Comparison of indocyanine green angiography and optical coherence tomographic angiography in polypoidal choroidal vasculopathy.

PurposeTo compare optical coherence tomographic angiography (OCTA) and indocyanine green angiography (ICGA) images for detecting polypoidal lesions (PLs) and branching vascular networks (BVNs), and to measure the polypoidal areas (PAs) in patients with polypoidal choroidal vasculopathy (PCV).MethodsAll patients underwent ICGA, optical coherence tomography (OCT), and OCTA. We compared the detection sensitivity for PL and BVN, as evaluated by the ICGA and OCTA images. Furthermore, PA measured by ICGA was divided into two groups: one in which the area could be measured by OCTA (ICGA+OCTA+) and the other in which the area could not be measured by OCTA (ICGA+OCTA-).ResultsTwenty-one consecutive eyes of 21 patients (mean age, 73.8±9.8 years) were included. ICGA detected PL in all eyes (100%), whereas OCTA detected PL in 16 eyes (75.2%); ICGA detected BVN in 15 eyes (71.4%), whereas OCTA detected BVN in 20 eyes (95.2%). The mean PA in ICGA+OCTA+ and ICGA+OCTA- was 0.24±0.04 and 0.14±0.01 mm2, respectively; a significant difference was observed between ICGA+OCTA+ PA and ICGA+OCTA- PA (P<0.0001). In addition, the mean PA in the ICGA+OCTA+ group measured by ICGA and OCTA was 0.24±0.04 was 0.19±0.04 mm2, respectively; these values were significantly different (P=0.0046).ConclusionsOCTA might detect more BVNs and fewer PLs compared with ICGA, and PL detected by OCTA might be smaller than those detected by ICGA.

Pilot Assessment of the Repeatability of Indocyanine Green Fluorescence Imaging and Correlation with Traditional Foot Perfusion Assessments.

BACKGROUND: Ankle brachial index (ABI), toe pressures (TP), and transcutaneous oxygen pressure (TcPO2) are traditionally used in the assessment of critical limb ischemia (CLI). Indocyanine green (ICG) fluorescence imaging can be used to evaluate local circulation in the foot and to evaluate the severity of ischemia. This prospective study analyzed the suitability of a fluorescence imaging system (photodynamic eye [PDE]) in CLI. MATERIAL AND METHODS: Forty-one patients with CLI were included. Of the patients, 66% had diabetes and there was an ischemic tissue lesion in 70% of the limbs. ABI, toe pressures, TcPO2 and ICG-fluorescence imaging (ICG-FI) were measured in each leg. To study the repeatability of the ICG-FI, each patient underwent the study twice. After the procedure, foot circulation was measured using a time-intensity curve, where T1/2 (the time needed to achieve half of the maximum fluorescence intensity) and PDE10 (increase of the intensity during the first 10 s) were determined. A time-intensity curve was plotted using the same areas as for the TcPO2 probes (n=123). RESULTS: The mean ABI was 0.43, TP 21 mmHg, TcPO2 23 mmHg, T1/2 38 s, and PDE10 19 AU. Time-intensity curves were repeatable. In a Bland-Altman scatter plot, the 95% limits of agreement of PDE10 was 9.9 AU and the corresponding value of T1/2 was 14 s. Correlation between ABI and TP was significant (R=.73, p<.001), and it was weaker in diabetic patients (R=.47, p=.048) compared with non-diabetic patients (R=.89, p=.002). Correlations between ABI and TcPO2 and TP and TcPO2 were weak (R=.37, p=.05 and R=.43, p=.037, respectively). Correlation between TcPO2 and PDE10 was strong in diabetic patients (R=.70, p=.003). CONCLUSIONS: According to this pilot study, ICG-FI with PDE can be used in the assessment of blood supply in the ischemic foot.

Multicenter observational study comparing sedation/analgesia protocols for laser photocoagulation treatment of retinopathy of prematurity.

