Absence of ectopic epithelial inclusions in 3,904 axillary lymph nodes examined in sentinel technique

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 μm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.

Efalizumab-associated papular psoriasis

BACKGROUND: Efalizumab is a human anti-CD11a monoclonal antibody used in the treatment of patients with moderate to severe plaque psoriasis. Some of the patients develop new papular lesions during treatment, which are predominantly located in the flexural regions. OBSERVATION: Four patients with recalcitrant psoriasis undergoing treatment with efalizumab presented with erythematous, partly scaly papules and small plaques on previously unaffected areas after 4 to 10 weeks of efalizumab therapy. Tissue sections of biopsy specimens were stained with hematoxylin-eosin, and immunohistochemical staining was performed using monoclonal antibodies against CD3, CD4, CD8, T-cell-restricted intracellular antigen 1, granzyme B, neutrophil elastase, CD68, CD1a, CD11c, HLA-DR, CD25, CD20, and CD56. Histopathological and immunohistochemical examination of the lesions showed features consistent with psoriasis and activation of various leukocyte subtypes including T cells, dendritic cells, macrophages, and neutrophils. CONCLUSIONS: Papular eruptions appearing during efalizumab therapy represent new psoriatic lesions and could be referred to as efalizumab-associated papular psoriasis (EAPP). They usually do not necessitate termination of efalizumab therapy and may optionally be treated with topical corticosteroids. Dermatologists should be aware of these lesions and inform their patients accordingly.

Interface membrane fibroblasts around aseptically loosened endoprostheses express MMP-13

The objective of this article was to assess whether matrix metalloproteinase-13 (MMP-13) is produced by cells of the peri-implant interface tissues and to further characterize these cells. Tissue specimens were collected from the bone-prosthesis interface at the time of revision surgery of clinically loosened hip and knee arthroplasties (n = 27). Synovial tissues from osteoarthritic patients and young patients with mild joint deformity were used as controls (n = 6). Tissue samples were fixed in 4% PFA, decalcified with EDTA, and embedded in paraffin. Sections (4 microm) were stained with hematoxylin/eosin and for the osteoclastic marker enzyme tartrate resistant acid phosphatase. Monocytes/macrophages were characterized with a monoclonal antibody against CD68 and mRNAs encoding MMP-13 and alpha(1) collagen I (COL1A1) were detected by in situ hybridization. Cells expressing transcripts encoding MMP-13 were found in 70% of the interface tissues. These cells colocalized with a cell population expressing COL1A1 mRNA, and were fibroblastic in appearance. MMP-13 expressing cells were found in the close vicinity of osteoclasts and multinuclear giant cells. No signals for transcripts encoding MMP-13 were detected in multinuclear giant cells or in osteoclasts. Control tissues were negative for transcripts encoding MMP-13 mRNA. Fibroblasts of the interface from aseptically loosened endoprostheses selectively express MMP-13. By the expression and the release of MMP-13, these fibroblastic cells may contribute to the local degradation of the extracellular matrix and to bone resorption.

Molecular investigation of lymph nodes in colon cancer patients using one-step nucleic acid amplification (OSNA): a new road to better staging?

A new diagnostic system, called one-step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases.

Neurokinin-2 receptor levels correlate with intensity, frequency, and duration of pain in chronic pancreatitis

