Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease.
Background
Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease.Methods From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000.
Methods
From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000.Results Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy (Relative Risk 0.14, 95%CI 0.00 to 0.80, p = 0.041). Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected.
Results
Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy (Relative Risk 0.14, 95%CI 0.00 to 0.80, p = 0.041). Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected.Conclusions This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size.
Conclusions
This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size.

Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation [ 1 ]. ROP remains as one of the most frequent cause of blindness in children, in particular in countries with infant mortality rates between 10 and 60/1000 [ 2 , 3 ]. Among 177 students attending schools for children with visual impairment in the city were this study was conducted, 107 (60.5%) had ROP [ 4 ]. The incidence of both any acute ROP, and of the more severe stages, varies inversely with gestational age at birth. ROP is unusual (except in the mildest forms) in infants of greater than 31 weeks gestation, and severe complications such as retinal detachment occur in less than one half of one percent of infants greater than 31 weeks gestation. However, more than 80% of infants less than 28 weeks gestation develop some ROP, and around 10% develop "threshold ROP. In threshold ROP more that 40% of the cases progresses to retina folds or detachment, with its consequent blindness. In this stage, ablative surgery (cryotherapy or laser photocoagulation) to the peripheral avascular retina is recommended to reduce the risk of disease progression to retinal detachment [ 5 - 7 ]. Pre-threshold stage has been linked to bad results in the visual function: reduction of visual acuity, short-sightedness, amblyopic, etc. [ 8 , 9 ]. A number of strategies have been developed to try to diminish the progress of ROP, but with limited success. These strategies include antioxidants such as vitamin E [ 10 ], D penicillamine [ 11 ] and allopurinol [ 12 ], reduction of exposure to light [ 13 ] and supplementation with oxygen [ 14 ]. The active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive [ 15 - 17 ]. In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin [ 18 ], dexamethasone [ 19 ], rofecoxib [ 20 ], and bucillamine [ 21 ], and increased activity of cyclooxygenase 2 (COX2) was also demonstrated in vessels of neo-proliferation in retina and its inhibition decreases the neovascularization in 37% [ 20 ]. Ketorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses [ 22 - 24 ]. Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure [ 24 ]. In addition it is effective in uveitis induced by tumors necrosis factor (TNF) [ 25 ]. Ketorolac is unquantifiable in plasma when administered in ophthalmic drops [ 24 ]. Ketorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning [ 26 - 28 ]. The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia [ 24 , 29 ]. On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects.
Background
Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation [ 1 ]. ROP remains as one of the most frequent cause of blindness in children, in particular in countries with infant mortality rates between 10 and 60/1000 [ 2 , 3 ]. Among 177 students attending schools for children with visual impairment in the city were this study was conducted, 107 (60.5%) had ROP [ 4 ]. The incidence of both any acute ROP, and of the more severe stages, varies inversely with gestational age at birth. ROP is unusual (except in the mildest forms) in infants of greater than 31 weeks gestation, and severe complications such as retinal detachment occur in less than one half of one percent of infants greater than 31 weeks gestation. However, more than 80% of infants less than 28 weeks gestation develop some ROP, and around 10% develop "threshold ROP. In threshold ROP more that 40% of the cases progresses to retina folds or detachment, with its consequent blindness. In this stage, ablative surgery (cryotherapy or laser photocoagulation) to the peripheral avascular retina is recommended to reduce the risk of disease progression to retinal detachment [ 5 - 7 ]. Pre-threshold stage has been linked to bad results in the visual function: reduction of visual acuity, short-sightedness, amblyopic, etc. [ 8 , 9 ]. A number of strategies have been developed to try to diminish the progress of ROP, but with limited success. These strategies include antioxidants such as vitamin E [ 10 ], D penicillamine [ 11 ] and allopurinol [ 12 ], reduction of exposure to light [ 13 ] and supplementation with oxygen [ 14 ].The active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive [ 15 - 17 ].In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin [ 18 ], dexamethasone [ 19 ], rofecoxib [ 20 ], and bucillamine [ 21 ], and increased activity of cyclooxygenase 2 (COX2) was also demonstrated in vessels of neo-proliferation in retina and its inhibition decreases the neovascularization in 37% [ 20 ].Ketorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses [ 22 - 24 ].Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure [ 24 ]. In addition it is effective in uveitis induced by tumors necrosis factor (TNF) [ 25 ]. Ketorolac is unquantifiable in plasma when administered in ophthalmic drops [ 24 ].Ketorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning [ 26 - 28 ].The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia [ 24 , 29 ].On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects.Methods This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac. There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP [ 30 ] was used to define the stages of the disease and verified by more than one observer. During the period in which ketorolac tromethamine was used, when risk signs for ROP were identified (zone I incomplete vascularization, vessels only in zone of transition I-II or anomalous ramification and equatorial incurvation of vessels in the avascular – vascular junction) [ 31 ] treatment was started with a drop of ketorolac tromethamine (0.25 mgrs.) every 8 hours in each eye. The treatment continued until they presented signs of threshold ROP and cryotherapy was indicated, or till the resolution of the condition. Parents of these children gave their consent so that their children could receive treatment. The variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed. Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates [ 32 ]. Statistical analysis was carried out using the statistical software SPSS version 8.0, Stats Direct, and STATA version 8.0.