OBJECTIVE: The aim of this study was to identify the best sedation/analgesia protocol for laser photocoagulation (PC) of retinopathy of prematurity (ROP). STUDY DESIGN: This multicenter observational study included five hospitals, each using a specific sedation/analgesia protocol: local anesthesia with oxybuprocaine hydrochloride (Group L); intravenous pentazocine (Group P); intravenous fentanyl (Group F); air, oxygen and sevoflurane (AOS) inhalation (Group I). The groups were compared for pain responses, vital signs and adverse events. RESULTS: Heart rates and systemic blood pressures were elevated by PC in Groups L and P and Groups L, P and F, respectively. Moreover, poor analgesic efficacy was recognized in Groups L, P and F. In contrast, Group I experienced hypothermia, enteral feeding intolerance and apnea more frequently. CONCLUSION: From the viewpoint of sedation/pain relief, AOS anesthesia should be the best protocol. However, considering all the various factors together, the most reasonable one can be varied based on the patient's condition and hospital.

Macrophage- and RIP3-dependent inflammasome activation exacerbates retinal detachment-induced photoreceptor cell death.

Detachment of photoreceptors from the retinal pigment epithelium is seen in various retinal disorders, resulting in photoreceptor death and subsequent vision loss. Cell death results in the release of endogenous molecules that activate molecular platforms containing caspase-1, termed inflammasomes. Inflammasome activation in retinal diseases has been reported in some cases to be protective and in others to be detrimental, causing neuronal cell death. Moreover, the cellular source of inflammasomes in retinal disorders is not clear. Here, we demonstrate that patients with photoreceptor injury by retinal detachment (RD) have increased levels of cleaved IL-1ß, an end product of inflammasome activation. In an animal model of RD, photoreceptor cell death led to activation of endogenous inflammasomes, and this activation was diminished by Rip3 deletion. The major source of Il1b expression was found to be infiltrating macrophages in the subretinal space, rather than dying photoreceptors. Inflammasome inhibition attenuated photoreceptor death after RD. Our data implicate the infiltrating macrophages as a source of damaging inflammasomes after photoreceptor detachment in a RIP3-dependent manner and suggest a novel therapeutic target for treatment of retinal diseases.

Bilateral central retinal artery occlusion and vein occlusion complicated by severe choroidopathy in systemic lupus erythematosus.

Severe retinal vascular occlusions resulting in blindness is a rare occurrence in patients with systemic lupus erythematosus (SLE). Herein, we report a case of a 33-year-old female who developed combined central retinal artery occlusion, retinal vein occlusion, and choroidopathy and rapidly became completely blind in both eyes within a week. The electroretinogram revealed a severely attenuated a-wave and b-wave, indicating a profound dysfunction of both choroidal and retinal circulation, respectively. The current case demonstrates objectively the functional impact of severe choroidopathy in SLE for the first time. Patients with unilateral blindness due to combined retinal/choroidal vascular obstructions should be monitored carefully to ensure adequate anticoagulant therapy in an attempt to guard the vision in the fellow eye.

Displacement of foveal area toward optic disc after macular hole surgery with internal limiting membrane peeling.

PURPOSE: To determine whether there is a displacement of the fovea toward the optic disc after successful macular hole (MH) surgery with internal limiting membrane (ILM) peeling. METHODS: The medical records of 54 eyes of 53 patients that had undergone pars plana vitrectomy with ILM peeling and gas or air tamponade for an idiopathic MH were evaluated. Spectral-domain optical coherence tomography (OCT) had been performed before and >6 months after the surgery. The preoperative distances between the center of the MH and the optic disc (MH-OD), center of the MH and the bifurcation or crossing of retinal vessels (MH-RV) were measured in the OCT images. In addition, the postoperative distance between the center of the fovea and optic disc (F-OD) and the center of the fovea and the same bifurcation or crossing of retinal vessels (F-RV) were measured in the OCT images. RESULTS: The F-OD was 2.67±0.33 disc diameters (DD), which was significantly shorter than that of the MH-OD of 2.77±0.33 DD (P<0.001). The F-RV was also significantly shorter than the MH-RV on the inner nasal area (from 0.85±0.16DD to 0.79±0.15DD; P<0.001), the inner temporal area (from 0.82±0.15DD to 0.77±0.14DD; P<0.001), and outer nasal area (from 1.70±0.31DD to 1.65±0.32DD; P<0.001), but it was significantly longer than the MH-RV in the outer temporal area (from 1.65±0.29DD to 1.68±0.29DD; P<0.001). CONCLUSION: Our results showed that successful closure of a MH by vitrectomy with ILM peeling and gas tamponade leads to a displacement of the center of the macula toward the optic disc.