OBJECTIVE: Generation and maintenance of pain in chronic pancreatitis (CP) have been shown to be partially attributable to neuroimmune interactions, which involve neuropeptides such as substance P (SP). So far, expression of SP receptors NK-2R, NK-3R, the SP-encoding gene preprotachykinin A (PPT-A), and the SP degradation enzyme neutral endopeptidase (NEP) and their relation to pain in CP have not been determined. METHODS: Tissue samples from patients with CP (n = 25) and from healthy donors (n = 20) were analyzed for PPT-A, NK-2R, NK-3R, and NEP expression using quantitative RT-PCR. NEP protein levels were examined by immunoblot analysis and its localization was determined using immunohistochemistry. A scoring system was used to grade the extent of fibrosis on hematoxylin and eosin- and Masson-Trichrome-stained sections. Messenger RNA levels and the extent of pain were analyzed for correlations. RESULTS: In CP tissues, NK-2R and PPT-A expression was increased, whereas NK-3R and NEP mRNA levels were comparable with normal pancreas. Overexpression of NK-2R was related to the intensity, frequency, and duration of pain in CP patients. NK-1R and NEP expression was significantly related to the extent of fibrosis. CONCLUSIONS: Expression of NK-2R and PPT-A is increased in CP and is associated with pain. Failure to up-regulate NEP may contribute to the disruption of the neuropeptides loop balance in CP and thus may exacerbate the severe pain syndrome.

Quality, size, and composition of pediatric endobronchial biopsies in cystic fibrosis

BACKGROUND: Studies on airway remodeling in children with cystic fibrosis (CF) may be hampered by difficulty in obtaining evaluable endobronchial biopsy specimens because of large amounts of mucus and inflammation in the CF airway. We prospectively assessed how the quality of biopsy specimens obtained from children with CF compare with those from children with other airway diseases. METHODS: Fiberoptic bronchoscopy with endobronchial biopsy was performed in 67 CF children (age range, 0.2 to 16.8 years), 34 children with wheeze/asthma (W/A), and 64 control children with chronic respiratory symptoms. Up to three biopsy specimens were taken and stained with hematoxylin and eosin. Biopsy specimen size and structural composition were quantified using stereology. RESULTS: At least one evaluable biopsy specimen was obtained in 72% of CF children, in 79% of children with W/A, and in 72% of control subjects (difference was not significant). The use of large biopsy forceps (2.0 mm) rather than small biopsy forceps (1.0 mm) [odds ratio (OR), 5.8; 95% confidence interval (CI), 1.1 to 29.8; p = 0.037] and the number of biopsy specimens taken (odds ratio, 2.6; 95% confidence interval, 1.3 to 5.2; p = 0.006) significantly contributed to the success rate. Biopsy size and composition were similar between groups, except that CF children and those patients with W/A had a higher percentage of the biopsy specimen composed of muscle than did control subjects (median 6.2% and 9.7% vs 0.9%, respectively; p = 0.002). CONCLUSIONS: Biopsy size and quality are adequate for the study of airway remodeling in CF children as young as 2 months of age. Researchers should use large forceps when possible and take at least two biopsy specimens per patient. An increased airway smooth muscle content of the airway mucosa may contribute to the pathophysiology of CF lung disease.

Gene therapy with adenovirus-mediated VEGF enhances skin flap prefabrication

We investigated the feasibility in rats of enhancing skin-flap prefabrication with subdermal injections of adenovirus-encoding vascular endothelial growth factor (Ad-VEGF). The left saphenous vascular pedicle was used as a source for vascular induction. A peninsular abdominal flap (8 x 8 cm) was elevated as distally based, keeping the epigastric vessels intact on both sides. After the vascular pedicle was tacked underneath the abdominal flap, 34 rats were randomly divided into three groups according to treatment protocol. The implantation site around the pedicle was injected with Ad-VEGF in group I (n = 10), with adenovirus-encoding green fluorescent protein (Ad-GFP) in control group I (n = 14), and with saline in control group II (n = 10). All injections were given subdermally at four points around the implanted vessel by an individual blinded to the treatment protocol. The peninsular flap was sutured in its place, and 4 weeks later, an abdominal island flap based solely on the implanted vessels was elevated. The prefabricated island flap was sutured back, and flap viability was evaluated on day 7. Skin specimens were stained with hematoxylin and eosin for histological evaluation. In two rats from each group, microangiography was performed to visualize the vascularity of the prefabricated flaps. There was a significant increase in survival of prefabricated flaps in the Ad-VEGF group compared to the control groups: Ad-VEGF, 88.9 +/- 6.1% vs. Ad-GFP, 65.6 +/- 9.4% (P < 0.05) and saline, 56.0 +/- 3.4% (P < 0.05). Sections from four prefabricated flaps treated with Ad-GFP revealed multiple sites of shiny deposits of green fluorescent protein around the area of local administration 1 day and 3 weeks after gene therapy. Histological examination done under high-power magnification (x400) with a light microscope revealed increased vascularity and mild inflammation surrounding the implanted vessel in all groups. However, we were unable to demonstrate any significant quantitative difference with respect to vascularity and inflammatory infiltrates in prefabricated flaps treated with Ad-VEGF compared with controls. Microangiographic studies showed increased vascularity around the implanted pedicle, which was similar in all groups. However, vascularization was distributed in a larger area in the prefabricated flaps treated with Ad-VEGF. In this study, the authors demonstrated that adenovirus-mediated VEGF gene therapy increased the survival of prefabricated flaps, suggesting that it may allow prefabrication of larger flaps and have the potential to reduce the time required for flap maturation.

