Conventional Histopathological Techniques Research Articles

Step Serial Sectioning in Head and Neck Squamous Cell Carcinoma of Unknown Primary

Patients with suspected head and neck squamous cell carcinoma of unknown primary (HNSCCUP) may undergo tonsillectomy and tongue base mucosectomy (TBM) to help identify clinicoradiologically occult primary disease. It is hypothesized that when these diagnostic specimens are analyzed, conventional histopathological (CH) techniques risk missing small primary tumors that may be hidden in the tissue blocks. To establish the outcomes of a step serial sectioning (SSS) histopathological technique vs CH when analyzing diagnostic tissue specimens from TBM and tonsillectomy performed for HNSCCUP. The MOSES prospective multicenter noninterventional cohort study was conducted over a 25-month period from November 2019 at secondary and tertiary care ear, nose, and throat departments in the United Kingdom and included adults with clinicoradiologically occult HNSCCUP who were undergoing TBM. Conventional histopathological techniques performed on TBM and tonsillectomy specimens at participating centers, followed by SSS performed at the central laboratory. Identification of cancer on central histopathological review of TBM and tonsillectomy specimens. Tissue from 58 eligible patients was analyzed (median [range] age, 58 [47-82] years; 10 women [17%]), with 20 480 sections cut in the laboratory and 4096 sections directly examined by a pathologist (median [range], 64 [28-135] per patient). The overall identification rate for TBM following SSS according to study protocol was 50.0% (95% CI, 37.5%-62.5%) and by subgroups was 42.9% (95% CI, 21.4%-67.4%) when performed following a negative bilateral tonsillectomy, 46.7% (95% CI, 24.8%-69.9%) at the same time as bilateral tonsillectomy, and 57.1% (95% CI, 36.5%-75.5%) following historic tonsillectomy. Conventional histopathological techniques at central review identified 2 undiagnosed primary tumors and revised the diagnosis of 2 other cases (1 nonmalignant and another down staged). Step serial sectioning identified a single additional tumor: an ipsilateral synchronous tongue base tumor for which a contralateral tumor had been identified on CH. Multifocal disease was seen in 5 (8.6%); all were human papillomavirus-related and in the tongue base. In this multicenter cohort study of patients undergoing TBM for HNSCCUP, SSS was associated with added considerable histopathological workload with minimal additional diagnostic benefit. A second opinion for conventional histological techniques may be more beneficial. Synchronous primary disease should be considered when planning diagnostic oropharyngeal surgery for these patients.

Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

Association of tumors having Epstein-Barr virus in surrounding lymphocytes with poor prognosis.

Infection with certain viruses is an important cause of cancer. The Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium recently analyzed the whole-genome sequencing (WGS) data from 2656 cases across 21 cancer types, and indicated that Epstein-Barr virus (EBV) is detected in many different cancer cases at a higher frequency than previously reported. However, whether EBV-positive cancer cases detected by WGS-based screening correspond to those detected by conventional histopathological techniques is still unclear. In this study, to elucidate the involvement of EBV in various cancers, we reanalyzed the WGS data of the PCAWG cohort combined with the analysis of clinical samples of gastric and pancreatic cancer in our cohort. Based on EBV copy number in each case, we classified tumors into three subgroups: EBV-High, EBV-Low, and EBV-Negative. The EBV-High subgroup was found to be EBV-positive in the cancer cells themselves, whereas the EBV-Low subgroup was EBV-positive in the surrounding lymphocytes. Further, the EBV-Low subgroup showed a significantly worse prognosis for both gastric cancer and across cancer types. In summary, we classified tumors based on EBV copy number and found a unique cancer subgroup, EBV-positive in the surrounding lymphocytes, which was associated with a poor prognosis.

X-ray Dark-Field Imaging (XDFI)-a Promising Tool for 3D Virtual Histopathology.