Methods
This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac.There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP [ 30 ] was used to define the stages of the disease and verified by more than one observer.During the period in which ketorolac tromethamine was used, when risk signs for ROP were identified (zone I incomplete vascularization, vessels only in zone of transition I-II or anomalous ramification and equatorial incurvation of vessels in the avascular – vascular junction) [ 31 ] treatment was started with a drop of ketorolac tromethamine (0.25 mgrs.) every 8 hours in each eye. The treatment continued until they presented signs of threshold ROP and cryotherapy was indicated, or till the resolution of the condition. Parents of these children gave their consent so that their children could receive treatment.The variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed.Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates [ 32 ]. Statistical analysis was carried out using the statistical software SPSS version 8.0, Stats Direct, and STATA version 8.0.Results During the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups (see table  1 ). Table  1  presents the characteristic of those alive at discharge. Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing Enterocolitis (see table  1 ). The duration of oxygen therapy was significantly longer in the control group (see table  1 ). Table 1 Characteristics of newborns treated with ketorolac and historic controls. Historic controls Ketorolac p** Year 1999 – 2000 Year 2001–2002 N = 35† N = 43† n(%) n(%) Dead before discharge* 18 (34.0) 16 (27.1) 0.54 Birthweight (g) 0.28  <= 750 4 (11.4) 8 (18.6)  751–1000 13 (37.1) 9 (20.9)  1001–1250 18 (51.4) 26 (60.59 Apgar Score < 6 at 5 minutes 10 (28.6) 11 (25.5) 0.80 Oxygen (days) 0.01  0–10 9 (25.7) 21 (48.8)  11–27 10 (28.6) 15 (34.9)  >= 28 16 (45.7) 7 (16.3) Sepsis 16 (45.7) 24 (55.8) 0.50 Peri–intraventricular hemorrhage > grade III 8 (22.9) 6 (14.0) 0.38 Necrotizing Enterocolitis >= II degree 5 (14.3) 9 (20.9) 0.56 † Excludes newborns that died before discharge. * N = 53 in historic controls group and N = 59 in ketorolac group, that includes all eligible live newborn during the study period. ** P-value Fisher exact test. In the ketorolac group 45 children were alive at the first ophthalmic control, two died before discharge due to late sepsis, so 43 received treatment with ketorolac. Nineteen children were discharged from NICU with treatment indication, and continued with ketorolac up to 44 weeks of gestational age, when the ophthalmologists considered that the risk for developing ROP was very small. The incidence of threshold ROP in newborns treated with ketorolac was significantly lower (Relative Risk Reduction 86%) than in the control group (see table  2 ). Adjusting for duration of oxygen therapy did not significantly change these results (see table  2 ). Further exploration of this relationship showed that duration of oxygen therapy equal or higher than 28 days was not associated with severe ROP in this data (Relative Risk 1.04, 95%CI 0.25 to 4.51, p = 0.28). This finding explains why duration of oxygen therapy does not act as a confounder. Hemorrhages were not observed in the vitreous after treatment with ketorolac. In four cases hemorrhages in the vitreous were already present at the beginning of therapy and they disappeared after 14 days of treatment. No signs of local intolerance, or conjunctival infection were observed. We did not find hemorrhages in other organs attributable to the drug, or signs of renal failure. Treatment was not suspended in none of the cases and all preterm babies received ketorolac until its interruption due to the resolution of ROP or the indication of cryotherapy. Table 2 Incidence of threshold retinopathy of prematurity in newborns treated with ketorolac and historic controls. Only those alive at discharged included in the analyses. Historic Controls Ketorolac Relative Risk (95%CI) P-value n/N(%) n/N(%) Crude Adjusted* Threshold retinopathy of prematurity 6/35 (17.1) 1/43 (2.3) 0.14 (0.00 to 0.80) 0.041** 0.12 (0.02 to 0.92) 0.04 * Adjusted by oxygen administration using robust Poisson Regression. **Fisher exact test
Results
During the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups (see table  1 ). Table  1  presents the characteristic of those alive at discharge. Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing Enterocolitis (see table  1 ). The duration of oxygen therapy was significantly longer in the control group (see table  1 ).Characteristics of newborns treated with ketorolac and historic controls.Historic controlsKetorolacp**Year 1999 – 2000Year 2001–2002N = 35†N = 43†n(%)n(%)Dead before discharge*Birthweight (g)Apgar Score < 6 at 5 minutesOxygen (days)SepsisPeri–intraventricular hemorrhage > grade IIINecrotizing Enterocolitis >= II degree† Excludes newborns that died before discharge. * N = 53 in historic controls group and N = 59 in ketorolac group, that includes all eligible live newborn during the study period. ** P-value Fisher exact test.In the ketorolac group 45 children were alive at the first ophthalmic control, two died before discharge due to late sepsis, so 43 received treatment with ketorolac. Nineteen children were discharged from NICU with treatment indication, and continued with ketorolac up to 44 weeks of gestational age, when the ophthalmologists considered that the risk for developing ROP was very small.The incidence of threshold ROP in newborns treated with ketorolac was significantly lower (Relative Risk Reduction 86%) than in the control group (see table  2 ). Adjusting for duration of oxygen therapy did not significantly change these results (see table  2 ). Further exploration of this relationship showed that duration of oxygen therapy equal or higher than 28 days was not associated with severe ROP in this data (Relative Risk 1.04, 95%CI 0.25 to 4.51, p = 0.28). This finding explains why duration of oxygen therapy does not act as a confounder. Hemorrhages were not observed in the vitreous after treatment with ketorolac. In four cases hemorrhages in the vitreous were already present at the beginning of therapy and they disappeared after 14 days of treatment. No signs of local intolerance, or conjunctival infection were observed. We did not find hemorrhages in other organs attributable to the drug, or signs of renal failure. Treatment was not suspended in none of the cases and all preterm babies received ketorolac until its interruption due to the resolution of ROP or the indication of cryotherapy.Incidence of threshold retinopathy of prematurity in newborns treated with ketorolac and historic controls. Only those alive at discharged included in the analyses.Historic ControlsKetorolacRelative Risk (95%CI) P-valuen/N(%)n/N(%)CrudeAdjusted*Threshold retinopathy of prematurity* Adjusted by oxygen administration using robust Poisson Regression. **Fisher exact testDiscussion In this preliminary study we have shown that the incidence of severe ROP was significantly lower in very preterm newborns treated with ketorolac, compared with historic controls not receiving such treatment. These results suggest that administration of ketorolac as an ophthalmic solution might be an effective preventive strategy in patient at risk of developing severe ROP. The study was conducted on non-concurrent patient groups, and changes in the subjects risk profile or quality of care between the two study periods might have an impact in the risk of developing severe ROP. The magnitude of the effect of ketorolac on the incidence of severe ROP found in this study (from 17% in the control group to 2.3% in the ketorolac group) is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen. The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree [ 33 ], but similar to the one reported by other units in our country and in others countries with similar development [ 34 - 36 ] Ketorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed. The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization [ 17 ]. Recent research work with inhibitors of COX2 shows that anti-inflammatory drugs apparently have an impact on the evolution of ROP [ 20 ]. Neufeld and collaborators found high plasmatic profiles of TNF and other cytokines during the phase of installation of the pre-threshold stage for ROP [ 37 ]. COX has a strong angiogenic action in the normal development of retina. The inhibition of COX2 diminishes the angiogenesis in cancer and rheumatoid arthritis [ 38 ]. The ganglion cells of retina secrete PGs that would interact with angiogenic substances as VEGF and Insulin like growth factor. The neo-vessels express a greater concentration of COX2 and inhibitors of COX2 stop the angiogenesis mediated by VEFG, which is the principal factor involved in the neovascularization and which would be regulated by the PGs secreted by ganglion cells of retina and endothelial cells [ 20 ]. The hyperoxia induces liberation of TNF with a powerful inflammatory action; dexamethasone interferes with TNF production attenuating the manifestations of retinopathy by hyperoxia [ 19 ]. Topical ketorolac is very effective in neutralizing uveitis generated by experimental infection with monoclonal TNF or bacterial endotoxins [ 25 , 39 , 40 ]. ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed [ 41 ], but a preventive measure that could avoid the need of ablation would be much better.
