ATP measured at a potential between these two reversal potentials (e.g.10 mV, Fig. 6C) showed a combination of both the ACh-induced outward current and the ATP-induced inward current. These results suggest that the driving force for permeant ions during combined ACh and ATP is similar to that during the application of each ligand separately.
The question arises as to whether occlusion is specific just for Na+ ions or whether inward currents carried by cations other than Na+ can also contribute to occlusion during co-activation of nAChRs and P2XRs. To address this question, we measured IACh, IATP, and IACh +ATP in the presence of Na+-free (NMDG+) saline containing 10 mM Ca2+ (Fig. 7). NMDG+ does not permeate through nAChR (Sorimachi and Wakamori [16]), and hence IACh would be mediated by only Ca2+ influx, whereas IATP would be mediated via both NMDG+ and Ca2+ influx (Fig. 3). Under these conditions, IACh+ATP was again occluded, being 70 ± 3% of the predicted sum of IACh and IATP (N = 11; P < 0.01; Fig. 7).
Fig. 7Occlusion of IACh+ATP in the presence of Na+-free saline containing 10 mM Ca2+. IACh, IATP or IACh+ATP was recorded 20 s after switching to Na+-free (replaced by NMDG+) saline containing 10 mM Ca2+. The sequence of applications was repeated multiple times. Following each ligand application, the external solution was changed back to normal saline containing 150 mM Na+ before the next ligand application. Vh: −70 mV.
In a further attempt to distinguish whether channel activation or ion permeation was primarily responsible for occlusion of one channel by the other, we compared IATP with IACh+ATP under two experimental conditions, in which a higher concentration of ACh should activate nAChR but produce very little current. In the absence of Na+ and Ca2+ (replaced by NMDG+) application of ACh induces a negligible current (Sorimachi and Wakamori [16]), while ATP induces a substantial current under the same conditions (Fig. 3A). As shown in Fig. 8B, there was no occlusion in these conditions; the amplitude of IATP+ACh was 105 ± 1% (N = 10) of the predicted sum of IATP and IACh. We also investigated currents in response to the ligands at a potential close to the EACh (7 ± 2 mV in this experiment, N = 13), taking advantage of the more positive EATP (22.7 ± 0.9 mV), so that at this potential only the IATP was observed. The amplitude of IATP+ACh was 105 ± 3% (N = 13) of that of IATP, and IACh was close to zero (Fig. 8D). These results are, however, in contrast to that obtained in sympathetic neurons (Nakazawa[30]), in which IATP in the absence of both Na+ (replaced by glucosamine) and Ca2+ was inhibited in the presence of ACh, which caused no current (Nakazawa [30]). In these experiments, we only included data in which IATP was large enough to be clearly resolved, greater than 50 pA (corresponding to a control IATP at a Vh of −70 mV larger than 1.5 nA). These results suggest that actual current flow through both nAChR and P2XR is responsible for occlusion.
Fig. 8Lack of inhibition in response to co-application of ACh and ATP under two experimental conditions designed to virtually eliminate IACh. A) Control IACh recorded at a Vh of −70 mV in standard saline. B) Current responses recorded at −70 mV from 20 s after exchanging the standard saline for a Na+-free (replaced by NMDG+), Ca2+-free saline. This sequence was repeated twice or three times. Note the small amplitude of IACh, and the lack of occlusion of IACh+ATP. C) Control IACh recorded at a Vh of −70 mV in another neuron. D) Representative traces of IACh, IATP, and IACh+ATP recorded at a Vh of +7 mV, which is very close to EACh. Note the lack of occlusion at this potential, when current flow through the AChR is negligible.
Discussion
In this study, we found that ATP induced an inward current and increased [Ca]in in isolated rat AP neurons. The ATP-induced current and [Ca]in increase were mimicked by ATPγS and 2-methylthio-ATP, but not by α,β-methylene-ATP, β, γ-methylene-ATP nor ADP, and was inhibited by suramin and PPADS, suggesting that it was mediated by P2XRs. These results are in good agreement with previous histochemical findings, which demonstrated the presence of P2X2, P2X4, and P2X6 receptor mRNAs (Collo et al. [37]), and of P2X2 receptor immunoreactivity in AP neurons (Kanjhan et al. [38]; Atkinson et al. [39]).
The IATP showed strong inward rectification and the EATP was 22.7 ± 0.9 mV (Fig. 1B). The amplitude of IATP varied inversely with the extracellular Ca2+ concentrations (Fig. 1C). The inhibitory effect of increasing extracellular Ca2+ has also been reported in rat sensory neurons (Krishtal et al. [40]), PC-12 cells (Nakazawa et al. [18]), ventromedial hypothalamic neurons (Sorimachi et al. [41]), and in cells expressing recombinant P2X2 receptors (Evans et al. [42]; Virginio et al. [43]), where an allosteric alteration of the ATP binding sites has been suggested to be the underlying mechanism.
The IATP was markedly reduced, but still persisted when NMDG+ was substituted for external Na+ even in the absence of Ca2+ (Fig. 3A). Since ATP did not induce a current when sucrose was substituted for external NaCl, our result suggests that NMDG+ could permeate through P2XR. A substantial IATP has been similarly reported in PC-12 cells and sympathetic neurons when glucosamine was substituted for Na+ (Nakazawa et al. [18]; Nakazawa [30]). The addition of Ca2+ to NMDG-Cl- and sucrose-substituted saline increased the IATP (Fig. 3B and C, respectively), indicating that Ca2+ also permeates through P2XR channel. We quantified the relatively high Ca2+ permeability, obtaining a PCa/PNa ratio of 3.0. A relatively high permeability of P2XR to Ca2+ has been reported in previous studies (Nakazawa et al. [18]; Sorimachi et al. [41]; Evans et al. [42]; Virginio et al. [43]). Although direct influx of extracellular Ca2+ through P2XR channel may thus contribute to the ATP-induced [Ca]in increase, membrane depolarization and the secondary activation of voltage-dependent Ca2+ channels could also make a significant contribution to the [Ca]in increase. In fact, the high K+- and ATP-induced [Ca]in increases were substantially inhibited by a range of Ca2+ channel antagonists, including those which block L- and N-type Ca2+ channels (Table 1). This is in contrast to a previous study, which reported an absence of the L- and N-type Ca2+ channels in rabbit AP neurons (Hay et al. [12]). The discrepancies between their results and ours could be, at least in part, accounted for by the use of different AP preparations (cultured rabbit neurons vs. acutely dissociated rat neurons).