X-ray dark-field imaging (XDFI) utilizing a thin silicon crystal under Laue case enables visualizing three-dimensional (3D) morphological alterations of human tissue. XDFI uses refraction-contrast derived from phase shift rather than absorption as the main X-ray image contrast source to render 2D and 3D images of tissue specimens in unprecedented detail. The unique features of XDFI are its extremely high sensitivity (approximately 1000:1 compared to absorption for soft tissues under X-ray energy of around 20keV, theoretically) and excellent resolution (8.5μm) without requiring contrast medium or staining. Thus, XDFI-computed tomography can generate 3D virtual histological images equivalent to those of stained histological sections pathologists observe under low-power light microscopy as far as organs and tissues selected as samples in preliminary studies. This paper reviews the fundamental principles and the potential of XDFI, describes two optical setups for XDFI with examples, illustrates features of XDFI that are salient for histopathology, and presents XDFI examples of refraction-contrast images of atherosclerotic plaques, musculoskeletal tissue, neuronal tissue, and breast cancer specimens. Availability of this X-ray imaging in routine histopathological evaluations of tissue specimens would help guide clinical decision making by highlighting suspicious areas in unstained, thick sections for further sampling and analysis using conventional histopathological techniques. XDFI is a promising tool for 3D virtual histopathology.

Synchrotron Radiation-Based FTIR Microspectroscopic Imaging of Traumatically Injured Mouse Brain Tissue Slices.

Traumatic brain injury (TBI) is a health problem of global concern because of its serious adverse effects on public health and social economy. A technique that can be used to precisely detect TBI is highly demanded. Here, we report on a synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopic imaging technique that can be exploited to identify TBI-induced injury by examining model mouse brain tissue slices. The samples were first examined by conventional histopathological techniques including hematoxylin and eosin (H&E) staining and 2,3,5-triphenyltetrazolium chloride staining and then spectroscopically imaged by SR-FTIR. SR-FTIR results show that the contents of protein and nucleic acid in the injured region are lower than their counterparts in the normal region. The injured and normal regions can be unambiguously distinguished from each other by the principle component analysis of the SR-FTIR spectral data corresponding to protein or nucleic acid. The images built from the spectral data of protein or nucleic acid clearly present the injured region of the brain tissue, which is in good agreement with the H&E staining image and optical image of the sample. Given the label-free and fingerprint features, the demonstrated method suggests potential application of SR-FTIR spectroscopic mapping for the digital and intelligent diagnosis of TBI by providing spatial and chemical information of the sample simultaneously.

Towards virtual biopsies of gastrointestinal tissues using photoacoustic remote sensing microscopy.

Gastrointestinal (GI) tissue biopsies provide critical diagnostic information for a wide variety of conditions such as neoplastic diseases (colorectal, small bowel and stomach cancers) and non-neoplastic diseases (inflammatory disorders, infection, celiac disease). Endoscopic biopsies collect small tissue samples that require resource intensive processing to permit histopathological analysis. Unfortunately, the sparsely collected biopsy samples may fail to capture the pathologic condition because selection of biopsy sites relies on macroscopic superficial tissue features and clinician judgement. Here, we present the first all-optical non-contact label-free non-interferometric photoacoustic microscopy system capable of performing "virtual biopsies". A modular photoacoustic remote sensing (PARS™) architecture is used facilitating imaging of unprocessed tissues providing information similar to conventional histopathological staining techniques. Prospectively this would allow gastroenterologists to assess subcellular tissue morphology in situ when selecting biopsy location. Tested on preserved unstained human and freshly resected murine tissues, the presented PARS microscope rapidly retrieves images of similar area to current biopsies, while maintaining comparable quality to the current standard for histopathological analysis. Additionally, results show the first label free assessment of subsurface cellular morphology in FFPE GI tissue blocks. Clinically relevant features are recovered including cellular details such as lamina propria within colon tissue and cell nuclear structure in resected smooth muscle. Constructed with a modular architecture, this system facilitates the future development of compact imaging heads. The modular PARS system overcomes many of the challenges with imaging unstained thick tissue in situ, representing a significant milestone in the development of a clinical microscope providing virtual biopsy capabilities.