Discussion
In this preliminary study we have shown that the incidence of severe ROP was significantly lower in very preterm newborns treated with ketorolac, compared with historic controls not receiving such treatment. These results suggest that administration of ketorolac as an ophthalmic solution might be an effective preventive strategy in patient at risk of developing severe ROP. The study was conducted on non-concurrent patient groups, and changes in the subjects risk profile or quality of care between the two study periods might have an impact in the risk of developing severe ROP. The magnitude of the effect of ketorolac on the incidence of severe ROP found in this study (from 17% in the control group to 2.3% in the ketorolac group) is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen.The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree [ 33 ], but similar to the one reported by other units in our country and in others countries with similar development [ 34 - 36 ]Ketorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed.The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization [ 17 ]. Recent research work with inhibitors of COX2 shows that anti-inflammatory drugs apparently have an impact on the evolution of ROP [ 20 ]. Neufeld and collaborators found high plasmatic profiles of TNF and other cytokines during the phase of installation of the pre-threshold stage for ROP [ 37 ]. COX has a strong angiogenic action in the normal development of retina. The inhibition of COX2 diminishes the angiogenesis in cancer and rheumatoid arthritis [ 38 ]. The ganglion cells of retina secrete PGs that would interact with angiogenic substances as VEGF and Insulin like growth factor. The neo-vessels express a greater concentration of COX2 and inhibitors of COX2 stop the angiogenesis mediated by VEFG, which is the principal factor involved in the neovascularization and which would be regulated by the PGs secreted by ganglion cells of retina and endothelial cells [ 20 ]. The hyperoxia induces liberation of TNF with a powerful inflammatory action; dexamethasone interferes with TNF production attenuating the manifestations of retinopathy by hyperoxia [ 19 ]. Topical ketorolac is very effective in neutralizing uveitis generated by experimental infection with monoclonal TNF or bacterial endotoxins [ 25 , 39 , 40 ].ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed [ 41 ], but a preventive measure that could avoid the need of ablation would be much better.Conclusions This preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question.
Conclusions
This preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question.Competing interest None declared.
Competing interest
None declared.Authors' contributions MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript.
Authors' contributions
MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background We studied the expression of  DMBT1  (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle.
Background
We studied the expression of  DMBT1  (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle.DMBT1Methods Sections from 17 benign lesions and 55 carcinomas were immunostained with anti  DMBT1  antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions.  DMBT1  gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients.
Methods
Sections from 17 benign lesions and 55 carcinomas were immunostained with anti  DMBT1  antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions.  DMBT1  gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients.DMBT1DMBT1Results Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments.  DMBT1  protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive.
Results
Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments.  DMBT1  protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive.DMBT1Conclusions The redistribution and up-regulation of  DMBT1  in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.
Conclusions
The redistribution and up-regulation of  DMBT1  in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.DMBT1

Background DMBT1  (Deleted in Malignant Brain Tumor) at chromosome 10q25.3–26.1, was considered a tumour suppressor gene because frequent deletions and/or lack of expression had been found in malignant tumours of the brain [ 1 - 3 ], the gastrointestinal tract [ 4 , 5 ], and the lung [ 6 , 7 ]. However, recent evidence has shown that  DMBT1  is not a typical tumour suppressor gene because it is up-regulated in normal or inflamed epithelium adjacent to lung carcinomas as well as in some primary lung cancers and glioblastomas [ 8 ]. It was also shown that mutations in combination with loss of heterozygosity do not play a role in  DMBT1  inactivation in many tumours including breast cancer [ 9 , 10 ] DMBT1  is a multi-functional protein related to the Mac-2 binding protein and mucins that have a protective function and mediate cell-extracellular matrix interactions [ 8 , 11 ]. DMBT1 GP340 (glycoprotein-340) and DMBT1 SAG  (Salivary Agglutinin) are  DMBT1  variants, present in the respiratory tract and the oral cavity, respectively, that have been linked to innate host defense and epithelial regeneration [ 3 , 12 - 16 ]. In the respiratory tract, DMBT1 GP340 is involved in innate host defence by its interaction with the lectins surfactant protein D and A (Sp-D, Sp-A) and by its ability to stimulate alveolar macrophage migration [ 12 , 13 , 17 ]. Another lectin, mannan binding protein (MBP), has been shown to have an anti tumour effect against gliomas and colorectal carcinomas  in vitro  and  in vivo  [ 18 , 19 ]. While this anti tumor property remains to be demonstrated for Sp-A and Sp-D, we have previously reported the presence of surfactant protein A (Sp-A) in the ductal epithelium of the normal breast and its variable expression in mammary carcinomas. We found that in the normal ductal epithelium Sp-A immunoreactivity is present on the luminal side, whereas in carcinomas it is present both on the luminal side as well as on the baso-lateral surfaces. In carcinomas, the Sp-A immunoreactivity is not only redistributed but also decreased as the histological tumour grade increases [ 20 ]. Remarkably, this resembles the changes of  DMBT1  expression and localisation observed in other tumor types originating from mono-layered epithelia such as primary esophageal adeno-carcinomas and lung carcinomas [ 5 , 8 ]. Hensin, the rabbit homologue of DMBT1, triggers epithelial terminal differentiation by mediating cell-extracellular matrix (ECM) interactions, and this may apply also to  DMBT1  [ 3 , 21 - 24 ]. CRP-ductin,  DMBT1  homologue in the mouse, and a novel homologue in the rat ( DMBT1 4,7 kb ), have been linked to initiation of cell proliferation, differentiation and repair [ 25 , 26 ]. It has been proposed that translocation of  DMBT1  to the ECM triggers cellular differentiation, whereas a loss of expression of  DMBT1  favours tumor cell growth [ 8 ]. These findings led us to explore whether similar principles apply to breast epithelium. Here we examined the expression of  DMBT1  in the morphologically normal, hyperplastic, and neoplastic breast epithelium. We also examined in morphologically normal epithelium a possible association of  DMBT1  expression with that of an early proliferative marker such as MCM5 (Minichromosome Maintenance protein 5). MCM5 is one of the 6 pre-replicative complex proteins (MCM 2–7) [ 27 ] and its expression was compared with that of Ki 67 proliferation index.