The present results, in combination with our previous demonstration of nAChR on AP neurons (Sorimachi and Wakamori [16]), suggest that both ATP and ACh may act as excitatory neurotransmitters in AP neurons, although their release from presynaptic nerve terminals has not yet been reported. We also report a negative functional interaction between P2XRs and nAChRs in AP neurons, as has been observed in a variety of peripheral neurons and in recombinant expression systems (Nakazawa et al. [18]; Nakazawa [30]; Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Searl et al. [19]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). This is the first report, that we are aware of, of such interactions occurring in central neurons. When ACh was applied in the presence of ATP, there was a positive correlation between the peak amplitude of inward IATP and the amount of occlusion of inward IACh (Fig. 4A and B). The converse was also true when ATP was applied in the presence of ACh (Fig. 4C and D). Non-additivity of the IACh+ATP was observed even when the inward current was carried by NMDG+ and/or Ca2+ (Fig. 7). Co-application of two agonists did not seem to change the driving force for Na+, because at a Vh between EACh and EATP an outward current due to the activation of nAChR, followed by an inward current due to the activation of P2XR, was observed (Fig. 6C). Occlusion was also observed at a Vh of −10 mV, which is closer to EACh or EATP. Furthermore, the removal of external Ca2+ did not alter the occlusion, and thus a Ca2+-mediated mechanism does not contribute to the current occlusion.
The IACh+ATP occlusion was observed at all negative holding potentials when the current was inward but was not observed for outward currents at a Vh of +40 mV. Here the outward IACh+ATP was not different from the predicted sum of IACh and IATP (Fig. 6B). The same dependence on current direction has been reported in guinea-pig enteric and submucosal neurons (Zhou and Galligan [20]; Barajas-Lopez et al. [21]), suggesting that the current occlusion was triggered by the inward movement of cations through two channels.
Some investigators have shown that the amplitude of IACh+ATP is equal to or even smaller than that of either IACh or IATP. For instance, the concentration of one agonist causing little or no inward current produced dramatic occlusion of the inward current generated by the other agonist (Searl et al. [19]). Khakh et al. [22] using Xenopus oocytes co-expressing P2X2 and α3β4 nAChR channels, provided several lines of evidence which indicated that occlusion was mostly mediated by the inhibition of the nAChR channel by activation of the P2XR. However, we found that concomitant application of two agonists always caused a larger current than either agonist (Fig. 6A), and that the amount of occlusion of one channel current in the presence of the other channel agonist was correlated with the amplitude of current through the other channel (Fig. 4). Furthermore, we found that IACh+ATP desensitizes faster than IATP, but more slowly than IACh, and IACh+ATP at a Vh between EACh and EATP showed a combination pattern of early outward IACh and delayed inward IATP (Fig. 6C). These results both strongly suggest that in AP neurons, IACh+ATP are carried through both nAChR and P2XR channels, and that inhibition between these channels is reciprocal.
The IATP was not inhibited by co-application of ACh when the inward IACh was markedly reduced in the absence of permeant cations (Fig. 8B) or at a Vh very close to EACh (Fig. 8D), and the inhibited IATP in the presence of ACh (Fig. 5B) recovered as soon as the nAChR channel closed in the presence of dTc (Fig. 5C). These results thus suggest that the inhibitory interaction not only requires the activation of both receptor channels, but also requires a substantial current to flow through these channels. These results are in contrast to that obtained in sympathetic neurons, in which the ATP-induced glucosamine influx was inhibited in the presence of ACh (Nakazawa [30]). The cause of this inconsistency remained unknown, but may reflect a real difference in the underlying mechanisms of occlusion in these two types of neurons.
Altogether, our results have characterized the P2XR responses in AP neurons and the cross-inhibition between P2XRs and nAChRs in AP neurons. We show that the current flow through one receptor channel hinders the current flow through the other channel. These interactions support the notion that these channels are positioned very close to each other, as has been previously considered (Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Khakh et al. [22]; Boue-Grabot et al. [24]) and more recently demonstrated for recombinant channels (Khakh et al. [23]). These results will be important to consider when designing ligands to modify excitability of ATP neurons and may have some physiological function during co-activation of P2XRs and nAChRs by synaptically released transmitters.

negative interaction between nicotinic and p2x channels


Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer

Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A–M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10–20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.