Concomitant use of Congo red staining and confocal laser scanning microscopy to detect amyloidosis in oral biopsy: A clinicopathological study of 16 patients

ABSTRACTTwenty oral biopsies from 16 patients were analyzed both by traditional microscopy and by confocal laser scanning microscopy. Using conventional histopathological techniques, the diagnosis of amyloidosis was confirmed only in 15 biopsies. Using confocal laser scanning microscopy, amyloid deposits were detected in all of the samples. The current study shows that confocal laser scanning analysis helps to identify minimal amyloid deposits that could be overlooked using traditional microscopy, thus raising the sensitivity of oral biopsy up to 100%.

Comparison of magnetic nanoparticle and microwave hyperthermia cancer treatment methodology and treatment effect in a rodent breast cancer model

Purpose: The purpose of this study was to compare the efficacy of iron oxide/magnetic nanoparticle hyperthermia (mNPH) and 915 MHz microwave hyperthermia at the same thermal dose in a mouse mammary adenocarcinoma model. Materials and methods: A thermal dose equivalent to 60 min at 43 °C (CEM60) was delivered to a syngeneic mouse mammary adenocarcinoma flank tumour (MTGB) via mNPH or locally delivered 915 MHz microwaves. mNPH was generated with ferromagnetic, hydroxyethyl starch-coated magnetic nanoparticles. Following mNP delivery, the mouse/tumour was exposed to an alternating magnetic field (AMF). The microwave hyperthermia treatment was delivered by a 915 MHz microwave surface applicator. Time required for the tumour to reach three times the treatment volume was used as the primary study endpoint. Acute pathological effects of the treatments were determined using conventional histopathological techniques. Results: Locally delivered mNPH resulted in a modest improvement in treatment efficacy as compared to microwave hyperthermia (p = 0.09) when prescribed to the same thermal dose. Tumours treated with mNPH also demonstrated reduced peritumoral normal tissue damage. Conclusions: Our results demonstrate similar tumour treatment efficacy when tumour heating is delivered by locally delivered mNPs and 915 MHz microwaves at the same measured thermal dose. However, mNPH treatments did not result in the same type or level of peritumoral damage seen with the microwave hyperthermia treatments. These data suggest that mNP hyperthermia is capable of improving the therapeutic ratio for locally delivered tumour hyperthermia. These results further indicate that this improvement is due to improved heat localisation in the tumour.

Abstract 1523: MALDI mass spectrometry imaging for on-tissue spatial profiling of tumor-specific N-linked glycans and lipids in renal carcinomas.

Abstract Pathologic staging of renal clear cell carcinoma (ccRCC) provides useful but limited prognostic information suggesting that there are underlying molecular processes not readily identified through conventional histopathological techniques in the primary tumor. At the molecular level, transcript, metabolomic and protein expression patterns have indicated a striking Warburg Effect profile in ccRCC tissues, with major affects on sugar and lipid metabolism. Our group has been applying MALDI-MS imaging approaches to ccRCC tissues to profile lipids and glycans associated with disease progression. As both lipids and glycans can act as signaling molecules and have been associated with cancer progression, ccRCC tissues were selected by a pathologist to contain substantial tumor, non-tumor and tumor margin regions to characterize the differential localization of lipid and glycan species within the tissue. Lipid profiles from fresh-frozen tissue slides coated in DHB matrix were obtained on a dual source Bruker Solarix 70 FTICR mass spectrometer. Glycans were imaged in a similar fashion with a method developed by our lab using protein N-glycanase F to release protein bound N-glycans. There are clear patterns of specific lipid and glycan species that are only present along the margin of the tumor and non-tumor interface, or those primarily localized in non-tumor or tumor regions. Off-tissue extraction of lipids and glycans from similarly processed tissues and further mass spectrometry analysis was done to confirm structural designations. Linkage of differentially expressed lipid and glycan species with clinical data and other molecular markers of ccRCC is ongoing. This approach has the potential to not only improve prognostic assessment and enhance post-operative surveillance, but also to better define the underlying biology of ccRCC aggressiveness and identify new rational targets for therapeutic intervention. Citation Format: Richard R. Drake, Thomas Powers, Ellen Jones, Raymond S. Lance, Anand S. Mehta, Dean A. Troyer. MALDI mass spectrometry imaging for on-tissue spatial profiling of tumor-specific N-linked glycans and lipids in renal carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2013-1523

