Alan D. Proia

Estrogen-Independent Proliferation Is Present in Estrogen-Receptor HER2-Positive Primary Breast Cancer After Neoadjuvant Letrozole

PURPOSE To investigate the impact of human epidermal growth factor receptor (HER) 1 and HER2 gene amplification on endocrine therapy responsiveness, a fluorescence in situ hybridization (FISH) study was conducted on tumor samples from 305 postmenopausal patients with stage II and III estrogen receptor (ER) -positive (ER > or = 10%) breast cancers treated on two independent neoadjuvant endocrine therapy trials. PATIENTS AND METHODS FISH analysis focused on HER1 and/or HER2 immunohistochemistry (IHC) -positive patients and a random selection of HER1/2 IHC-negative patients. HER2 FISH status was correlated with response and changes in the proliferation marker Ki67. RESULTS HER1 was rarely amplified (< 1%), and HER2 amplification was observed in 9.2% of patients. Letrozole response by clinical measurement (71% HER2 FISH positive v 71% HER2 FISH negative), mammogram (44% HER2 FISH positive v 47% HER2 FISH negative), or ultrasound (47% HER2 FISH positive v 54% HER2 FISH negative) was not impaired by HER2 FISH-positive status. In contrast, HER2 FISH-positive tumors showed higher histologic grade (P = .009), higher pretreatment Ki67 (P = .005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = .0001). Similar observations regarding Ki67 were made in a smaller cohort of tamoxifen-treated tumors. CONCLUSION Neoadjuvant letrozole is clinically effective in ER-positive HER2 FISH-positive tumors, indicating sensitivity to short-term estrogen deprivation. However, continued proliferation despite ongoing letrozole or tamoxifen treatment in the majority of ER-positive HER2 FISH-positive samples (88%) could imply therapeutic resistance that may manifest later in the clinical course of the disease. Discordance between clinical and biomarker findings in this study serves to emphasize the need for surrogate end point validation in neoadjuvant endocrine trials through correlation with information on long-term outcomes.

Preoperative Mobilization of Circulating Dendritic Cells by Flt3 Ligand Administration to Patients With Metastatic Colon Cancer

PURPOSE To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.

The feasibility and safety of immunotherapy with dendritic cells loaded with CEA mRNA following neoadjuvant chemoradiotherapy and resection of pancreatic cancer

SummaryBackground. Resected pancreatic cancer has a high risk of recurrence and mortality despite the the use of chemoradiotherapy. Because pancreatic cancers express tumor antigens such as carcinoembryonic antigen (CEA), it may be possible to immunize patients to induce tumor antigen-specific immune responses. We hypothesize that high-frequency tumor antigen-specific immune responses will reduce recurrence and increase survival. Autologous dendritic cells (DCs) loaded with tumor antigens are particularly potent at inducing tumor antigen-specific immune responses. Methods. Three patients with resected pancreatic adenocarcinoma following neoadjuvant chemoradiotherapy received autologous, monocyte-derived DCs loaded with the mRNA encoding CEA monthly for 6 mo. Results. It was feasible to generate an adequate number of DC from these patients and to cryopreserve them for repeated use. The DC demonstrated the typical immature phenotype. The immunizations were well-tolerated without evidence of adverse events. All three developed injection site reactivity. All three are alive without evidence of disease at more than 21/2 yr from the original diagnosis. Conclusion. The postoperative period following neoadjuvant chemoradiotherapy and pancreaticoduo-denectomy for pancreatic cancer is an ideal environment to test novel immune-based therapies. DC-based immunotherapy in this setting is safe and feasible and may lead to prolonged survival.

Histopathological variation in keratoconus.