Introduction
Fanconi anemia (FA) is a recessive disease with both autosomal and X-linked inheritance. FA is associated with progressive pancytopenia, developmental defects, and marked predisposition to malignancies, especially acute myeloid leukemia and squamous cell carcinoma of the head and neck [1, 2]. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). FA is genetically heterogeneous and comprises at least 12 complementation groups (A–M). Eleven of the FA genes have been identified so far: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCJ, FANCL, and FANCM [3–18]. Eight FA proteins (A, B, C, E, F, G, L, and M) form a nuclear protein complex which is required for mono-ubiquitination of the downstream FA protein, FANCD2. Activation of FANCD2 by mono-ubiquitination is an essential step in the DNA damage response induced by MMC or ionizing irradiation [2, 10]. This DNA damage response pathway also includes the breast cancer susceptibility genes BRCA1 and BRCA2, also referred to as the FA-BRCA pathway. Following ionizing radiation, FANCD2 and BRCA1 accumulate and colocalize in nuclear foci, which reflect sites of DNA damage and repair [10, 19]. Like FA cells, cells lacking BRCA1/2 proteins are hypersensitive to DNA cross-linking agents. D’Andrea et al. showed that FANCD1 and BRCA2 are the same proteins. BRCA2 is a direct regulator of RAD51, a protein essential for homologous recombination repair [20]. Although BRCA1 is mainly involved in hereditary breast cancer [21], it has also been implicated in sporadic breast cancer [22]. In an immunohistochemical analysis, we have previously shown that FANCD2 is expressed in proliferating cells of different organs, including the premenopausal breast duct epithelium [23]. This is in line with the role of FANCD2 in DNA repair which is important to guarantee the integrity of the genome during cell replication [10]. As deregulation of proliferation is one of the crucial processes of carcinogenesis, these observations imply a potential role for FANCD2 in the pathogenesis of breast cancer. Indeed, FANCD2 knockout mice develop breast tumors [24]. These considerations prompted us to investigate the expression of FANCD2 in sporadic and hereditary invasive breast cancers by immunohistochemistry in relation to several other proliferation-related biomarkers and survival.
Materials and methods
Tissue microarray
Paraffin blocks containing formaldehyde-fixed breast cancer tissues of 129 cases of invasive breast cancer not selected for family history (further denoted “sporadic”) were obtained from the archives of the Department of Pathology of the VU University Medical Center, Amsterdam. For all breast cancer cases, age, lymph node status, and tumor size were documented. A second array block was constructed containing 24 cases with a proven BRCA1 germline mutation identified through the Family Cancer Clinic of the VU University Medical Center as previously described [25]. Patient characteristics are shown in Table 1. The hematoxylin–eosin stainings were used to identify representative areas of tumor tissue in the blocks. A tissue microarray was then constructed by transferring tissue cylinders of 4–5 mm from the representative tumor area of each donor block to the recipient block using a tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA) as described before [26]. A third tissue array block of 220 sporadic breast cancer patients with long-term follow-up was obtained from the archives of the Gerhard-Domagk Institute of Pathology, University of Muenster, as previously described [27].
Table 1Patient characteristics and histology of 120 sporadic and hereditary breast cancersSporadicBRCA1 mutationTotalNo. of patients9624120Age   Mean654262Lymph node status   Negative58967   Positive381149Tumor size   Mean2.342.782.40Histological type   Ductal8517102   Lobular718   Medullary123   Tubular1–1   Cribriform1–1   Apocrine1–1   Metaplastic–44
Sections of 4 μm were cut and transferred on SuperFrost+ (Menzel&Glaeser, Germany) slides for immunohistochemistry. Use of anonymous or coded left over material for scientific purposes is part of the standard treatment contract with patients in our hospital [28].
Immunohistochemistry
Immunohistochemical analysis had been previously performed on conventional sections for the following markers: Ki-67, Cyclin A, p21, p27, p53, estrogen receptor (ER), progesterone receptor (PR), HER-2/neu, and EGF-receptor [29].
Rabbit polyclonal antiserum against FANCD2 was generated as previously described [23]. Tissue sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with methanol/0.3% H2O2 (20 min). Sections were heated (30 min, 120°C) in 0.1 M citrate buffer pH 6. Unspecific binding was blocked with a 1:50 normal goat serum in PBS pH 7.4/1% BSA. Polyclonal rabbit anti-FANCD2 (200 μg/ml) was diluted 1:500 in PBS/1% BSA, and sections were incubated overnight (4°C) in a humidified chamber. Subsequently, sections were incubated with HRP-conjugated secondary antibodies (EnVision, DAKO) and diaminobenzidin (10 min), counterstained with hematoxylin (20 s), dehydrated, and cover-slipped. Appropriate positive controls were used throughout, and negative controls were obtained by omission of the primary antibodies.
Percentages of positively stained nuclei were estimated by an experienced observer (P.J.v.D.), except for HER-2/neu and EGF-receptor where membrane staining was scored as positive. In addition, FANCD2 intensity was scored semiquantitatively as 0–3, and an FANCD2 score was calculated for each case by multiplying the % FANCD2-positive cells by the staining intensity.
For FANCD2/Ki-67 double staining, anti-FANCD2 was diluted 1:150, incubated overnight, followed by incubation with swine anti-Rabbit HRP 1:200 (Dako, Glostrup, Denmark), and detected with the TSA™ Tetramethylrhodamine system (PerkinElmer Life Sciences, Boston, USA). This was immediately followed by incubation with mouse anti-Ki-67 1:50 (MIB1, Immunotech, Marseille, France) followed by rabbit anti-mouse FITC 1:40 (Dako, Glostrup, Denmark). Nuclei were counterstained by incubation with TO-PRO-3 (Molecular Probes, Eugene, OR, USA) 1:5,000 as previously described [30].
Statistics
Bivariate scatter plots were generated between the percentage of FANCD2-expressing cells and the other continuous features. For the proliferation-associated features Ki-67, Cyclin A, and MAI, the cases with no FANCD2 expression were excluded from the analysis, assuming that FANCD2 is, by some mechanisms, no longer expressed in these cases. By linear regression analysis, the correlation coefficient R and related p values were calculated. Student’s t test was used to compare FANCD2 expression levels between the low-level vs. high-level groups for HER-2/neu, the EGF-receptor, ER (cut off 10%) and PR (cut off 10%), p53 (cut off 10%), and Cyclin D1 (cut off 5%). Prognostic value of FANCD2 (Muenster cases) was assessed by computing Kaplan–Meier curves, and differences between the curves were evaluated with the log-rank test. Multivariate survival analysis was performed by Cox regression.