Background
DMBT1  (Deleted in Malignant Brain Tumor) at chromosome 10q25.3–26.1, was considered a tumour suppressor gene because frequent deletions and/or lack of expression had been found in malignant tumours of the brain [ 1 - 3 ], the gastrointestinal tract [ 4 , 5 ], and the lung [ 6 , 7 ].DMBT1However, recent evidence has shown that  DMBT1  is not a typical tumour suppressor gene because it is up-regulated in normal or inflamed epithelium adjacent to lung carcinomas as well as in some primary lung cancers and glioblastomas [ 8 ]. It was also shown that mutations in combination with loss of heterozygosity do not play a role in  DMBT1  inactivation in many tumours including breast cancer [ 9 , 10 ]DMBT1DMBT1DMBT1  is a multi-functional protein related to the Mac-2 binding protein and mucins that have a protective function and mediate cell-extracellular matrix interactions [ 8 , 11 ]. DMBT1 GP340 (glycoprotein-340) and DMBT1 SAG  (Salivary Agglutinin) are  DMBT1  variants, present in the respiratory tract and the oral cavity, respectively, that have been linked to innate host defense and epithelial regeneration [ 3 , 12 - 16 ]. In the respiratory tract, DMBT1 GP340 is involved in innate host defence by its interaction with the lectins surfactant protein D and A (Sp-D, Sp-A) and by its ability to stimulate alveolar macrophage migration [ 12 , 13 , 17 ]. Another lectin, mannan binding protein (MBP), has been shown to have an anti tumour effect against gliomas and colorectal carcinomas  in vitro  and  in vivo  [ 18 , 19 ]. While this anti tumor property remains to be demonstrated for Sp-A and Sp-D, we have previously reported the presence of surfactant protein A (Sp-A) in the ductal epithelium of the normal breast and its variable expression in mammary carcinomas. We found that in the normal ductal epithelium Sp-A immunoreactivity is present on the luminal side, whereas in carcinomas it is present both on the luminal side as well as on the baso-lateral surfaces. In carcinomas, the Sp-A immunoreactivity is not only redistributed but also decreased as the histological tumour grade increases [ 20 ]. Remarkably, this resembles the changes of  DMBT1  expression and localisation observed in other tumor types originating from mono-layered epithelia such as primary esophageal adeno-carcinomas and lung carcinomas [ 5 , 8 ].DMBT1DMBT1in vitroin vivoDMBT1Hensin, the rabbit homologue of DMBT1, triggers epithelial terminal differentiation by mediating cell-extracellular matrix (ECM) interactions, and this may apply also to  DMBT1  [ 3 , 21 - 24 ]. CRP-ductin,  DMBT1  homologue in the mouse, and a novel homologue in the rat ( DMBT1 4,7 kb ), have been linked to initiation of cell proliferation, differentiation and repair [ 25 , 26 ]. It has been proposed that translocation of  DMBT1  to the ECM triggers cellular differentiation, whereas a loss of expression of  DMBT1  favours tumor cell growth [ 8 ].DMBT1DMBT1DMBT1 4,7 kbDMBT1DMBT1These findings led us to explore whether similar principles apply to breast epithelium. Here we examined the expression of  DMBT1  in the morphologically normal, hyperplastic, and neoplastic breast epithelium. We also examined in morphologically normal epithelium a possible association of  DMBT1  expression with that of an early proliferative marker such as MCM5 (Minichromosome Maintenance protein 5). MCM5 is one of the 6 pre-replicative complex proteins (MCM 2–7) [ 27 ] and its expression was compared with that of Ki 67 proliferation index.DMBT1DMBT1Methods Tissue and cell-lines From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma  in situ  (DCIS) and 45 cases of infiltrating carcinoma (IC) (36 ductal, 2 lobular, 2 mixed lobular and ductal, 2 mucinous, 1 apocrine, 1 cribriform, 1 papillary). RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended. Immunohistochemistry Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody [ 3 ] as well as anti Ki67 Mib1 (Dako Cytomation DK). Double labelling with anti- DMBT1  and anti MCM5 monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle upon Tyne, U.K.) was performed on 17 benign lesions and a subset of 19  in situ  and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti- DMBT1  and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes.  DMBT1  and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [ 28 ], briefly: 1) first antigen retrieval 6 minutes in 0.5 M EDTA pH8 in MW oven three times, followed by 1 h incubation with anti DMBT1h12. The reaction was developed with alkaline phosphatase using Fucsin + (Dako) as chromogen. 2) same antigen retrieval followed by over-night incubation with anti MCM5 Ab and development with peroxydase and diaminobenzidine (DAB) as chromogen. All slides were incubated in an automated slides stainer (Biogenex, S.Ramon, CA, U.S.A.); reaction products were visualized by En Vision product (Dako). Negative and positive controls were added in each experiment. Three investigators (AM, PB, PGN) evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands. RNA extraction Total RNA was extracted from breast specimens using Trizol reagent, (Invitrogen s.r.l., S. Giuliano Milanese, Italy) according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry. cDNA synthesis Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer (50 mM KCl, 15 mM Tris-HCl, pH 8.3), 500 μM each deoxynucleotide triphosphate, 5 mM MgCl 2 , 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min. PCR amplification Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgCl 2 , 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of  DMBT1  was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [ 3 ]. The specificity of the PCR product was confirmed by sequencing with BigDyeTM terminator chemistry using the genetic analyser ABI Prism 310 (Applied Biosystems), β-2-microglobulin (as control for sample integrity) was amplified in the amplification mixture described above and using the following primers: 5'-TCTGGGTTTCATCCATCCGA-3' and 5'-CCCCAAATTCTAAGCAGAGTATGTAA-3'. The thermal cycling parameters were: initial step of 10 min at 94°C, 39 cycles at 94°C for 30 sec, 58°C for 30 sec, 72 °C for 30 sec, and a final step for 5 min at 72°C. All the PCR products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under UV transillumination. Amplification product of 602 bp (β-2-microglobulin) and 435 bp ( DMBT1 ) were obtained. Data analysis Statistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant. T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions.
Methods
Tissue and cell-lines From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma  in situ  (DCIS) and 45 cases of infiltrating carcinoma (IC) (36 ductal, 2 lobular, 2 mixed lobular and ductal, 2 mucinous, 1 apocrine, 1 cribriform, 1 papillary). RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended.
Tissue and cell-lines
From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma  in situ  (DCIS) and 45 cases of infiltrating carcinoma (IC) (36 ductal, 2 lobular, 2 mixed lobular and ductal, 2 mucinous, 1 apocrine, 1 cribriform, 1 papillary).in situRT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended.Immunohistochemistry Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody [ 3 ] as well as anti Ki67 Mib1 (Dako Cytomation DK). Double labelling with anti- DMBT1  and anti MCM5 monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle upon Tyne, U.K.) was performed on 17 benign lesions and a subset of 19  in situ  and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti- DMBT1  and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes.  DMBT1  and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [ 28 ], briefly: 1) first antigen retrieval 6 minutes in 0.5 M EDTA pH8 in MW oven three times, followed by 1 h incubation with anti DMBT1h12. The reaction was developed with alkaline phosphatase using Fucsin + (Dako) as chromogen. 2) same antigen retrieval followed by over-night incubation with anti MCM5 Ab and development with peroxydase and diaminobenzidine (DAB) as chromogen. All slides were incubated in an automated slides stainer (Biogenex, S.Ramon, CA, U.S.A.); reaction products were visualized by En Vision product (Dako). Negative and positive controls were added in each experiment. Three investigators (AM, PB, PGN) evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands.