Defining the molecular tumor margin regions of clear cell renal carcinoma tissues by MALDI-MS imaging of lipid and glycan species.

421 Background: The frequent tumor recurrence associated with clear cell renal cell carcinoma (ccRCC) suggests that there are underlying molecular processes present in the remaining tissue following nephrectomy that are not identified through conventional histopathological techniques. At the molecular level, transcript, metabolomic, and protein expression patterns have indicated a striking Warburg Effect profile in ccRCC tissues, with major affects on sugar and lipid metabolism. Our group has been applying MALDI mass spectrometry imaging approaches to uniquely profile lipids and glycans associated with disease progression directly in frozen tissue slides. Methods: Frozen ccRCC tissues with tumor, non-tumor adjacent, and tumor margin regions were selected by a pathologist. Lipid profiles from fresh-frozen tissue slides coated in DHB matrix were obtained on a dual source Bruker Solarix 70 FTICR mass spectrometer. Glycans were imaged in a similar fashion in ethanol-washed tissues using on-tissue protein N glycanase F digestion to release surface N-glycans. Detected lipid and glycan ion intensities were converted to a color pixel scale for creating an image of individual peaks, linked directly to the histopathology of the tissue. Results: Five groups of lipid and N-glycan species were identified following MALDI tissue imaging, those present in the immediate margin area of non-tumor tissue adjacent to tumor; only in non-tumor regions; only in tumor regions; primarily in tumor regions but extended beyond the margin; and present throughout the tissue. Specific lipid and glycan species associated with margin and tumor regions are being correlated with disease progression and pathology data. Conclusions: Analysis of the periphery of the tumor tissue and the normal parenchyma or capsule regions at this biomolecule level may better define the metastatic potential of the tumor as compared to analysis of the central tumor region. This approach has the potential to not only improve prognostic assessment and treatment choices, but also to inform on the underlying biology of ccRCC metastasis and new rational targets for therapeutic intervention.

Toward Routine Use of 3D Histopathology as a Research Tool

Three-dimensional (3D) reconstruction and examination of tissue at microscopic resolution have significant potential to enhance the study of both normal and disease processes, particularly those involving structural changes or those in which the spatial relationship of disease features is important. Although other methods exist for studying tissue in 3D, using conventional histopathological features has significant advantages because it allows for conventional histopathological staining and interpretation techniques. Until now, its use has not been routine in research because of the technical difficulty in constructing 3D tissue models. We describe a novel system for 3D histological reconstruction, integrating whole-slide imaging (virtual slides), image serving, registration, and visualization into one user-friendly package. It produces high-resolution 3D reconstructions with minimal user interaction and can be used in a histopathological laboratory without input from computing specialists. It uses a novel method for slice-to-slice image registration using automatic registration algorithms custom designed for both virtual slides and histopathological images. This system has been applied to >300 separate 3D volumes from eight different tissue types, using a total of 5500 virtual slides comprising 1.45 TB of primary image data. Qualitative and quantitative metrics for the accuracy of 3D reconstruction are provided, with measured registration accuracy approaching 120 μm for a 1-cm piece of tissue. Both 3D tissue volumes and generated 3D models are presented for four demonstrator cases.