Results
Successful FANCD2 staining was performed in 96 of the 129 cases of the VUmc sporadic array block. The drop outs were caused by damaged or detached cores during cutting, mounting, or staining, or did not contain tumor. Eighteen of these cases (19%) were completely FANCD2-negative. The other cases showed variable staining from 1 to 85% of the nuclei. In 21 of the 24 BRCA1 cases, FANCD2 staining was performed successfully. In two of these (9.5%), FANCD2 expression was completely negative. Figure 1 shows some representative examples of FANCD2 staining.
Fig. 1Examples of FANCD2 staining in sporadic (A/B, A = negative control) and BRCA1-related breast cancers (C/D, C = negative control)
The mean percentage of FANCD2-expressing cells was significantly higher in ER-negative patients (p = 0.019), PR-negative patients (p = 0.016), EGFR-positive patients (p = 0.002), Cyclin-D1-negative patients (p = 0.002), and p53-positive patients (p < 0.001) (Table 2). No statistically significant difference was seen for HER-2/neu. When analyzing the sporadic and hereditary subgroups, similar associations were seen. FANCD2 staining intensity yielded no useful correlations and no prognostic value, and the FANCD2 score yielded essentially the same correlations and prognostic value as the % FANCD2-positive cells (data not shown).
Table 2Mean percentage of FANCD2-positive cells in high- and low-level expression groups of hormone receptors, growth factor receptors, Cyclin D1, and p53 in sporadic and hereditary breast cancersNumber (%)Mean (SE) of FANCD2%p Value (t test)Total117 (100)ER   Low56 (48)14 (2.5)0.019   High61 (52)8 (1.6)PR   Low81 (69)17 (1.9)0.016   High36 (31)12 (1.9)HER-2/neu   Negative93 (79)16 (1.6)0.797   Positive24 (21)17 (3.5)EGF receptor   Negative78 (66)13 (1.5)0.002   Positive39 (33)19 (3.1)Cyclin D1   Low85 (73)12 (1.9)0.002   High32 (27)7 (2.6)p53   Low88 (75)8 (1.3)<0.001   High29 (25)19 (4.3)ER estrogen receptor, PR progesterone receptor
In linear regression analysis (Table 3), the percentage of FANCD2-expressing cells was significantly positively correlated to Ki-67 (R = 0.502, p < 0.0001), Cyclin A (R = 0.482, p < 0.0001), MAI (R = 0.506, p < 0.001), and p53 (R = 0.379, p < 0.0001), and significantly negatively correlated to age (R = 0.197, p = 0.033), ER (R = 0.221, p = 0.017), and PR (R = 0.204, p = 0.028). There was no correlation between FANCD2 and the other continuous features. Figure 2 shows examples of FANCD2/Ki-67 immunofluorescence double staining, underlining the coexpression of FANCD2 and Ki-67 in invasive breast cancers cells. In the Muenster cases, most of these correlations could be reproduced (age: p = 0.063, Ki-67: p = 0.001, p53: p = 0.003, ER: p = 0.034). Only PR was not significant here, and Cyclin A was not performed.
Table 3Correlation between mean percentage of FANCD2-expressing cells and other continuous clinicopathological variables in sporadic and hereditary breast cancersFeatureRp ValueMAIa0.506<0.001Ki-67a0.502<0.001Cyclin Aa0.482<0.001Age−0.1970.033ER−0.2210.017PR−0.2040.028Tumor size0.0180.852p27−0.0740.430p21−0.1710.065p530.379<0.001Cyclin D1−0.1260.176ER estrogen receptor, PR progesterone receptoraFANCD2-negative cases excludedFig. 2FANCD2/Ki-67 immunofluorescence double staining in a representative case of invasive breast cancer. Top left: Ki-67 staining. Top right: FANCD2 staining. Bottom left: TO-PRO staining. D: Triple exposure showing coexpression of FANCD2 and Ki-67
In the regression analysis between the percentages of FANCD2-expressing cells and Ki-67 and Cyclin A, the completely negative FANCD2 cases were excluded. These FANCD2-negative cases had Ki-67 values between 1 and 65 (mean 16%), Cyclin A values between 0 and 50 (mean 10%), and MAI values between 0 and 37 (mean 11), indicating that these cases had (sometimes even high) FANCD2-independent proliferation.
In survival analysis (sporadic Muenster cases), high FANCD2 expression appeared to be prognostically unfavorable (p = 0.03). Figure 3 shows the survival curves. In Cox regression including tumor size, lymph node status, ER, and grade, FANCD2 staining appeared to have independent prognostic value for overall survival (p = 0.026).
Fig. 3Prognostic value of FANCD2 expression in sporadic invasive breast cancer. Low expressors have a better survival than high expressors (p = 0.03, N = 122)
Discussion
The aim of this study was to investigate the expression of FANCD2 in sporadic and hereditary breast cancers. This was inspired by several observations. First, FANCD2 and BRCA1/2 are functionally closely linked in the DNA repair response, and BRCA1 and BRCA2 are implicated in hereditary and sporadic breast cancers [21, 22]. Second, targeted deletion of FANCD2 in mice resulted in an increased rate of breast tumors [24]. Third, we have shown that FANCD2 is expressed in proliferating cells in the duct epithelium of the normal breast [23].