Immunohistochemistry
Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody [ 3 ] as well as anti Ki67 Mib1 (Dako Cytomation DK). Double labelling with anti- DMBT1  and anti MCM5 monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle upon Tyne, U.K.) was performed on 17 benign lesions and a subset of 19  in situ  and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti- DMBT1  and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes.  DMBT1  and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [ 28 ], briefly: 1) first antigen retrieval 6 minutes in 0.5 M EDTA pH8 in MW oven three times, followed by 1 h incubation with anti DMBT1h12. The reaction was developed with alkaline phosphatase using Fucsin + (Dako) as chromogen. 2) same antigen retrieval followed by over-night incubation with anti MCM5 Ab and development with peroxydase and diaminobenzidine (DAB) as chromogen. All slides were incubated in an automated slides stainer (Biogenex, S.Ramon, CA, U.S.A.); reaction products were visualized by En Vision product (Dako). Negative and positive controls were added in each experiment.DMBT1in situDMBT1DMBT1Three investigators (AM, PB, PGN) evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands.RNA extraction Total RNA was extracted from breast specimens using Trizol reagent, (Invitrogen s.r.l., S. Giuliano Milanese, Italy) according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry.
RNA extraction
Total RNA was extracted from breast specimens using Trizol reagent, (Invitrogen s.r.l., S. Giuliano Milanese, Italy) according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry.cDNA synthesis Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer (50 mM KCl, 15 mM Tris-HCl, pH 8.3), 500 μM each deoxynucleotide triphosphate, 5 mM MgCl 2 , 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min.
cDNA synthesis
Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer (50 mM KCl, 15 mM Tris-HCl, pH 8.3), 500 μM each deoxynucleotide triphosphate, 5 mM MgCl 2 , 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min.PCR amplification Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgCl 2 , 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of  DMBT1  was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [ 3 ]. The specificity of the PCR product was confirmed by sequencing with BigDyeTM terminator chemistry using the genetic analyser ABI Prism 310 (Applied Biosystems), β-2-microglobulin (as control for sample integrity) was amplified in the amplification mixture described above and using the following primers: 5'-TCTGGGTTTCATCCATCCGA-3' and 5'-CCCCAAATTCTAAGCAGAGTATGTAA-3'. The thermal cycling parameters were: initial step of 10 min at 94°C, 39 cycles at 94°C for 30 sec, 58°C for 30 sec, 72 °C for 30 sec, and a final step for 5 min at 72°C. All the PCR products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under UV transillumination. Amplification product of 602 bp (β-2-microglobulin) and 435 bp ( DMBT1 ) were obtained.
PCR amplification
Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgCl 2 , 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of  DMBT1  was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [ 3 ]. The specificity of the PCR product was confirmed by sequencing with BigDyeTM terminator chemistry using the genetic analyser ABI Prism 310 (Applied Biosystems), β-2-microglobulin (as control for sample integrity) was amplified in the amplification mixture described above and using the following primers: 5'-TCTGGGTTTCATCCATCCGA-3' and 5'-CCCCAAATTCTAAGCAGAGTATGTAA-3'. The thermal cycling parameters were: initial step of 10 min at 94°C, 39 cycles at 94°C for 30 sec, 58°C for 30 sec, 72 °C for 30 sec, and a final step for 5 min at 72°C. All the PCR products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under UV transillumination. Amplification product of 602 bp (β-2-microglobulin) and 435 bp ( DMBT1 ) were obtained.DMBT1DMBT1Data analysis Statistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant. T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions.
Data analysis
Statistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant.T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions.Results Immunohistochemistry Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface [Figure  1a ]; b) a strong surface decoration of luminal cells [Figure  1b ], c) intra-luminal immuno-reactive material [Figure  1c ], d) multiple, randomly distributed peri-nuclear, irregular dots [Figure  1d ], e) marked supra-nuclear and/or baso-lateral accumulation of reaction product [Figure  1e ] and f) strong diffuse granular intra-cytoplasmic decoration [Figure  1f ]. Morphologically normal ducts showed the pattern of immunopositivity described in section a). In addition to pattern a), the epithelium in benign lesions showed immunoreactivity patterns b) and c). Hyperplastic tissues flanking carcinoma frequently showed depolarization of immuno-reaction of patterns d), e), and f). Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of  DMBT1  at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure  1g ]; in the second case only few cells had cytoplasmic positivity (immunopositivity score = 1) [Figure  1h ] and in the third case only weak granular decoration in a small clusters of cells was present (immunopositivity score = 1). All other carcinomas were completely negative (immunopositivity score = 0) [Figure  1i ]. Down-regulation of  DMBT1  expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma  in situ  (DCIS) (1/10 versus 33/42 normal and/or hyperplastic epithelia; p = 0.0001) and invasive carcinoma (IC) (2/45 versus 33/42 normal and/or hyperplastic epithelia; p < 0.0001). The results of  DMBT1  expression at protein and mRNA level are summarized in table  1 . According to the 4 category scoring system, there was no statistically significant difference of  DMBT1  immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions,  in situ  and invasive carcinomas (Table  2 ). Double immunostaining with DMBTh12 Ab and anti MCM5 Ab revealed a co-expression of the two antigens, respectively, in the cytoplasm and the nucleus of the same cells in morphologically normal breast epithelium (Figure  2a,2b ). Conversely, glands that were negative with anti  DMBT1  were negative also with anti MCM5 antibody (Figure  2b ).  DMBT1  and MCM5 positive or negative glands were morphologically undistinguishable. In comparison to MCM5, very few morphologically normal epithelial cells labelled by Anti  DMBT1  Ab, expressed Ki67 (Figure  3a,3b,3c ). 30–50% of glands were positive with anti MCM5 Ab with a mean number of 4,5 positive cells per positive glandular structure versus less than 10% of positive glands with a mean number of 1,46 positive cell per positive glandular structure with anti Ki 67 Ab. As expected for a pre-replicative marker, anti MCM5 Ab decorated diffusely all benign proliferative and malignant lesions. The percentage of Ki 67 positive cells of carcinomas varied and was assessed as part of the protocol for the surgical pathology diagnosis of breast neoplasia together with other prognostic parameters i.e. hormonal receptors status and Her2 Neu positivity. These data did not correlate with  DMBT1  expression. Expression analysis by RT-PCR In 28/39 cases (72%) RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification (Table  1 ). In two of these discordant cases we could document the presence in the sample of residual normal epithelial cells.  DMBT1  mRNA was detected in both the breast carcinoma cell lines analysed: Hs 578T (Figure  4 ) and T47D (not shown). The RT-PCR positivity of samples did not correlate with histological parameters or with IHC expression of hormonal receptors, proliferation index Ki 67 and Her 2/Neu.