During examination of 131 penetrating keratoplasty specimens from patients with keratoconus obtained in an 11- year period, we observed two histopathologic variants based on the appearance of Bowmans layer and the corneal epithelium. “Typical” keratoconus specimens had multiple breaks in Bowmans layer and central epithelial thinning, whereas “atypical” corneas lacked breaks in Bowmans layer and had less thinning of the central epithelium. Ninety-five corneas were from patients who underwent grafting in only one eye. Seventy-six (80%) of these corneas were “typical” and 19 corneas (20%) were “atypical” in appearance. Both variants had similar degrees of central stromal thinning. Patients with “typical” and “atypical” corneas differed demographically by race only; 49% of “typical” and 95% of “atypical” corneas were from white individuals. Thirty-six corneas were from 18 patients who underwent bilateral penetrating keratoplasty. The histologic appearance of these corneal pairs was concordant in 13 patients and discordant (one “typical” and one “atypical” cornea) in five patients. Statistical analysis indicated that this distribution is not significantly different from that predicted by chance and suggests that “typical” and “atypical” corneas are manifestations of the same disease process

Improvement in efficacy of corticosteroid therapy in an animal model of proliferative vitreoretinopathy by pretreatment.

Intraocular injection of the corticosteroid triamcinolone acetonide reduces the incidence of retinal detachment in rabbit eyes injected with tissue-cultured fibroblasts. When the steroid was injected simultaneously with the cells, a reduction of retinal detachment from 93% (control) to 75% (treated) was achieved on day 28. When the steroid was injected 24 h preceding cell injection, the reduction of retinal detachment was from 85% (control) to 43% (treated). The development of retinal detachment is caused by proliferation of injected fibroblasts. Reduction of this proliferation is probably achieved partially through direct inhibition of mitosis, but more important may be the reduction of the reactive inflammatory process.

Malignant Syringoma of the Eyelid

Two cases of malignant syringoma of the eyelid are reported. The first patient, a 20-year-old woman, presented with a one year history of a slowly growing nodule in the left lower eyelid. The lesion recurred five months after excision, slowly grew over the ensuing two and one-half years, and was reexcised. The second case was an 18-year-old man who was first examined by an ophthalmologist because of a slowly enlarging, painless induration of his right lower eyelid for two years. Histologic examination of the excised lesions from both patients disclosed proliferated ducts lined by two layers of epithelium infiltrating the dermis and subcutaneous tissue; there was skeletal muscle and perineural invasion, but only minimal cellular atypia. Malignant syringomas are rare forms of sweat gland carcinoma, which most often occur on the upper lip and to our knowledge have not been reported on the eyelid. These tumors probably arise from eccrine ducts, are more common in women, and have a peak incidence in the fifth decade. The histologic feature that best indicates prognosis is the degree of cellular atypia. Patients with cytologically well-differentiated tumors do well following complete local excision. Features that help to distinguish malignant syringoma from its benign counterpart are the larger size, solitary nature, and subcutaneous, muscular, and perineural invasion.

Interferon γ–Inducible Protein-10 (IP-10) and Eotaxin as Biomarkers in Age-Related Macular Degeneration

PURPOSE To analyze serum cytokine levels in subjects with different stages of AMD and to study the expression of salient cytokines in postmortem eyes with AMD. METHODS A suspension array system was used to analyze sera (n = 18 to 20/group) from control subjects and those with early AMD (AREDS stage 1), intermediate dry AMD (AREDS stage 3), advanced AMD with geographic atrophy (GA), or neovascular AMD (CNV). Postmortem eyes with AMD or control eyes were examined immunohistochemically for expression of IP-10 and eotaxin (n = 4 to 8/group). RESULTS Serum eotaxin and IP-10 levels were significantly elevated in all stages of AMD, except for eotaxin levels in neovascular AMD (P < 0.07). The peak of serum IP-10 concentration was at intermediate dry AMD. In donor eyes, IP-10 and eotaxin expressions were increased in the RPE of eyes with early AMD, GA, and CNV. Eotaxin accumulated within the layer of basal linear/laminar deposits in all stages of AMD, while IP-10 was mainly in eyes with GA and CNV. IP-10 was abundant in the connective tissue matrix associated with CNV, and eotaxin was usually present but more focally and with less intense staining. Both IP-10 and eotaxin were expressed by neovascular endothelial cells. Both IP-10 and eotaxin were expressed in the neurosensory retina, but there was no detectable difference in staining between eyes with or without AMD. CONCLUSIONS IP-10 and eotaxin may be early biomarkers in AMD. The authors hypothesize that the relative balance between levels of IP-10 and eotaxin is critical in regulating the neovascular response.