It appeared that 19% of sporadic breast cancers completely lacked FANCD2 expression. Yet, these FANCD2-negative cases had high mean Ki-67, Cyclin A, and MAI values, indicating that the low FANCD2 levels in these cases cannot be explained by low proliferation. The fact that these FANCD2-negative cases stained for other proteins makes it quite unlikely that the FANCD2 negativity is due to fixation problems. In the FANCD2-negative cases, FANCD2 inactivation may, in view of its important function, have been a hit in carcinogenesis. BRCA1 germline mutation-related breast cancers showed lack of FANCD2 expression in only 9.5% of cases, which fits with the concept that a major hit in an important pathway (in these cases the BRCA1 germline mutation) is usually not associated with further hits in this pathway. It is yet unclear what the mechanism behind the lack of FANCD2 expression in these cases is. It needs to be further studied whether there are inactivating somatic mutations in these cases or whether promoter methylation plays a role.
FANCD2 expression was strongly correlated with expression of the proliferation-associated features Ki-67, Cyclin A, and MAI, and FANCD2 and Ki-67 were coexpressed in invasive cancer cells. This is likely a reflection of the physiological function of FANCD2 in DNA repair of proliferating cells, rather than an independent overexpression of an altered gene, in line with our previous study where we found a coexpression of FANCD2 and Ki-67 in proliferating cells of various normal human tissues [23]. The observation that high FANCD2 expression indicated poor prognosis fits within the same concept, as rate of proliferation (and thereby FANCD2 expression) is a major cell biological phenomenon determining prognosis [31–33]. FANCD2 had prognostic value independent of stage and grade, which can be explained by the fact that proliferation and stage are not strongly correlated, and that grade includes nuclear atypia and tubule formation besides rate of proliferation as measured by mitotic index.
Although heterozygosity for p53 was shown to accelerate epithelial tumor formation in Fancd2 knockout mice [34], a functional link between p53 and Fancd2 has not been described to explain the association found in the present study, which is likely caused by the fact that p53 mutated and thereby p53 protein accumulated tumors show, in general, higher proliferation and therefore more proliferating FANCD2-expressing cells. The same may also hold for the relation between FANCD2 and EGFR expressions, for which also no functional relationship has been described.
The negative relation between FANCD2 and age can likely be explained by the fact that BRCA1-related patients that have higher FANCD2 expression are younger.
Within the light of the above observations, the question remains why FANCD2 patients do not seem to be predisposed to breast cancer in clinical practice. FA itself is a rare genetic disease where the complementation group D2 constitutes only 1–2% of all FA cases and these patients generally have a more severe clinical course. They may therefore simply not live to get breast cancer in an apparent increased frequency. Our results do not indicate that somatic (epi)genetic changes in FANCD2 are a frequent secondary carcinogenetic event in BRCA1 germline-mutated patients, although this needs to be confirmed in a larger study group.
In conclusion, FANCD2 expression is absent in 10–20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.

fancd2
breast cancer
brca1
proliferation
immunohistochemistry
prognosis


The Impact on Post-surgical Treatment of Sentinel Lymph Node Biopsy of Internal Mammary Lymph Nodes in Patients with Breast Cancer

Background Since the introduction of the sentinel lymph node (SLN) biopsy in breast cancer patients there is a renewed interest in lymphatic drainage to the internal mammary (IM) chain nodes. We evaluated the frequency of lymphatic drainage to the IM chain, the rate of SLNs that contain metastases and the clinical implications of IM LN metastases.
The internal mammary (IM) chain is well known as a site of metastases in patients with breast cancer. The presence of IM lymph node (LN) metastases is a poor prognostic sign as reflected by the UICC-TNM staging system that classifies IM LN metastases as stage N3.1 In earlier times dissection of the IM LNs, as part of an extended mastectomy with IM node dissection, was advocated by some authors but randomized trials failed to show any beneficial effect of the procedure that was accompanied by significant morbidity.2–4 Since then, interest in the IM LN chain has waned.
Since the introduction of the sentinel lymph node (SLN) biopsy in breast cancer patients there has been a renewed interest in the IM LNs. Drainage to this basin is frequently seen on preoperative lymphoscintigraphy, while the localization of the primary tumor and the nanocolloid injection technique appear to influence the frequency of visualized IM nodes.5–7 Various authors advocate removal of these IM SLNs,8,9 but the clinical implications of IM LN biopsy are still unclear.
In this study we evaluated the frequency of lymphatic drainage to the IM chain, the rate of metastases in the IM SLN and the clinical implications of IM LN metastases.
PATIENTS AND METHODS
Between June 1999 and April 2005, 523 consecutive patients underwent surgical treatment including SLN biopsy as a staging procedure for clinically stage T1-2N0 breast cancer. Data regarding all procedures were collected prospectively in a database of the nuclear medicine department.
A diagnosis of invasive breast cancer was established preoperatively by fine-needle aspiration or image-guided large core needle biopsy. Sixteen patients who underwent SLN biopsy as a secondary operative procedure following previous excisional biopsy of the primary tumor and one patient who eventually turned out to have multicentric breast cancer were excluded from the study.
The study cohort consisted of 506 patients. At the time of the introduction of the sentinel node procedure the ethical committee of the hospital approved the routine use of the SLN biopsy as a staging procedure. All patients received written information regarding the SLN procedure and the possibility of SLNs in the IM chain. The unknown clinical implications of surgically removing IM LNs were discussed with the patients.
On the day of the operation, all patients received a combination of peritumoral intraparenchymal and subcutaneous injections of an average of 77.7 MBq (53–150 MBq) of 99mTc nanocolloid in a total volume of 0.6 ml of physiologic saline, given in two to three equal doses. In case of a nonpalpable breast tumor the tracer was injected around the tumor using a 7.5-MHz ultrasound probe (Aloka). After injection the area was massaged until the appearance of the SLN. Continuous visualization was done and imaging started as soon as lymphatic drainage was visualized on the persistence scope and at 2–3 h after injection in both the anterior and lateral direction. Images were obtained during a 2-min imaging time on the Toshiba 901 HG single-head gamma camera, using low-energy high-resolution collimators. A skin marker was placed on the projection of the SN using a handheld γ-ray detection probe (Europrobe, PI Medical diagnostic equipment BV).