Scleral thickness in highly myopic eyes measured by enhanced depth imaging optical coherence tomography.

PURPOSE: The purpose of this study was to determine the subfoveal scleral thickness in highly myopic eyes by enhanced depth imaging spectral-domain optical coherence tomography (EDI-OCT) and to identify the ocular parameters significantly associated with the scleral thickness. METHODS: The subfoveal scleral thickness of myopic eyes (≥-8 diopters (D) or axial length ≥26.5 mm) was examined by EDI-OCT. The correlations between the thickness and the best-corrected visual acuity (BCVA), refractive error, axial length (AL), the subfoveal retinal thickness, choroidal thickness, and posterior staphyloma height 2 mm from the fovea were investigated. RESULTS: A total of 75 eyes of 54 patients (21 men, 33 women; mean age, 62.3±11.3 years; mean AL, 30.2±1.68 mm) were studied. Eighteen eyes had no pathological retinochoroidal lesions, and 57 eyes had retinochoroidal lesion, that is, myopic schisis, choroidal neovascularization, and other retinochoroidal pathologies. The mean subfoveal scleral thickness was 284.0±70.4 µm, and the thickness was significantly correlated negatively with the absolute value of the nasal and overall average posterior staphyloma height (P<0.05 and P<0.01, respectively). The subfoveal scleral thickness was also significantly correlated negatively with the relative value of the superior, nasal, and overall average posterior staphyloma height (P<0.05, P<0.01, and P<0.001, respectively). Stepwise analyses showed that the factor most significantly associated with the scleral thickness was the average relative posterior staphyloma height (F=16.0, P<0.001). The scleral thickness was not significantly different between eyes with and without myopic retinochoroidal pathologies (P>0.05). CONCLUSION: Posterior staphyloma formation was a key factor associated with a posterior scleral thinning in highly myopic eyes.

Retinal remodeling.

Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and optogenetic approaches that target appropriate classes of neurons in the remnant neural retina.

Vascular laboratory for critical limb ischaemia.

This is a narrative review on vascular assessment for critical limb ischaemia in the past and present combining Finnish and Japanese experience.

Half-dose vs one-third-dose photodynamic therapy for chronic central serous chorioretinopathy.

PURPOSE: To prospectively compare the effects of half-dose verteporfin (3 mg/m(2)) photodynamic therapy (1/2 PDT) with those of one-third-dose verteporfin (2 mg/m(2)) PDT (1/3 PDT) for chronic central serous chorioretinopathy (CSC). METHODS: Sixteen eyes of 16 consecutive patients with chronic CSC were enrolled and followed up for a 3-month study period. The first 10 patients received 1/2 PDT and the next 6 patients received 1/3 PDT. The resolution rate of subretinal fluid (SRF) was compared between the two groups. The changes in the choroidal thickness inside and outside the PDT-applied area in both groups were also evaluated. RESULTS: SRF disappeared in all eyes (100%) in the 1/2 PDT group and in two eyes (33%) in the 1/3 PDT group. In the 1/2 PDT group, choroidal thickness inside and outside the PDT-applied area reduced significantly from the baseline (inside, from 387 ± 24 to 325 ± 25 µm; outside, from 292 ± 25 to 249 ± 19 µm; both P=0.005). In the 1/3 PDT group, choroidal thickness decreased in two eyes where SRF disappeared (inside, 87.2 and 90.9% of the baseline; outside, 91.4 and 92.6% of the baseline), but did not change in the other four eyes where SRF remained (inside, 104.1, 100.0, 105.1, and 100.5% of the baseline; outside, 98.9, 103.0, 100.0, and 99.0% of the baseline). CONCLUSIONS: 1/2 PDT is more effective than 1/3 PDT in the resolution of SRF for chronic CSC. Decrease in the choroidal thickness after PDT may be related to the resolution of SRF in chronic CSC.