HER-2 Gene Amplification Correlates with Higher Levels of Angiogenesis and Lower Levels of Hypoxia in Primary Breast Tumors

Purpose: This study investigated the connection among HER-2 gene amplification, HER-2 protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors. Experimental Design: From 1988 to 1995, 425 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate “vascular hot spots” using image analysis. Tumor samples were also stained for HER-2 by IHC, HER-2 gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined. Results: There was a significant positive correlation between HER-2 gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-2 gene amplification and expression of the tumor hypoxia marker CA-9 (χ2 P = 0.02). The level of HER-2 gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.021). Interestingly, tumors with HER-2 by IHC had decreased amounts of VEGF staining (χ2 = 5.81; P = 0.01). There was no correlation between HER-2 by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.0128) predicted disease-free survival (Cox univariate model). Conclusions: HER-2-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

Autopsy findings in late-onset Pompe disease: A case report and systematic review of the literature

BACKGROUND Late-onset Pompe disease (LOPD) is a rare cause of declining proximal muscle strength and respiratory function that can also affect other organ systems. The development of enzyme replacement therapy has made it one of the few inherited muscle disorders with treatment, but clinical response is difficult to assess due to the variable and often slow progression of illness. A better understanding of the diseases systemic effects can be gleaned through autopsy findings. PURPOSE The purpose of this study was to: (1) describe the histological findings observed in LOPD, (2) provide correlations between reported histological and clinical findings, and (3) review the literature on autopsy findings in LOPD. METHODS Histological evaluation of autopsy tissues from a 62-year-old woman with LOPD was conducted. A clinical history was obtained by review of the medical records. The literature was reviewed for previously reported histological and clinical findings in LOPD. Based on this case report and information from prior publications, histological and clinical findings for the disease were correlated. RESULTS Histologic examination revealed mostly mild vacuolar myopathy typical of glycogen accumulation within skeletal and smooth muscle cells. The most prominent vacuolar myopathy was in quadriceps muscle, which also exhibited chronic myositis with degenerating and regenerating muscle fibers. Transmission electron microscopy disclosed lysosomal glycogen accumulation within skeletal, cardiac, and vascular smooth muscle cells, correlating with published case reports of basilar artery and ascending aortic aneurysms and carotid artery dissection. Organs containing smooth muscle cells (the bladder, intestine, and esophagus) were also affected, explaining reports of symptoms such as urinary incontinence and dysphagia. In addition to glycogen accumulation, there was obvious damage to the contraction apparatus of myofibrils within cardiac and skeletal muscle cells. These histological and ultrastructural findings correlate with the clinical manifestations of LOPD. CONCLUSIONS This study is the first to describe histological findings of LOPD utilizing both traditional paraffin-processed tissues and epoxy resin embedded tissues for high-resolution light microscopy. The findings are similar to those seen in previous studies, but with improved morphological detail and glycogen preservation. This patient exhibited histological involvement of multiple organs, correlating with the clinical features of LOPD. With the advent of definitive therapy for Pompe disease, it is important to be aware of these findings and use them to develop methods for tracking therapeutic response.

Retinal toxicity of cyanoacrylate tissue adhesive in the rabbit.

N-butyl-2-cyanoacrylate tissue adhesive was injected into the preretinal space of rabbit eyes to study potential toxicity to the retina. Application of 3.3-10.0 ul of cyanoacrylate tissue adhesive showed localized but definite retinal toxicity. White halos appeared surrounding the preretinal cyanoacrylate immediately after injection with a gradual evolution of the white areas into pigmentary scars by 1 month. Histological examination confirmed severe focal necrosis of the retina. No identifiable distant toxic effects or electrophysiologic changes were observed during the 6-month follow-up period.