The operative procedure was carried out in the afternoon of the same day of the 99mTc nanocolloid injection. A γ-ray detection probe and a peritumoral injection of patent blue dye (Bleu patenté V, Laboratoire Guerbet, Aulnay-sous-Bois, France) were intraoperatively used for SLN identification. During the operation axillary sentinel nodes were retrieved first and frozen section analysis was done to enable axillary dissection during the same operative procedure in case of LN metastases. When no axillary SLN was visualized on preoperative lymphoscintigraphy, the axilla was nonetheless explored in search for a blue dye containing SLN. When no axillary SLN was identified by either means, axillary LN dissection was performed. Following retrieval of axillary SLNs, surgical exploration for IM SLNs was done. When an IM SLN was visualized on preoperative lymphoscintigraphy, the γ-ray detection probe was used to guide a parasternal intercostal incision through the best-suited intercostal space to harvest the visualized node. A partial rib resection was not routinely done for sentinel IM nodes that were localized behind the ribs.
In addition to frozen section analysis of the axillary SLNs, all collected SLNs were formalin-fixed, paraffin embedded and cut at five levels of 250 μm. Pathological evaluation followed hematoxylin-eosin and immunohistochemical cytokeratin-8 staining. The presence of axillary and IM LN metastases was classified according to the 2002 version of the UICC-TNM-classification.1
To assess the additional operative time of IM SLN biopsy we compared the time between incision and skin closure needed for lumpectomy and mastectomy with and without IM SLN biopsy. We selected four groups of ten patients: those who underwent lumpectomy with or without IM SLN biopsy and those who had had mastectomy with or without IM SLN biopsy. In all patients the operative procedure had started with axillary SLN biopsy. We only evaluated patients who did not have axillary dissection to avoid the potential bias of extra time awaiting the result of frozen section analysis. For each category we selected the last ten patients to avoid the learning curve effect.
To evaluate the impact of IM LN metastases on subsequent systemic treatment we assessed the proposed adjuvant treatment strategy in the absence and presence of metastases in these nodes, applying the Dutch national guidelines for treatment of breast cancer (version 2005, http//www.oncoline.nl, summarized in Table 1). Locoregional radiotherapy, comprising the affected breast and/or thoracic wall, and the ipsilateral axillary, periclavicular and parasternal fields was indicated in patients with four or more metastatic axillary LNs. In patients with IM LN metastases with 0–3 tumor-positive axillary LNs a parasternal irradiation was given: in combination with irradiation of the breast in patients who had breast-conserving therapy or as parasternal radiotherapy following mastectomy.
TABLE 1.Indications for adjuvant chemo- and hormonal systemic therapy according to the Dutch national guidelines 2005Axillary lymph node metastasesPrimary tumor characteristicsTumor >3 cmTumor >2 cm and BR grade IITumor >1 cm and BR grade IIIOther conditionsAge <35 years: always systemic therapy 60-69 years: chemotherapy when ER- or ≥4 axillary lymph node metastases ≥70 years: no chemotherapyER statusHormonal therapy for the aforementioned indications at all ages if the tumor is ER receptor positive.BR, Bloom-Richardson; ER, estrogen receptor.
Chi-square analysis was performed to evaluate differences in IM SLN visualization rates between groups of patients with various clinicopathological variables and to explore the relation between IM and axillary LN metastases. The ANOVA test was used to explore the relation between age and the visualization of IM SLNs, as well as for the analysis of operative time differences.
RESULTS
The median age of the 506 patients was 60 years (range 24–92 years) and there were three male patients (0.8%). The distribution of primary tumor characteristics is summarized in Table 2.
TABLE 2.Comparison of characteristics of patients who had visualized internal mammary sentinel lymph nodes (IM SLNs) versus those who had not visualized IM SLNs on preoperative lymphoscintigraphyAll patientsIM SLN visualizedIM SLN not visualizedPn = 506n = 109n = 397Median age (years) 60 (range 24–92) 57 (range 30–91)61 (range 24–92)0.016*Gender0.36  Male 3(0.6)0(0)3(0.8)  Female 503(99.4)109(100)394(99.2)T-stage0.47  T1316(62.5)73(67.0)243(61.2)  T2184(36.4)36(33.0) 148(37.3)  T34(0.8)0(0)4(1.0)  Tx2(0.4)0(0)2(0.5)Tumor localization <0.001  Cranial47(9.3)11(10.1)36(9.1)  Craniolateral230(45.5)30(27.5)200(50.4)  Lateral25(4.9)2(1.8)23(5.8)  Caudolateral40(7.9)10(9.2)30(7.6)  Caudal18(3.6)1(3.6)17(4.3)  Caudomedial30(5.9)13(11.9)17(4.3)  Medial11(2.2)4(3.7)7(1.8)  Craniomedial80(15.8)34(31.2)46(11.6)  Central25(4.9)4(3.7)21(5.3)Malignancy grade0.63  BRI206(40.7)48(44.0)158(39.8)  BRII196(38.7)38(34.9)158(39.8)  BRIII104(20.6)23(21.1)81(20.4)Estrogen receptor status 0.24  Positive427(84.4)88(80.7)339(85.4)  Negative79(15.6)21(19.3)58(14.6)Axillary lymph node involvement0.58  No axillary metastases296(58.5)68(62.4)228(57.4)  1–3 lymph node metastases174(34.4)35(32.1)139(35.0)  *4 lymph node metastases 36(7.1)6(5.5)30(7.6)Values in parentheses are percentages.BR, Bloom-Richardson grade.* Age difference between the groups was compared by ANOVA.