Granular cells in a benign parotid gland tumour: an attempt to understand its probable histogenesis

Tumours that occur in the oral cavity may contain granular cells as a component of their pathology. A more common granular cell lesion occurring in the head and neck region is the granular cell tumour (GCT) that usually arises in the tongue or the buccal mucosa. Granular cell tumours are very rare in the parotid gland with only 11 cases previously reported in the English literature. We report a case of a benign tumour involving the parotid gland of a young female patient. The case was diagnostically challenging due to the large proportion of granular cells masking the underlying pathology. Histopathological features and immunohistochemical analysis favoured a diagnosis of a benign GCT. The present report provides an insight into the differential diagnosis and attempts to characterise the granular cells with the use of the wellestablished immunohistochemical markers and conventional histopathological techniques.

HISTOLOGICAL AND HISTOMETRIC ANALYSIS OF SOLITARY AND NON-SYNDROME ASSOCIATED MULTIPLE ODONTOGENIC KERATOCYSTS

Objectives: Odontogenic keratocysts are common, locally destructive lesions of the head and neck region. They can occur as solitary or multiple lesions, and if multiple, they are mostly associated with Gorlin-Goltz syndrome. This study aims to categorize the OKCs which behave more aggressively, using conventional histopathological techniques. Methodology: The present study exploits conventional histopathological techniques to gain an insight into the behavior of the OKC (both solitary and non-syndrome associated multiple OKCs). The histological features (keratinization pattern, presence of satellite cysts / odontogenic islands, epithelial infoldings and corrugations) and histometric parameters (total number of nuclei, epithelial height, total nuclear density, number of basal nuclei, basement membrane length, basal nuclear density and mitotic index) were compared between the two groups of cysts. Result: Multiple OKCs exhibited increase in number of infoldings, corrugations, mitotic index, reduced epithelial height, total number of nuclei, number of basal nuclei, and basal nuclear density. This information may be used to predict their biologic behavior and thus serve as a basis for diagnostic and therapeutic strategies. Conclusion: Conventional histopathological techniques can provide valuable information which may be useful to categorize those OKCs that have more aggressive biological behavior and helps in deciding the treatment protocol.

Oxidized regenerated cellulose does not prevent the formation of experimental postoperative perineural fibrosis assessed by digital analysis.

It is difficult to prevent and treat intra- and peri-neural fibrosis after peripheral nerve surgery. Many authors have attempted to develop and verify the effectiveness of substances to decrease the formation of adherences in different tissues. this study aimed to assess the effectiveness of a barrier of oxidized regenerated cellulose (ORC) to reduce adherence and perineural fibrosis in a model of surgical perineural induced fibrosis in rat sciatic nerve in 40 rats. After tissue aggression, the nerve of the right rear limb was wrapped in ORC and the left limb served as control. Animals were killed at 3 and 6 weeks, and nerves and muscle mass were extracted en bloc. Connective tissue was quantified by conventional histopathological techniques and Fibrosis HR(R) automatic image analysis. No significant differences were found in intra- or peri-neural induced fibrosis between control nerves (6.88% and 8.90%, respectively) and treated nerves (6.57% and 9.90%) at 3 or 6 weeks (10.41% and 12.51% in controls; 11.85% and 15.72% in treated nerves). Inflammatory phenomena and granulomatous reactions were more frequent in treated animals. ORC conferred no advantage in prevention of nerve fibrosis and might have interfered with healing.