Decreased visual function after patchy loss of retinal pigment epithelium induced by low-dose sodium iodate

PURPOSE: To correlate damage to the retinal pigment epithelium (RPE) with decreased visual function after the systemic administration of sodium iodate (NaIO(3)). METHODS: Damage was produced in mice by injection of 15, 25, or 35 mg/kg NaIO(3). Visual function was assessed with the cued water maze (WM) behavioral test and the optokinetic reflex (OKR) measurement at different times after injection. Autofluorescence in whole eye flatmounts was quantified, and hematoxylin and eosin staining of paraffin sections was performed to assess changes in the outer retina. RESULTS: After 15 mg/kg NaIO(3), cued WM test results were normal, whereas OKR measurements were significantly decreased at all times. Focal RPE loss began on day 21, but no significant damage to the outer nuclear layer was observed. After 25 mg/kg NaIO(3), the cued WM test was transitionally reduced and the OKR measurement again decreased at all times. Large areas of RPE loss occurred on day 14 with a reduced outer nuclear layer on the same day. With 35 mg/kg NaIO(3), the cued WM test was reduced beginning on day 14 with complete obliteration of the OKR beginning on day 3, large areas of RPE loss on the same day, and a reduced outer nuclear layer on day 7. CONCLUSIONS: Stable, patchy RPE loss was observed with a low concentration of NaIO(3). The OKR measurement showed changes in visual function earlier than the cued WM test and before histologic findings were observed.

Dissection of the Appendix with Ultrasound-Activated Scalpel: An Experimental Study in Pediatric Laparoscopic Appendectomy

Abstract Background: The aim of this study was to examine mechanical, microbiologic, and morphologic changes of the appendicle rim to assess if it is appropriate to dissect the appendix with the ultrasound-activated scalpel (UAS) during laparoscopic appendectomy. Materials and Methods: After laparoscopic resection of the appendix, using conventional Roeder slings, we investigated 50 appendicle rims with an in vitro procedure. The overall time of dissection of the mesoappendix with UAS was noted. Following removal, the appendix was dissected in vitro with the UAS one cme from the resection rim. Seal-burst pressures were recorded. Bacterial cultures of the UAS-resected rim were compared with those of the scissors resected rim. Tissue changes were quantified histologically with hematoxylin and eosin (HE) stains. Results: The average time to dissect the mesoappendix was 228 seconds (25-900). Bacterial culture growths were less in the UAS-resected probes (7 versus 36 positive probes; (p > 0.01). HE-stained tissues revealed mean histologic changes in the lamina propria muscularis externa of 2 mm depth. The seal-burst pressure levels of the appendicle lumen had a mean of 420 mbar. Seal-burst pressures and depths of histologic changes were not dependent on the different stages of appendicitis investigated, gender, or age groups. Seal-burst pressure levels were not related to different depths of tissue changes (P = 0.64). Conclusions: The UAS is a rapid instrument for laparoscopic appendectomy and appears to be safe with respect to stability, sterility and tissue changes. It avoids complex time consuming instrument change manoeuvres and current transmission, which may induce intra- and postoperative complications. Our results suggest that keeping a safety margin of at least 5 mm from the bowel would be sufficient to avoid thermal damage.