Visualization and Retrieval of Sentinel Lymph Nodes in the IM Chain (Fig. 1)
SLNs were visualized on preoperative lymphoscintigraphy in 502 of the 506 patients (99%). Axillary SLNs were visualized in 499 of 502 patients (99%), while one or more IM SLNs were found in 109 patients (22%). Three of the 109 patients with visualized IM SLNs had IM sentinel nodes only (3%); in one of these latter patients, an axillary SN was removed following patent blue dye injection and axillary exploration. Location of the tumor in the craniomedial and caudomedial aspect of the breast was associated with drainage to the IM nodes (P < 0.001), and patients with visualized internal mammary nodes were younger (Table 2).
FIG. 1.Summary of search for internal mammary sentinel lymph nodes (IM SLNs).
IM SLNs could be retrieved through a parasternal intercostal incision in 85 of the 109 patients (78%). Parasternal exploration for IM SLNs added 16 min to the mean operative time in patients who underwent breast conservative therapy (P = 0.02). For patients who underwent mastectomy the operative time was not significantly prolonged (Table 3). Complications (pleural breeching, internal mammary vessel damage) of the intercostal surgical exploration were rare; in particular there were no pneumothoraxes or bleeding complications necessitating drainage or reoperation (Table 4).TABLE 3.Operative time of surgical exploration for internal mammary sentinel lymph nodes (IM SLNs) (n = 40)Mean operative time in minutes (range)PAxillary SLN biopsy/lumpectomy/IM SLN biopsy60(27–76)0.02Axillary SLN biopsy/lumpectomy/no IM SLN biopsy44(32–83)Axillary SLN biopsy/mastectomy/IM SLN biopsy72(42–104)0.8Axillary SLN biopsy/mastecomy/no IM SLN biopsy69(30–100)TABLE 4.Complications of surgical exploration for internal mammary sentinel lymph nodes (IM SLNs) (n = 109)nIntraoperative complicationsPleural breeching4(4)Internal mammary vessel damage6(6)Postoperative complicationsPneumothorax−Bleeding necessitating reoperation−Values in parentheses are percentages.
IM LN Metastases
Metastases in the IM SLN were observed in 20 of the 85 patients who underwent successful IM SLN exploration (24%), and 42% of the patients with axillary SLNs (210/499). There was a correlation between IM metastases and axillary metastases (P = 0.002). In 16 of the 20 patients with IM LN metastases, the axillary SLN contained metastases too (80%). Conversely, IM metastases were found in 1% of patients without axillary metastases (4/297), in 7% of patients with 1–3 axillary metastases (13/174) and in 8 % of patients with ≥4 axillary metastases (3/36).
Clinical Implications of Metastases in the IM SLN (Table 5)
Adjuvant systemic therapy was already indicated in 14 of the 20 patients with IM metastases due to concomitant axillary LN metastases or unfavorable primary tumor characteristics: ten patients would be candidates for postoperative chemotherapy and 14 would receive hormonal treatment. Based on tumor-positive IM SLNs six additional patients would receive systemic treatment. This proportion reflects 7% of the patients in whom IM SLNs were removed. In four patients chemotherapy was indicated and in five patients hormonal therapy.
TABLE 5.Clinical postsurgical implications of internal mammary lymph nodes IM LN metastases (n = 20)No.Tumor characteristicsAxillary metastasesPostsurgical treatmentTreatment changed due to IM metastasesIM SLN not consideredIM SLN consideredSize (cm)Grade (BR)ER(n)CTHTRTCTHTRT Axilla N4+/age<701) 63, BCT1.5I+7++LR++LRNo2) 45, BCT1.6III+4++LR++LRNo3) 47, mastectomy1.8I+4++LR++LRNoAxilla N1-3+/age<704) 39, mastectomy2.5II+3++BCT++BCT+PSRT5) 46, BCT3.5III−3+−BCT+−BCT+PSRT6) 58, BCT1.8II+2++BCT++BCT+PSRT7) 45, BCT2.4I+2++BCT++BCT+PSRTAxilla N1-N1a or unfavorable primary tumor characteristics/age<708) 43, BCT1.8I+1mi−−BCT++BCT+PSCT/HT/RT9) 54, BCT2.2II+1mi++BCT++BCT+PSRT10) 66, BCT2.5II+1mi−+BCT−+BCT+PSRT11) 50, BCT2.5III+1mi++BCT++BCT+PSRT12) 42, mastectomy3.0I+1++No++PSRTAxilla N0/favorable tumor characteristics/age<7012) 54, BCT2.1I+0−−BCT++BCT+PSCT/HT/RT14) 60, BCT2.5I+0−−BCT++BCT+PS CT/HT/RT15) 67, mastectomy0.8I+0−−No−+PSHT/RT16) 61, BCT1.1I−0−−BCT+−BCT+PSCT/RTAge>7017) 82, mastectomy2.8II+2−+No−+PS?RT?18) 71, BCT2.1II+1−+BCT−+BCT+PSRT19) 72, BCT2.1II+1mi−+BCT−+BCT+PSRT20) 85, mastectomy0.9I+1mi−−No−+PS?HT/RT?BCT, breast-conserving therapy; BR, Bloom-Richardson grade; ER, estrogen receptor status; Nax+, number of positive axillary lymph nodes; IM SLN, internal mammary sentinel lymph node; ST, systemic therapy; CT, chemotherapy; HT, hormonal therapy; RT, radiotherapy; PS, parasternal radiotherapy; 1mi, micrometastases.
In 3 of the 20 patients axillary tumor load (≥4 tumor-positive lymph nodes) was a reason for locoregional radiotherapy including the IM lymphatic chain, leaving 17 patients in whom the radiotherapy field was adjusted because of metastases in the IM SLN. These 17 patients in whom the radiotherapeutic strategy was changed made up 20% of the patients in whom IM sentinel nodes were visualized.