Results
Immunohistochemistry Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface [Figure  1a ]; b) a strong surface decoration of luminal cells [Figure  1b ], c) intra-luminal immuno-reactive material [Figure  1c ], d) multiple, randomly distributed peri-nuclear, irregular dots [Figure  1d ], e) marked supra-nuclear and/or baso-lateral accumulation of reaction product [Figure  1e ] and f) strong diffuse granular intra-cytoplasmic decoration [Figure  1f ]. Morphologically normal ducts showed the pattern of immunopositivity described in section a). In addition to pattern a), the epithelium in benign lesions showed immunoreactivity patterns b) and c). Hyperplastic tissues flanking carcinoma frequently showed depolarization of immuno-reaction of patterns d), e), and f). Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of  DMBT1  at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure  1g ]; in the second case only few cells had cytoplasmic positivity (immunopositivity score = 1) [Figure  1h ] and in the third case only weak granular decoration in a small clusters of cells was present (immunopositivity score = 1). All other carcinomas were completely negative (immunopositivity score = 0) [Figure  1i ]. Down-regulation of  DMBT1  expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma  in situ  (DCIS) (1/10 versus 33/42 normal and/or hyperplastic epithelia; p = 0.0001) and invasive carcinoma (IC) (2/45 versus 33/42 normal and/or hyperplastic epithelia; p < 0.0001). The results of  DMBT1  expression at protein and mRNA level are summarized in table  1 . According to the 4 category scoring system, there was no statistically significant difference of  DMBT1  immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions,  in situ  and invasive carcinomas (Table  2 ). Double immunostaining with DMBTh12 Ab and anti MCM5 Ab revealed a co-expression of the two antigens, respectively, in the cytoplasm and the nucleus of the same cells in morphologically normal breast epithelium (Figure  2a,2b ). Conversely, glands that were negative with anti  DMBT1  were negative also with anti MCM5 antibody (Figure  2b ).  DMBT1  and MCM5 positive or negative glands were morphologically undistinguishable. In comparison to MCM5, very few morphologically normal epithelial cells labelled by Anti  DMBT1  Ab, expressed Ki67 (Figure  3a,3b,3c ). 30–50% of glands were positive with anti MCM5 Ab with a mean number of 4,5 positive cells per positive glandular structure versus less than 10% of positive glands with a mean number of 1,46 positive cell per positive glandular structure with anti Ki 67 Ab. As expected for a pre-replicative marker, anti MCM5 Ab decorated diffusely all benign proliferative and malignant lesions. The percentage of Ki 67 positive cells of carcinomas varied and was assessed as part of the protocol for the surgical pathology diagnosis of breast neoplasia together with other prognostic parameters i.e. hormonal receptors status and Her2 Neu positivity. These data did not correlate with  DMBT1  expression.
Immunohistochemistry
Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface [Figure  1a ]; b) a strong surface decoration of luminal cells [Figure  1b ], c) intra-luminal immuno-reactive material [Figure  1c ], d) multiple, randomly distributed peri-nuclear, irregular dots [Figure  1d ], e) marked supra-nuclear and/or baso-lateral accumulation of reaction product [Figure  1e ] and f) strong diffuse granular intra-cytoplasmic decoration [Figure  1f ]. Morphologically normal ducts showed the pattern of immunopositivity described in section a). In addition to pattern a), the epithelium in benign lesions showed immunoreactivity patterns b) and c). Hyperplastic tissues flanking carcinoma frequently showed depolarization of immuno-reaction of patterns d), e), and f).Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of  DMBT1  at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure  1g ]; in the second case only few cells had cytoplasmic positivity (immunopositivity score = 1) [Figure  1h ] and in the third case only weak granular decoration in a small clusters of cells was present (immunopositivity score = 1). All other carcinomas were completely negative (immunopositivity score = 0) [Figure  1i ]. Down-regulation of  DMBT1  expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma  in situ  (DCIS) (1/10 versus 33/42 normal and/or hyperplastic epithelia; p = 0.0001) and invasive carcinoma (IC) (2/45 versus 33/42 normal and/or hyperplastic epithelia; p < 0.0001).DMBT1DMBT1in situThe results of  DMBT1  expression at protein and mRNA level are summarized in table  1 .DMBT1According to the 4 category scoring system, there was no statistically significant difference of  DMBT1  immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions,  in situ  and invasive carcinomas (Table  2 ).DMBT1in situDouble immunostaining with DMBTh12 Ab and anti MCM5 Ab revealed a co-expression of the two antigens, respectively, in the cytoplasm and the nucleus of the same cells in morphologically normal breast epithelium (Figure  2a,2b ). Conversely, glands that were negative with anti  DMBT1  were negative also with anti MCM5 antibody (Figure  2b ).  DMBT1  and MCM5 positive or negative glands were morphologically undistinguishable. In comparison to MCM5, very few morphologically normal epithelial cells labelled by Anti  DMBT1  Ab, expressed Ki67 (Figure  3a,3b,3c ). 30–50% of glands were positive with anti MCM5 Ab with a mean number of 4,5 positive cells per positive glandular structure versus less than 10% of positive glands with a mean number of 1,46 positive cell per positive glandular structure with anti Ki 67 Ab. As expected for a pre-replicative marker, anti MCM5 Ab decorated diffusely all benign proliferative and malignant lesions. The percentage of Ki 67 positive cells of carcinomas varied and was assessed as part of the protocol for the surgical pathology diagnosis of breast neoplasia together with other prognostic parameters i.e. hormonal receptors status and Her2 Neu positivity. These data did not correlate with  DMBT1  expression.DMBT1DMBT1DMBT1DMBT1Expression analysis by RT-PCR In 28/39 cases (72%) RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification (Table  1 ). In two of these discordant cases we could document the presence in the sample of residual normal epithelial cells.  DMBT1  mRNA was detected in both the breast carcinoma cell lines analysed: Hs 578T (Figure  4 ) and T47D (not shown). The RT-PCR positivity of samples did not correlate with histological parameters or with IHC expression of hormonal receptors, proliferation index Ki 67 and Her 2/Neu.