Visual function 5 years or more after macular translocation surgery for myopic choroidal neovascularisation and age-related macular degeneration.

PURPOSE: To evaluate the changes in the best-corrected visual acuity (BCVA) after 1 year and after ≥ 5 years after macular translocation for age-related macular degeneration (AMD) or myopic choroidal neovascularisation (mCNV). METHODS: The medical records of 61 consecutive patients who underwent macular translocation with 360° retinotomy for AMD (35 eyes) or mCNV (26 eyes) were reviewed. Overall, 40 patients, 17 mCNV and 23 AMD, were followed for at least 5 years. BCVA and area of the Goldmann visual field (VF) measured before, 12 months after surgery, and at the final visit. RESULTS: In the 23 AMD eyes followed for ≥ 5 years, the mean preoperative BCVA was 1.149 ± 0.105 logMAR units, which significantly improved to 0.69 ± 0.06 logMAR units at 1 year (P<0.001). This BCVA was maintained at 0.633 ± 0.083 logMAR units on their final examination. In the 17 eyes with mCNV followed for ≥ 5 years, the mean preoperative BCVA was 1.083 ± 0.119 logMAR units, which was significantly improved to 0.689 ± 0.121 logMAR units at 1 year (P = 0.001). This BCVA was maintained at 0.678 ± 0.142 logMAR units on their final examination. The area of the VF was significantly decreased at 12 months and did not change significantly thereafter. CONCLUSIONS: Our results show that macular translocation surgery significantly improves the BCVA and significantly decreases the VF area of eyes with mCNV or AMD after first 1 year. The BCVA and VF area do not change significantly from the values at 1 year for at least 5 years.

Retinal remodeling in the Tg P347L rabbit, a large-eye model of retinal degeneration.

Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photoreceptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.

Correlation of autoantibodies and CD5+ B cells in ocular adnexal marginal zone B cell lymphomas.

AIM: To determine the clinicopathological properties of ocular adnexal marginal zone B cell lymphomas (MZBLs) with CD5+ B cells. METHODS: This study determined the clinicopathological properties of MZBL samples from 97 patients with ocular adnexal MZBLs and searched for hallmarks of systemic autoimmunity in these patients. RESULTS: Two elderly female patients were found to have ocular adnexal MZBLs with CD5+ B cells; flow cytometry analysis suggested that one of these MZBLs had CD5+ B cell clonal proliferation. The levels of anti-single stranded (SS)-DNA and anti-SS-A/Ro antibodies in these two patients were significantly higher than those in controls that were matched for age, gender and disease (2/2 versus 0/14; p = 0.008) and controls without MZBL (2/2 versus 0/30; p = 0.002). The genes from the immunoglobulin heavy-chain variable region for one of the patients showed a V3-21 segment. In addition, another patient with ocular adnexal reactive lymphoid hyperplasia with CD5+ B cells also had anti-SS-DNA antibodies. CONCLUSION: Patients with ocular adnexal MZBLs with CD5+ B cells may have a background of systemic conditions with CD5+ B-cell-related autoantibodies.

Survival and axonal regeneration of off-center retinal ganglion cells of adult cats are promoted with an anti-glaucoma drug, nipradilol, but not BDNF and CNTF.