Conversely, there were three patients with ≥4 axillary metastases and IM SLNs without metastases, and in these patients parasternal irradiation was omitted. In addition, parasternal irradiation could also be omitted in 30 patients with ≥4 axillary metastases who had no IM lymphatic drainage on preoperative lymphoscintigraphy.
DISCUSSION
In the present study, SLNs in the IM chain were visualized in approximately one-fifth of the patients who underwent surgery for primary breast cancer. Retrieving these nodes by parasternal intercostal exploration was feasible in the majority of patients. One-fifth of the retrieved IM LNs contained metastases and radiation treatment was adjusted in most of these patients.
In this prospective cohort of patients who underwent SLN biopsy for clinically T1-2N0 breast cancer, IM SLNs were seen on the preoperative lymphoscintigraphy in 22% of the patients.5,6,8,10,11 The visualization rate in our study was rather high and the likely result of the tracer injection technique (Table 6). We used a combination of a parenchymatous peritumoral and a subcutaneous injection technique of the 99mTc nanocolloid, and this technique is associated with a higher visualization rate of IM SLNs than the subcutaneous or periareolar injection of the radiotracer.5–7,12,13 Apart from the effect of the injection technique we observed a higher frequency of lymphatic drainage to the IM LNs in patients with cranio- and caudomedially located tumors, as reported by others,8,14,15 as well as an effect of age: IM SLNs were more common in young patients.
TABLE 6.Visualization and surgical extirpation rate of internal mammary sentinel lymph nodes (IM SLNs) in breast cancer patientsAuthorYear nMethod of tracer injectionVisualized IM SLNs (%)Surgically removed IM SLNs (%)*Madsen et al.2006506PT and SC2278Leidenius et al.102005984IT1488Paredes et al.62005383SC, later IT/PT 0–1773Farrus et al.52004225SC, later IT/PT 11–1769Estourgie et al.82003681IT2287van der Ent et al.112002256PT2563* Proportion of the visualized IM SLNs. SC, subcutaneous; PT, peritumoral; IT, intratumoral.
Retrieving SLNs from the IM chain does not appear to be very troublesome. Approximately 15 min extraoperative time is needed during breast conservative surgery, while extra time is negligible in patients who undergo mastectomy. Although the success rate was lower than for SLNs in the axilla, 78% of the visualized IM nodes could be harvested by the described parasternal intercostal exploration. Others reported similar ‘surgical identification’ rates (69–88%, see Table 6).5,6,8,10,11 It can be difficult to retrieve the usually very small IM SLNs when they are localized directly behind one of the ribs. ‘Blind’ retrocostal dissection is not without risks, and we consider a rib resection not justified as long as the place of parasternal lymph node exploration is not well defined. Although the pleural cavity was breeched occasionally during the procedure and a number of patients had postoperative hematomas, postoperative drainage of a pneumothorax or a reoperation for a bleeding complication of the parasternal wound was never necessary. Other studies also reported low morbidity rates.8,9,11,16,17
In the present study IM LN metastases were found in 24% of the patients who underwent surgical extirpation of these SLNs, while others usually reported lower rates of metastases containing IM SLNs (9–26%).8–11,18 In patients with IM metastases axillary metastases were common: 80% had concomitant axillary metastases.19 Conversely, axillary metastases were accompanied by IM metastases in 8% of the cases. Isolated IM metastases were rare affecting 5% of the patients with harvested IM SLNs and approximately 1% of patients without axillary LN metastases.8,10,19
Successful exploration of SLNs from the IM chain had a substantial impact on subsequent radiotherapeutic treatment. In 20% of these patients adjustment of radiotherapy was considered necessary. Although randomized data about the use of radiotherapy in patients with IM metastases are lacking, adding a parasternal irradiation in these patients appears conceivable. After all radiotherapy does affect locoregional control in other high-risk patient groups such as patients with ≥4 axillary metastases and node-negative patients with young age, poor tumor differentation and large tumor size.20 We consider patients with IM metastases at risk for locoregional recurrence and therefore feel that parasternal irradiation is indicated in patients with IM metastases. Conversely, absence of IM lymphatic drainage or metastases in retrieved IM SLNs justified omission of parasternal radiotherapy in 7% of all the patients.
Systemic treatment strategy was rarely influenced by IM metastases. Due to axillary metastases and unfavorable primary tumor characteristics, half of the patients already would have received chemotherapy and even more would have had hormonal therapy. In the remaining group of patients, old age and negative estrogen receptor status further limited the proportion of patients that would receive adjuvant systemic therapy based on IM LN metastases. However, since prognosis of patients with both axillary and IM metastases is poor when compared with axillary or IM metastases alone,3 patients with IM and axillary metastases might need different chemotherapy schedules. That would increase the proportion of patients in whom adjuvant systemic treatment would be adjusted.
Is IM SLN biopsy worthwhile? One may argue that more extensive radiotherapy was indicated in less than 5% of all patients and additional systemic treatment in only 1%, and thus consider IM SLN biopsy hardly “worth the effort.”10 We feel that the group of patients with visualized SLNs should be taken as a reference, and that the proportion of patients in whom less radiotherapy was given should also be taken into account. As a consequence, we do consider the clinical implications of IM SLN biopsy substantial.
In conclusion, lymphatic drainage of breast cancer to IM LNs is a common feature and retrieving these nodes is relatively easy. The clinical impact of metastases in IM lymph nodes is substantial and justifies surgical exploration for these nodes. We advocate routine parasternal intercostal exploration for IM SLNs whenever preoperative lymphoscintigraphy visualizes IM SLNs. For that purpose we also advocate the (additional) intraparenchymatous tracer injection to optimize the visualization of IM SLNs.

internal mammary lymph nodes
breast cancer
sentinel node
metastasis