Expression analysis by RT-PCR
In 28/39 cases (72%) RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification (Table  1 ). In two of these discordant cases we could document the presence in the sample of residual normal epithelial cells.  DMBT1  mRNA was detected in both the breast carcinoma cell lines analysed: Hs 578T (Figure  4 ) and T47D (not shown).DMBT1The RT-PCR positivity of samples did not correlate with histological parameters or with IHC expression of hormonal receptors, proliferation index Ki 67 and Her 2/Neu.Discussion In the present study we investigated  DMBT1  expression by immunohistochemistry and RT-PCR in benign and malignant breast epithelial lesions. By immunohistochemistry we found a variable DMBTh12 positivity in the morphologically normal and hyperplastic epithelium adjacent to benign (88%) and malignant (78%) lesions. Among 55 carcinomas we could detect immunopositivity only in 3 tumours (5%); in one case of infiltrating carcinoma the immunopositivity was widespread with a pattern similar to that seen in hyperplastic lesions near carcinoma; in the other two cases (one carcinoma  in situ  and one infiltrating ductal carcinoma), it was limited to few tumour cells.  DMBT1  expression was detected by RT-PCR in 13 of 35 (37%) infiltrating carcinomas and in 2 breast cancer cell lines. Eleven tumours, that were negative with anti-DMBTh12, revealed PCR-products after RT-PCR amplification. This discordance between IHC and RT-PCR results may be due to residual normal epithelial cells present in the sample. Furthermore other possible reasons of this discrepancy could be: 1) the greater sensitivity of molecular technique like RT-PCR in detecting very low expression levels of  DMBT1  that were below detection by immunohistochemistry, 2) sampling problems i.e. the presence of few positive tumour cells only in the frozen samples or 3) few immunoreactive cells have been missed by microscopic examination and finally 4)  DMBT1  protein could have an altered epitope not recognized by its paratope in the antibody. By either immunohistochemistry or molecular technique we found no correlation between  DMBT1  expression and tumor histotype, grade, hormonal receptors status, proliferation index, Her2 Neu expression or lymph-nodal metastases. In a recent paper on breast carcinogenesis, qualitatively similar data were reported for  DMBT1  in human breast cancer, but an indirect weak correlation between the degree of differentiation of the breast carcinomas and the extent of the immunoreactivity was found [ 10 ]. The discrepancy in the results between our and this study may be due to sampling problems, the size of the sample and heterogeneous expression of  DMBT1  in breast cancer or to other poorly-defined factors such as race, age or hormonal status of the population under study. Mollenhauer et al. point out that  DMBT1  expression may be up-regulated in pathophysiological conditions such as inflammation, liver injury or after carcinogen administration and it is down-regulated in tumors to achieve a growth advantage in a dynamic and complex model. Also according to these authors, the possibility that  DMBT1  loss may be a consequence rather than a primary event in carcinogenesis is unlikely (10). Although the normal mammary gland has been reported to have low  DMBT1  expression by RT-PCR [ 12 ], we found variable DMBTh12 immunopositivity in breast epithelium flanking benign and malignant lesions. Immunopositive epithelial cells in our material appeared either morphologically normal and indistinguishable from immunonegative cells or hypertrophic with abundant cytoplasm and large elongated nuclei oriented perpendicularly to the basement membrane [Figure  1i ]. Moreover, DMBTh12 immunoreactivity was also maintained in hyperplastic epithelium flanking carcinomas. This resembles the  DMBT1  expression patterns observed in the human respiratory tract and in associated inflammation and carcinomas [ 8 ] and suggest that the variable  DMBT1  expression in the normal mammary gland epithelium may result from the necessity of  DMBT1  induction. Indeed, in parallel  in vivo  studies, it was found that  DMBT1  is strongly up-regulated in the mammary gland tissue of the mouse shortly after carcinogen-administration and prior to the onset of histo-pathologically visible symptoms [ 10 ]. It has been reported that Sp-D and Sp-A are ligands of  DMBT1  products [ 29 ]. We recently demonstrated that Sp-A, in breast carcinoma tissues, has a pattern of expression resembling that of its interaction partner [ 20 ]; thus, we hypothesize that co-ordinated induction of both SP-A and  DMBT1  in breast tissue may be part of a protective response as suggested by Kang et al [ 30 ] for  DMBT1  and Sp-D in pulmonary epithelium. While this interaction may be attributed to lumenally secreted  DMBT1 , the depolarisation of the staining patterns indicates that  DMBT1  may also be secreted to other destinations, i.e. the ECM, by the mammary gland epithelial cells. In this regard, the second putative function of  DMBT1 , i.e. triggering of epithelial differentiation, is noteworthy. It may prevent hyperproliferative epithelial cells from out-of control proliferation and progression to the tumor state. This view is supported by two present findings. The correlation between MCM5 and  DMBT1  expression may point to a coupling to cell-cycle processes: basolateral relocation and hyper-expression of  DMBT1  at the ECM may provide cells that are licensed to proliferate with protection from unregulated proliferation. On the other hand loss of  DMBT1  expression in a significant numbers of DCISs and ICs is consistent with the notion that  DMBT1  inactivation may offer growth advantages to neoplastic cells [ 10 ]. The loss of  DMBT1  expression appears to take place early, i.e. already at the time of transition from hyperplastic lesions to carcinoma, as it has been reported in oesophageal squamous cell carcinomas [ 5 ]. The exact mechanisms for  DMBT1  inactivation remain unclear. An inactivation by mutation appears not to be a major mechanism for  DMBT1  inactivation in tumors [ 10 , 31 ] The studies of Munoz and co-workers suggest that epigenetic silencing of either  DMBT1  or genes that regulate  DMBT1  expression takes place in some tumors [ 32 ]. Accordingly, except for direct epigenetic silencing of the  DMBT1  promotor, loss of expression or mutation of  DMBT1 -regulatory genes might represent alternative candidate mechanisms.
Discussion
In the present study we investigated  DMBT1  expression by immunohistochemistry and RT-PCR in benign and malignant breast epithelial lesions. By immunohistochemistry we found a variable DMBTh12 positivity in the morphologically normal and hyperplastic epithelium adjacent to benign (88%) and malignant (78%) lesions. Among 55 carcinomas we could detect immunopositivity only in 3 tumours (5%); in one case of infiltrating carcinoma the immunopositivity was widespread with a pattern similar to that seen in hyperplastic lesions near carcinoma; in the other two cases (one carcinoma  in situ  and one infiltrating ductal carcinoma), it was limited to few tumour cells.  