Abstract 2954: Overexpression of specific genes in surgical margins of head and neck squamous cell carcinoma patients may predict a significantly increased risk of recurrence

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is a high incidence and mortality disease. Despite significant improvements in therapeutic strategies, the overall survival rate in 5 years is around 50%. The presence of microscopic tumor in surgical margins is associated with treatment failure, locoregional recurrence and reduction of overall survival. For this reason, it is vital to ensure tumor-free surgical margins in these patients. Commonly, the extension of surgical resection is determined during the surgery through a low sensitivity histological exam of frozen tissue stained with Hematoxylin and Eosin. Since genetic alterations precede histological alterations, molecular markers may be useful to identify cells that have already started malignant transformation without having developed a histological phenotype that could be detected through conventional histopathological techniques. Also, novel molecular techniques make possible to obtain molecular data of surgical margins in a period of time compatible with the surgery, helping the surgeon in the resection of positive molecular margins in the same surgery and thereby reducing the risk of local recurrence. This study aimed to determine the gene expression pattern of specific markers in HNSCC surgical margins and verify the utility of these markers in detecting the presence of tumor cells in these tissues and predict tumor recurrence. Nine selected genes were evaluated and a panel of 5 genes showed a high specificity and good sensibility. Overexpression of at least one of these genes was found in 50% (23/46) of histologically free surgical margins analyzed and was associated with local recurrence. Patients with positive molecular margins were 7.3-fold more likely to have local recurrence. These preliminary results suggest that the approach used in this analysis is more sensible than traditional histopathological exams and could be useful for the intraoperative diagnosis of the presence of tumor cells in HNSCC surgical margins and as an informative tool to identify and monitor subsets of HNSCC patients with an increased risk of recurrence and consequently reduced survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2954.

Identification of Microbial Biofilms in Osteonecrosis of the Jaws Secondary to Bisphosphonate Therapy

Biofilm theory has emerged to explain the etiology of the chronic infections that have come to constitute between 65% to 80% of the microbial diseases treated by physicians in the developed world. The purpose of this article is to report for the first time the observation of multispecies microbial biofilms on affected bone in patients with osteonecrosis of the jaws (ONJ) secondary to bisphosphonate therapy. A program has been established at the University of Southern California to monitor and evaluate patients with ONJ as a multidisciplinary collaboration between the School of Dentistry, Center for Biofilms, Center for Craniofacial Molecular Biology and the Keck School of Medicine. From this cohort, 4 patients with active ONJ who were scheduled for necessary treatment in the form of sequestrectomy gave informed consent for this study. Bone samples were evaluated using conventional histopathologic techniques and scanning electron microscopy, a technique applicable to biofilm characterization. Bone specimens from affected sites in all patients showed large areas occluded with biofilms comprising mainly bacteria, and occasionally yeast, embedded in extracellular polymeric substance. The number of bacterial morphotypes in the biofilms ranged from 2 to 15, and they included species from the genus Fusobacterium, bacillus, actinomyces, staphylococcus, streptococcus, Selenomonas, and 3 different types of treponemes. The yeast identified was consistent with Candida species. Co-aggregation was observed between different species within the biofilms. These findings have important clinical and therapeutic implications and may suggest a role for microbial biofilms in the disease process of ONJ.

Micrometastases detected by cytokeratin 19 expression in sentinel lymph nodes of patients with early-stage cervical cancer

The purpose of this study was to detect micrometastases in sentinel lymph nodes (SLNs) by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) analyses for cytokeratin 19 (CK19) expression in early-stage cervical cancer. One hundred twenty-five SLNs were collected from 46 patients with early-stage cervical cancer. Conventional histopathologic techniques revealed 14 metastatic SLNs from 11 out of 46 patients. CK19 expression was detected by RT-PCR and IHC in all the 125 SLNs. Cervical cancer tissues from nine patients and five pelvic lymph nodes from the patients without tumor were utilized as positive and negative controls, respectively. All the metastastic SLNs on conventional histopathologic techniques were positive by either RT-PCR or IHC analyses, while all the positive controls were positive and all the negative controls were negative as expected. Of 35 patients without metastatic SLNs on conventional histopathologic techniques, the detection rate of micrometastases was 42.85% by RT-PCR and 20% by IHC analyses. RT-PCR and IHC were more sensitive to identify micrometastases in SLNs of patients with early-stage cervical cancer than routine pathology. These findings demonstrated that micrometastasis could be identified by molecular technique such as RT-PCR and IHC analyses for CK19 expression. RT-PCR was more sensitive to detect micrometastases in SLNs than IHC in patients with early-stage cervical cancer. Therefore, molecular assessment of the SLNs may be a valuable tool to complement routine histologic examination of cervical cancer. The importance of micrometastases in SLNs is under close clinical observation to determine whether it can be used as a predicting factor to help us make decision whether to proceed with whole-pelvic lymph node dissection or as a prognostic factor for clinical outcome.