OFF-center retinal ganglion cells (RGCs) occupy a smaller proportion than ON RGCs when RGCs regenerate axons into a transplanted peripheral nerve. We examined whether the regeneration ability of OFF RGCs in adult cats was promoted when the numbers of regenerating RGCs were increased with brain-derived neurotrophic factor (BDNF)+ciliary neurotrophic factor (CNTF)+forskolin (BCF) or 3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran (nipradilol), an anti-glaucoma drug. ON or OFF RGCs were morphologically determined on the basis of their dendritic ramification in the inner plexiform layer using computational analysis. In the normal intact retina the ratio of ON and OFF RGCs (ON/OFF ratio) was 1.25 (55%/44%); whereas, it was 2.61 in regenerating RGCs with saline injection (control) 6 weeks after peripheral nerve transplantation. Estimated numbers of regenerating ON and OFF RGCs were 2149 and 895, respectively. An injection of BCF increased only numbers of ON RGCs into 5766 (2.7-fold to control) but not that of OFF RGCs, n=858. Nipradilol increased both estimated numbers of ON (11,518, 5.4-fold to control) and OFF RGCs (7330, 8.2-fold to control). In the retinas with optic nerve (OpN) transection and intravitreal saline-, BCF- or nipradilol-injection, numbers of ON and OFF RGCs surviving axotomy showed similar trend to that in regenerating RGCs. Thus, nipradilol promoted the survival and regeneration abilities of both of ON and OFF RGCs whereas BCF only did the abilities of ON RGCs. The distribution of tropo-myosin-related kinase B, BDNF receptor, was sparser in the outer two thirds of inner plexiform layer. The lower surviving ability of OFF-RGCs may be attributed in part to the distribution.

Seasonal variations of acute massive submacular haemorrhage associated with age-related macular degeneration.

AIMS: To determine whether there is a seasonal variation in the onset of acute, massive submacular haemorrhage associated with age-related macular degeneration. METHODS: Sixty eyes of 59 patients diagnosed between April 1998 and March 2005, were studied retrospectively. For each patient, the month and season of onset of the submacular haemorrhage and the mean monthly ambient temperature in Nagoya were analysed. Any history of systemic hypertension was also recorded, and the seasonal variations were also investigated in hypertensive and non-hypertensive groups. RESULTS: The number of cases peaked in winter with a trough in summer, and this seasonal variation was significant (Roger's R = 12.03, p<0.01). The monthly incidence was inversely correlated with the temperature (Spearman's rank correlation coefficient r = 0.89, p<0.01). The seasonal variations were significant in the hypertensive group but not in the non-hypertensive group. CONCLUSION: The considerable seasonal variations suggests that the mechanism for the haemorrhage is strongly correlated with the systemic blood pressure.

Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer.

BACKGROUND: The techniques to isolate and purify retinal pigment epithelial (RPE) cells from small piece of autologous tissues are extremely difficult, and it is important to develop an efficient cell culture technique for RPE cells. The purpose of this study was to investigate the effect of 3T3-J2 cells and conditioned medium from 3T3-J2 cells on the proliferation of cultured RPE cells. METHODS: RPE cells from pigmented rabbits and a human RPE-derived cell line, ARPE-19, were used. First, the effects of co-culturing RPE cells with 3T3-J2 cells on the growth of the cells were analyzed. Second, the effects of the conditioned medium from 3T3-J2 cells on the proliferation of both types of cells were investigated. And third, the effects of the conditioned medium on RPE cell culture from a surgically removed choroidal neovascular (CNV) membrane were investigated. RESULTS: The 3T3-J2 cells increased the proliferation of both rabbit RPE cells and ARPE-19 cells. The number of rabbit RPE cells cultured in a mixture of the conditioned medium from 3T3-J2 cells was significantly higher than that in the reported optimal condition, and a similar tendency was observed for ARPE-19 cells. The results from enzyme-linked immunosorbent assay showed the presence of PDGF-AB, VEGF and IGF-I in the conditioned medium. The conditioned medium also promoted selective growth of human RPE cells from CNV. DISCUSSION: The results from this study present the conditions for efficient and selective culture of primary RPE cells.

Polypoidal choroidal vasculopathy treated with macular translocation: clinical pathological correlation.