Melan-a-positive "pseudomelanocytic nests": a pitfall in the histopathologic and immunohistochemical diagnosis of pigmented lesions on sun-damaged skin

We encountered recently 3 cases with a histopathologic diagnosis of melanoma in situ on sun-damaged skin (male = 2, female = 1; median age: 59 years; range: 52-60 years). The diagnosis was based mainly on the finding of actinic elastosis in the dermis and increased number of melanocytes in the epidermis and was confirmed by strong positivity for Melan-A in single cells and in small nests ("pseudomelanocytic nests"), located at the dermoepidermal junction. Indeed, examination of slides stained with hematoxylin and eosin revealed the presence of marked hyperpigmentation and small nests of partially pigmented cells at the dermoepidermal junction, positive for Melan-A. The histologic and especially the immunohistochemical features were indistinguishable from those of melanoma in situ on chronic sun-damaged skin. In addition, a variably dense lichenoid inflammation was present. Clinicopathologic correlation, however, showed, in all patients, the presence of a lichenoid dermatitis (phototoxic reaction, 1 case; lichen planus pigmentosus, 1 case; and pigmented lichenoid keratosis, 1 case). Our cases clearly show the histopathologic pitfalls represented by lichenoid reactions on chronic sun-damaged skin. Immunohistochemical investigations, especially if performed with Melan-A alone, may lead to confusing and potentially disastrous results. The unexpected staining pattern of Melan-A in cases like ours raises concern about the utility of this antibody in the setting of a lichenoid tissue reaction on chronic sun-damaged skin. It should be underlined that pigmented lesions represent a paradigmatic example of how immunohistochemical results should be interpreted carefully and always in conjunction with histologic and clinical features.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

Assessment of tumor regression of esophageal adenocarcinomas after neoadjuvant chemotherapy: comparison of 2 commonly used scoring approaches.

Histopathologic determination of tumor regression provides important prognostic information for locally advanced gastroesophageal carcinomas after neoadjuvant treatment. Regression grading systems mostly refer to the amount of therapy-induced fibrosis in relation to residual tumor or the estimated percentage of residual tumor in relation to the former tumor site. Although these methods are generally accepted, currently there is no common standard for reporting tumor regression in gastroesophageal cancers. We compared the application of these 2 major principles for assessment of tumor regression: hematoxylin and eosin-stained slides from 89 resection specimens of esophageal adenocarcinomas following neoadjuvant chemotherapy were independently reviewed by 3 pathologists from different institutions. Tumor regression was determined by the 5-tiered Mandard system (fibrosis/tumor relation) and the 4-tiered Becker system (residual tumor in %). Interobserver agreement for the Becker system showed better weighted κ values compared with the Mandard system (0.78 vs. 0.62). Evaluation of the whole embedded tumor site showed improved results (Becker: 0.83; Mandard: 0.73) as compared with only 1 representative slide (Becker: 0.68; Mandard: 0.71). Modification into simplified 3-tiered systems showed comparable interobserver agreement but better prognostic stratification for both systems (log rank Becker: P=0.015; Mandard P=0.03), with independent prognostic impact for overall survival (modified Becker: P=0.011, hazard ratio=3.07; modified Mandard: P=0.023, hazard ratio=2.72). In conclusion, both systems provide substantial to excellent interobserver agreement for estimation of tumor regression after neoadjuvant chemotherapy in esophageal adenocarcinomas. A simple 3-tiered system with the estimation of residual tumor in % (complete regression/1% to 50% residual tumor/>50% residual tumor) maintains the highest reproducibility and prognostic value.

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.

The impact of neural invasion severity in gastrointestinal malignancies: a clinicopathological study.

OBJECTIVES Because neural invasion (NI) is still inconsistently reported and not well characterized within gastrointestinal malignancies (GIMs), our aim was to determine the exact prevalence and severity of NI and to elucidate the true impact of NI on patient's prognosis. BACKGROUND The union internationale contre le cancer (UICC) recently added NI as a novel parameter in the current TNM classification. However, there are only a few existing studies with specific focus on NI, so that the distinct role of NI in GIMs is still uncertain. MATERIALS AND METHODS NI was characterized in approximately 16,000 hematoxylin and eosin tissue sections from 2050 patients with adenocarcinoma of the esophagogastric junction (AEG)-I-III, squamous cell carcinoma (SCC) of the esophagus, gastric cancer (GC), colon cancer (CC), rectal cancer (RC), cholangiocellular cancer (CCC), hepatocellular cancer (HCC), and pancreatic cancer (PC). NI prevalence and severity was determined and related to patient's prognosis and survival. RESULTS NI prevalence largely varied between HCC/6%, CC/28%, RC/34%, AEG-I/36% and AEG-II/36%, SCC/37%, GC/38%, CCC/58%, and AEG-III/65% to PC/100%. NI severity score was uppermost in PC (24.9±1.9) and lowest in AEG-I (0.8±0.3). Multivariable analyses including age, sex, TNM stage, and grading revealed that the prevalence of NI was significantly associated with diminished survival in AEG-II/III, GC, and RC. However, increasing NI severity impaired survival in AEG-II/III and PC only. CONCLUSIONS NI prevalence and NI severity strongly vary within GIMs. Determination of NI severity in GIMs is a more precise tool than solely recording the presence of NI and revealed dismal prognostic impact on patients with AEG-II/III and PC. Evidently, NI is not a concomitant side feature in GIMs and, therefore, deserves special attention for improved patient stratification and individualized therapy after surgery.

Tumor budding in colorectal cancer revisited: results of a multicenter interobserver study.

Tumor budding in colorectal cancer (CRC) is recognized as a valuable prognostic factor but its translation into daily histopathology practice has been delayed by lack of agreement on the optimal method of assessment. Within the context of the Swiss Association of Gastrointestinal Pathology (SAGIP), we performed a multicenter interobserver study on tumor budding, comparing hematoxylin and eosin (H&E) with pan-cytokeratin staining using a 10 high power field (10HPF) and hotspot (1HPF) method. Two serial sections of 50 TNM stage II-IV surgically treated CRC were stained for H&E and pan-cytokeratin. Tumor buds were scored by independent observers at six participating centers in Switzerland and Austria using the 10HPF and 1HPF method on a digital pathology platform. Pearson correlation (r) and intra-class correlation coefficients (ICC) comparing scores between centers were calculated. Three to four times more tumor buds were detected in pan-cytokeratin compared to H&E slides. Correlation coefficients for tumor budding counts between centers ranged from r = 0.46 to r = 0.91 for H&E and from r = 0.73 to r = 0.95 for pan-cytokeratin slides. Interobserver agreement across all centers was excellent for pan-cytokeratin [10HPF: ICC = 0.83 and 1HPF: ICC = 0.8]. In contrast, assessment of tumor budding on H&E slides reached only moderate agreement [10HPF: ICC = 0.58 and 1HPF: ICC = 0.49]. Based on previous literature and our findings, we recommend (1) pan-cytokeratin staining whenever possible, (2) 10HPF method for resection specimens, and (3) 1HPF method for limited material (preoperative biopsy or pT1). Since tumor budding counts can be used to determine probabilities of relevant outcomes and as such more optimally complement clinical decision making, we advocate the avoidance of cutoff scores.