Inorganic particles in the skin of inhabitants of volcanic areas of Central America: their possible immunomodulatory influence in leishmaniasis and leprosy

We have evaluated biopsies from patients with atypical nodular and typical ulcerated lesions of cutaneous leishmaniasis, from leishmanin reactions and skin from normal individuals from Nicaragua, Honduras and Guatemala for the presence of inorganic particles using confocal microscopy with a polarised light source and conventional histopathological techniques. Analysis by semiquantitative confocal microscopy permitted the demonstration of significantly larger numbers of particles in atypical lesions. Silica and aluminium, important components of these particles, were less abundant in particles from normal skin. The histology of these atypical lesions, characterised by 'naked' sarcoidal granulomas with epithelioid differentiation but very few lymphocytes, was very similar to the histological reaction observed after 14 days in persisting inflammation at leishmanin skin test sites. The presence of these unusual lesions in areas of Central American countries characterised by the presence of large amounts of volcanic ash, as well the unexpectedly low prevalence of leprosy in Central America, suggest that environmental factors may contribute significantly to the frequency and clinical manifestations of these infections. Among possible environmental features, the presence of inorganic particles with immunomodulatory properties in the skin may be a significant factor.

Do basal cell carcinomas recur after complete conventional surgical excision?

For 1378 patients treated in the 11 years 1988-1998 by conventional excision of 1635 basal cell carcinomas, 1516 first index lesions were histologically completely excised. All patients having more than one BCC excised were identified from the data base from 1988 to 2003 to give minimum 5 years follow for last treated primary lesions in 1998. Measured clearance margins around the initial lesions at or near sites of presumptive recurrent lesions were noted and the lesions recorded photographically. All incompletely excised lesions whether or not re-excised were excluded. The median age for all patients was 70 years. Over minimum 5 years follow up, six patients developed nine subsequent lesions contiguous with the scar or graft repair of primary index lesion excision site (probable recurrences). The median interval to recurrence was 41 months (4 months-8 years 10 months), with median lateral clearance margin around the primary tumour of 2 mm (0.3-6.8 mm). A further nine patients developed 11 new lesions near (within 1cm of) the scar or graft of primary index lesion excision site (possible recurrences). The median interval to recurrence was 59 months (1 year-8 years 6 months). The median lateral clearance margin around the primary tumour was 4.1 mm (0.8-5.8 mm). For the two groups combined the maximum recurrence rate expressed as a percentage of index lesions was 1.3% (20/1516). Two thirds of possible and probable recurrences occurred in the temple and forehead, although these sites represented only 22% of all lesions, which may rather suggest new lesions in an area of field change as opposed to residual disease. The measured clearance margins reported here perhaps suggest that some original lesions may well have been completely excised primarily and many 'recurrences' were new primaries. These figures indicate there is a low order of probability for the incidence of recurrent basal cell carcinoma during minimum 5 years follow period after conventional surgical excision and conventional histological assessment of tumour resection margins.

Metastatic Testicular Carcinoma From the Colon With Clinical, Immunophenotypical, and Molecular Characterization: Report of a Case

We report a case of testicular metastasis from a colonic adenocarcinoma. The presentation of testicular metastasis, diagnosis, management, and possible modes of spread are reported. In addition to conventional investigations and histopathologic techniques, a molecular study of the testicular metastasis and colon primary, using microsatellite analysis, was performed to confirm the primary origin. Its potential uses are discussed.