AIMS: To report the histopathology of two specimens of polypoidal choroidal vasculopathy (PCV) obtained from two eyes of Japanese patients. METHODS: Specimens were obtained under direct visualisation during macular translocation surgery with 360 degree retinotomy. The clinical findings were correlated with the light microscopic findings of the two specimens. RESULTS: One specimen from a 77 year old man was the central portion of the lesion that lay under the sensory retina on the retinal pigment epithelium (RPE). The specimen was made up mainly of fibrous tissue with small, thin walled vessels. Indocyanine green angiography after surgery revealed that active leaking polypoidal element remained under the RPE. Another specimen obtained from a 62 year old man was made up of a fibrovascular membrane situated within Bruch's membrane. The part of this specimen inferior to the foveal region included a collection of dilated, thin walled blood vessels without pericytes, surrounded by macrophages that stained positive for CD68. The dilated vessels appeared to be correlated with the orange coloured polyps observed by ophthalmoscopy, the polypoidal structure seen in indocyanine green angiograms, and the pyramidal elevation with intermediate reflectivity by optical coherence tomography. CONCLUSION: Polypoidal structures are located within Bruch's space. They are composed of clusters of dilated, thin walled blood vessels surrounded by macrophages and fibrin material. The positive immunohistochemical staining for vascular endothelial growth factor in the RPE and the vascular endothelial cells suggests that this fibrovascular complex is a subretinal choroidal neovascularisation.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Detection of HER-2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression.

BACKGROUND: Fluorescent in situ hybridization (FISH) has been shown to be one of the most reliable methods with which to estimate the status of the HER-2/neu (or c-erb B-2) oncogene at the DNA level. METHODS: To study interobserver reproducibility and to determine more clinically correlated criteria for HER-2/neu alterations, two observers independently estimated HER-2/neu DNA status. The correlation between the consensus HER-2/neu DNA status by FISH and HER-2/neu protein status detected by immunohistochemistry (IHC) using a polyclonal antibody was studied in 216 surgically resected breast carcinomas and 34 noncancerous tissues. RESULTS: According to the HER-2/CEP17 ratio and mean HER-2 copies per nucleus, agreement level of HER-2/neu amplification was shown to be nearly perfect between two observers (kappa statistic (kappa) = 0.94 and kappa = 0.84). Finally, 40 tumors (19%) were judged to have HER-2/neu DNA amplification, with 6 having low-level amplification (> or = 2 but < 3 folds) and 34 having high-level amplification (> or = 3 folds). One hundred seventy-six other tumors, including 3 tumors that only 1 of the observers determined to be low-level amplifiers, and 34 noncancerous tissues had no detected amplification. The DNA amplification status was concordant between invasive and intraductal components in 14 carcinomas. HER-2/neu protein overexpression of moderate (2+) or high (3+) intensity based on IHC was detected in 51 carcinomas (24%), and was 2+ in 20 carcinomas and 3+ in 31 carcinomas. The HER-2/CEP17 ratio of > or = 2 was concordant with IHC findings of 2+/3+ in 91% of carcinomas (195 of 215 carcinomas), with a sensitivity of 70% (35 of 50 carcinomas) and a specificity of 97% (160 of 165 carcinomas). High-level amplification was detected in 29 of 31 IHC 3+ cases (94%), but in only 5 of 20 IHC 2+ cases (25%) and 0 in 165 IHC 0/1+ cases. All 34 cases with high-level amplification showed an IHC score of 3+ (29 cases) or an IHC score of 2+ (5 cases), but only 1 case was found to have an IHC score of 3+ and the remainder were IHC 0/1+ in 6 low-amplification cases. The concordance rate of the high-level amplification with an IHC score of 3+ was 97% (208 of 215 cases), with a sensitivity of 94% (29 of 31 cases) and a specificity of 97% (179 of 184 cases). CONCLUSIONS: The results of the current study indicated that high-level HER-2/neu amplification and an IHC score of 3+ nearly optimally identified breast carcinomas with clinically and biologically significant HER-2/neu activation. Conversely, it was confirmed that careful interpretation of test results is required in the case of low-level amplification and/or an IHC score of 2+.