DMBT1  expression was detected by RT-PCR in 13 of 35 (37%) infiltrating carcinomas and in 2 breast cancer cell lines. Eleven tumours, that were negative with anti-DMBTh12, revealed PCR-products after RT-PCR amplification. This discordance between IHC and RT-PCR results may be due to residual normal epithelial cells present in the sample. Furthermore other possible reasons of this discrepancy could be: 1) the greater sensitivity of molecular technique like RT-PCR in detecting very low expression levels of  DMBT1  that were below detection by immunohistochemistry, 2) sampling problems i.e. the presence of few positive tumour cells only in the frozen samples or 3) few immunoreactive cells have been missed by microscopic examination and finally 4)  DMBT1  protein could have an altered epitope not recognized by its paratope in the antibody. By either immunohistochemistry or molecular technique we found no correlation between  DMBT1  expression and tumor histotype, grade, hormonal receptors status, proliferation index, Her2 Neu expression or lymph-nodal metastases.DMBT1in situDMBT1DMBT1DMBT1DMBT1In a recent paper on breast carcinogenesis, qualitatively similar data were reported for  DMBT1  in human breast cancer, but an indirect weak correlation between the degree of differentiation of the breast carcinomas and the extent of the immunoreactivity was found [ 10 ]. The discrepancy in the results between our and this study may be due to sampling problems, the size of the sample and heterogeneous expression of  DMBT1  in breast cancer or to other poorly-defined factors such as race, age or hormonal status of the population under study. Mollenhauer et al. point out that  DMBT1  expression may be up-regulated in pathophysiological conditions such as inflammation, liver injury or after carcinogen administration and it is down-regulated in tumors to achieve a growth advantage in a dynamic and complex model. Also according to these authors, the possibility that  DMBT1  loss may be a consequence rather than a primary event in carcinogenesis is unlikely (10).DMBT1DMBT1DMBT1DMBT1Although the normal mammary gland has been reported to have low  DMBT1  expression by RT-PCR [ 12 ], we found variable DMBTh12 immunopositivity in breast epithelium flanking benign and malignant lesions. Immunopositive epithelial cells in our material appeared either morphologically normal and indistinguishable from immunonegative cells or hypertrophic with abundant cytoplasm and large elongated nuclei oriented perpendicularly to the basement membrane [Figure  1i ]. Moreover, DMBTh12 immunoreactivity was also maintained in hyperplastic epithelium flanking carcinomas. This resembles the  DMBT1  expression patterns observed in the human respiratory tract and in associated inflammation and carcinomas [ 8 ] and suggest that the variable  DMBT1  expression in the normal mammary gland epithelium may result from the necessity of  DMBT1  induction. Indeed, in parallel  in vivo  studies, it was found that  DMBT1  is strongly up-regulated in the mammary gland tissue of the mouse shortly after carcinogen-administration and prior to the onset of histo-pathologically visible symptoms [ 10 ]. It has been reported that Sp-D and Sp-A are ligands of  DMBT1  products [ 29 ]. We recently demonstrated that Sp-A, in breast carcinoma tissues, has a pattern of expression resembling that of its interaction partner [ 20 ]; thus, we hypothesize that co-ordinated induction of both SP-A and  DMBT1  in breast tissue may be part of a protective response as suggested by Kang et al [ 30 ] for  DMBT1  and Sp-D in pulmonary epithelium.DMBT1DMBT1DMBT1DMBT1in vivoDMBT1DMBT1DMBT1DMBT1While this interaction may be attributed to lumenally secreted  DMBT1 , the depolarisation of the staining patterns indicates that  DMBT1  may also be secreted to other destinations, i.e. the ECM, by the mammary gland epithelial cells. In this regard, the second putative function of  DMBT1 , i.e. triggering of epithelial differentiation, is noteworthy. It may prevent hyperproliferative epithelial cells from out-of control proliferation and progression to the tumor state. This view is supported by two present findings. The correlation between MCM5 and  DMBT1  expression may point to a coupling to cell-cycle processes: basolateral relocation and hyper-expression of  DMBT1  at the ECM may provide cells that are licensed to proliferate with protection from unregulated proliferation. On the other hand loss of  DMBT1  expression in a significant numbers of DCISs and ICs is consistent with the notion that  DMBT1  inactivation may offer growth advantages to neoplastic cells [ 10 ]. The loss of  DMBT1  expression appears to take place early, i.e. already at the time of transition from hyperplastic lesions to carcinoma, as it has been reported in oesophageal squamous cell carcinomas [ 5 ].DMBT1DMBT1DMBT1DMBT1DMBT1DMBT1DMBT1DMBT1The exact mechanisms for  DMBT1  inactivation remain unclear. An inactivation by mutation appears not to be a major mechanism for  DMBT1  inactivation in tumors [ 10 , 31 ] The studies of Munoz and co-workers suggest that epigenetic silencing of either  DMBT1  or genes that regulate  DMBT1  expression takes place in some tumors [ 32 ]. Accordingly, except for direct epigenetic silencing of the  DMBT1  promotor, loss of expression or mutation of  DMBT1 -regulatory genes might represent alternative candidate mechanisms.DMBT1DMBT1DMBT1DMBT1DMBT1DMBT1Conclusions We have shown that  DMBT1  expression and cellular location in breast epithelium varies in normal, hyperplastic and neoplastic cells. In normal or hyperplastic tissue flanking carcinomas it is up-regulated and polarized toward glandular lumen or the basolateral membrane while it is down-regulated in carcinomas. These findings closely resemble the patterns observed for its interaction partner SP-A in an earlier study. Additionally we have shown that  DMBT1  is co-expressed with MCM5 in normal and hyperplastic tissue. We hypothesize that hyper-expression and basolateral relocation of  DMBT1  in these epithelia prevents the out of control growth that characterizes neoplastic epithelium.
Conclusions
We have shown that  DMBT1  expression and cellular location in breast epithelium varies in normal, hyperplastic and neoplastic cells. In normal or hyperplastic tissue flanking carcinomas it is up-regulated and polarized toward glandular lumen or the basolateral membrane while it is down-regulated in carcinomas. These findings closely resemble the patterns observed for its interaction partner SP-A in an earlier study. Additionally we have shown that  DMBT1  is co-expressed with MCM5 in normal and hyperplastic tissue. We hypothesize that hyper-expression and basolateral relocation of  DMBT1  in these epithelia prevents the out of control growth that characterizes neoplastic epithelium.DMBT1DMBT1DMBT1List of abbreviations used DMBT1: Deleted in Malignant Brain Tumor 1, MCM5: Minichromosome Maintenance protein, 5 RT-PCR: Reverse Transcriptase Polymerase Chain Reaction GP 340: Glycoprotein 340 SAG: salivary Agglutinin Sp-A, Sp-D: Surfactant Protein A, D MBP: Mannan Binding Protein ECM: Extracellular Matrix FD: Fibrocystic Disease GM: gynecomastia DCIS: Ductal Carcinoma  In Situ  IC: Invasive Carcinoma MW: Micro Wave EDTA Ethylenediaminetetraacetic Acid DAB: Diaminobenzidine
List of abbreviations used
DMBT1: Deleted in Malignant Brain Tumor 1,MCM5: Minichromosome Maintenance protein, 5RT-PCR: Reverse Transcriptase Polymerase Chain ReactionGP 340: Glycoprotein 340SAG: salivary AgglutininSp-A, Sp-D: Surfactant Protein A, DMBP: Mannan Binding ProteinECM: Extracellular MatrixFD: Fibrocystic DiseaseGM: gynecomastiaDCIS: Ductal Carcinoma  In SituIn SituIC: Invasive CarcinomaMW: Micro WaveEDTA Ethylenediaminetetraacetic AcidDAB: DiaminobenzidineCompeting interests None declared.
Competing interests
None declared.Autors Contributions PB designed the study, participated in the evaluation of immunohistochemical results and drafted the manuscript. PGN participated in the evaluation of immunohistochemical results and performed statistical analysis. JM participated in sequence alignment, criticism and produced DMBTh12 antibody. AP participated in sequence alignment and data evaluation. CP Carried out RT-PCR. AM participated in the evaluation of histologic samples and immunohistochemical results. GB participated in data evaluation and sequence alignment GC participated in study design and data evaluation. SB participated in study design, sequence alignment, criticism and coordinated molecular study. GGP participated in study design, criticism and general revision of paper.
Autors Contributions
PB designed the study, participated in the evaluation of immunohistochemical results and drafted the manuscript. PGN participated in the evaluation of immunohistochemical results and performed statistical analysis. JM participated in sequence alignment, criticism and produced DMBTh12 antibody. AP participated in sequence alignment and data evaluation. CP Carried out RT-PCR. AM participated in the evaluation of histologic samples and immunohistochemical results. GB participated in data evaluation and sequence alignment GC participated in study design and data evaluation. SB participated in study design, sequence alignment, criticism and coordinated molecular study. GGP participated in study design, criticism and general revision of paper.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: