Rolf Pfannl

Multicolored stain-free histopathology with coherent Raman imaging

Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.

Adjuvant Gamma Knife radiosurgery following surgical resection of brain metastases: a 9-year retrospective cohort study

Given the potential morbidity of whole brain radiation therapy (WBRT), there has been an increasing trend to defer WBRT and deliver Gamma Knife stereotactic radiosurgery (GKS) to cerebral metastatic lesions. We analyzed our experience delivering GKS to the tumor cavity following surgical resection of brain metastases and compared our results to patients receiving WBRT after surgical resection of a metastatic lesion. We performed a retrospective review of patients undergoing surgical resection of at least one brain metastasis between December 1999 and December 2008. Both univariate and multivariate Cox proportional hazards regression were utilized to analyze the influence of various prognostic factors on survival. Twenty-five patients had a metastatic lesion resected followed by adjuvant GKS to the resection cavity while another 18 had surgical resection followed by WBRT. Aside from a disparity in gender distribution (72% of GKS patients were female while women only constituted 28% of the WBRT group), no significant differences existed between groups. The median survival for patients receiving GKS was 15.00xa0months as compared to 6.81xa0months among those receiving WBRT (Pxa0=xa00.08). Univariate Cox regression analysis identified the number of metastases (HR 1.65, 95% CI 1.07–2.54, Pxa0=xa00.02) and regional recurrence (RR 5.23, 95% CI 1.78–15.38, Pxa0=xa00.003) as poor prognostic factors. Multivariate regression analysis showed that regional recurrence (HR 5.17, 95% CI 1.69–15.78, Pxa0=xa00.004) was again strongly associated with worse survival. Although limited by the retrospective nature of our study and lack of some clinical measures, patients undergoing GKS to the resection cavity had a trend towards longer median survival.

Distinctive association of p16INK4a overexpression with penile intraepithelial neoplasia depicting warty and/or basaloid features: a study of 141 cases evaluating a new nomenclature.

From the pathogenic point of view, penile cancers may be grouped in human papillomavirus-related and unrelated tumors, each one of them with distinctive morphologic features. The former are predominantly composed of small, undifferentiated basaloid cells, with more or less prominent koilocytic changes, and the latter of keratinizing differentiated squamous cells. The same cellular types are observed in precancerous lesions. On the basis of these observations, we constructed a novel nomenclature for penile precancerous lesions and classified them as penile intraepithelial neoplasia (PeIN) of differentiated, warty, basaloid, and warty-basaloid types. The aim of this study was to test the usefulness of immunohistochemical p16INK4a overexpression, considered as a surrogate for high-risk human papillomavirus infection, using this classification system. We pathologically evaluated 141 patients with PeIN, associated (123 cases) and unassociated (18 cases) with invasive cancer. Distribution of PeIN types was: differentiated, 72%; basaloid, 9%; warty-basaloid, 7%; warty, 4%; and mixed, 7%. There was a striking similarity in the morphology of in situ and invasive squamous cell carcinomas. Differentiated PeIN was commonly associated with usual, verrucous, papillary, and other low-grade keratinizing variants of squamous cell carcinoma whereas in basaloid and warty carcinomas the presence of in situ lesions with similar morphology was habitual. We evaluated p16INK4a overexpression using a 4-tiered (0, 1, 2, and 3) pattern-based system. To properly distinguish differentiated PeIN from in situ lesions with warty and/or basaloid features only pattern 3, which requires full-thickness staining in all epithelial cells, was considered positive. Using this approach, there was a significant association of the negative patterns and differentiated PeIN and of the positive pattern and warty, basaloid, and warty-basaloid PeIN (P<0.0001). Basaloid variant had the strongest association. The sensitivity rate of p16INK4a positivity for discriminating types of PeIN was of 82%, with a specificity of 100% and an accuracy of 95%. Lichen sclerosus was identified in 42 cases and their epithelial component was p16INK4a negative in all cases. Although more studies are necessary to confirm these observations, p16INK4a overexpression seems to be a useful tool for discriminating differentiated from warty, basaloid, and warty-basaloid PeIN.

Histologic Grade in Penile Squamous Cell Carcinoma: Visual Estimation Versus Digital Measurement of Proportions of Grades, Adverse Prognosis With any Proportion of Grade 3 and Correlation of a Gleason-like System With Nodal Metastasis

Histologic grade has been reported as an important pathologic parameter predictive of nodal metastases and outcome in patients with penile squamous cell carcinoma. There is no consensus about the criteria for grading and the proportion of anaplastic cells required to classify a tumor as high grade. The incidence and management of heterogeneous tumors (tumors harboring more than 1 histologic grade) are not well established. The purposes of this study were to present a grading model for penile cancer, to test the practicality of the system by comparing a visual (“naked-eye”) estimation of the proportions of grades with a digitally guided measuring system and to determine the influence on nodal metastasis of the various proportions of grades. A total of 117 penectomy and circumcision specimens with bilateral inguinal lymph node dissections were studied and 62 heterogeneous tumors were identified (53%). The following steps were taken: (1) design of a grading system model; (2) determination of proportions of histologic grades by naked-eye evaluation and by digital measurement; (3) evaluation of metastasis according to proportions of grades; (4) determination of the influence of site of grade 3 in nodal metastasis; (5) design of a Gleason-like scoring system; and (6) statistical evaluation. We designed a 3-tier grading system. Grade 1: well-differentiated cells, almost undistinguishable from normal squamous cells except for the presence of minimal basal/parabasal cell atypia. Grade 3: tumors predominantly composed of anaplastic cells. Grade 2: all tumors not fitting into criteria described for grade 1 or 3. A visual and digital-based (slides scanned and the corresponding areas measured with an image-editing software) proportions of grades were estimated and the metastatic rate between them were confronted using different proportions of grade 3. To evaluate the influence of site of grade 3 on nodal metastasis, we selected 20 heterogeneous tumors. We established 3 sites: superficial, at or within the main tumor and deep at front of invasion. There was no significant difference between the visual estimation and the digital measurement systems. Heterogeneous tumors comprised about half of penile squamous cell carcinomas. The majority of the heterogeneous tumors were composed of a combination of grades 2 and 3 (68%). No statistical differences were noted in the incidence of nodal metastasis when comparing tumors with various proportions of anaplastic cells (P>0.10 in all cases). Metastatic rate was significantly more frequent in tumors harboring any proportion of grade 3 as compared with tumors without grade 3 (58% vs. 14%, P=0.04). Any proportion of grade 3 was equally associated with a significant risk of nodal metastasis. A Gleason-like system showed a correlation of higher scores and rate of nodal metastasis. No predictive advantage was found when comparing the Gleason-like model with the proposed 3-tier grading system. The proposed grading system emphasized both ends of the differentiation spectrum and was based on easily recognizable morphologic criteria. When histologically evaluating penile carcinomas, we recommend a careful search of areas of grade 3. Any focus of grade 3 should be sufficient to grade the neoplasm as a high-grade tumor.

Anatomic levels: important landmarks in penectomy specimens: a detailed anatomic and histologic study based on examination of 44 cases.

The majority of squamous cell carcinomas of the penis arise from the glans, and the prognosis is related significantly to the depth of invasion of crucial anatomic landmarks. Accurate information related to this can only be obtained when specimens are carefully evaluated grossly. Most pathologists in developed countries encounter resected specimens of penile carcinoma infrequently, and gross evaluation is occasionally suboptimal, potentially preventing obtaining reliable prognostic information. The four distinct levels of the glans penis are the epithelium, lamina propria, corpus spongiosum, and corpus cavernosum. A simple method for pathologic evaluation of the glans is presented. Noteworthy findings in our study of a South American population were that the distance from the lamina propria to tunica albuginea ranged from 7 to 13 to 6 mm at the dorsal, central, and ventral areas of the corpus spongiosum, respectively. The most distal portion of the corpus cavernosum was located within the glans in 34 of 44 cases and in the body of the penis in only 10. The corpus spongiosum was thinner in the former cases. These anatomic variations may bear on prognosis.

Distinctive immunohistochemical profile of penile intraepithelial lesions: A study of 74 cases

Several classification schemes for penile precancerous lesions have been proposed, but none of them seems to correlate with the current understanding of penile cancer pathogenesis. Recently, a system, which takes into account morphologic features and purported etiopathogenesis, was proposed, separating penile intraepithelial neoplasia (PeIN) in differentiated and warty/basaloid subtypes. This study was designed to seek an immunohistochemical profile that can be helpful in the classification and differential diagnosis of penile epithelial abnormalities and precancerous lesions using the aforementioned system. The immunohistochemical panel included stains for p16INK4a, p53, and Ki-67. For p16INK4a immunostaining, only full-thickness positivity in all epithelial cells was considered as positive; for p53 and Ki-67 immunostaining, patchy or diffuse nuclear positivity above the basal layer was considered as positive. Seventy-four lesions in 59 patients were selected and classified as follows: differentiated PeIN, 34 cases; squamous hyperplasia (SH), 21 cases; basaloid PeIN, 15 cases; and warty PeIN, 4 cases. The mean age of patients was 64 years. Forty-two lesions (56.8%) were located in the glans and 32 (43.2%) in the foreskin. Overexpression of p16INK4a was useful for distinguishing SH from warty/basaloid PeINs (0% vs. 94.7%, P<0.0001) but not SH from differentiated PeINs (0% vs. 5.9%, P=0.519). In addition, p16INK4a allowed the distinction of differentiated and warty/basaloid PeINs (5.9% vs. 94.7%, P<0.0001). Immunohistochemistry results for p53 allowed the separation of SH and differentiated PeIN (9.5% vs. 44.1%, P=0.0078) and SH and warty/basaloid PeIN (9.5% vs. 55.6%, P=0.0042). Ki-67 immunostain was useful for distinguishing SH from differentiated PeIN (52.6% vs. 89.7%, P=0.0062) and SH from PeIN with warty and/or basaloid features (52.6% vs. 100%, P=0.0011). There seems to be a distinctive immunohistochemical profile for associated and precursor epithelial lesions of the penis. SH was p16INK4a and p53 negative, with variable Ki-67 positivity. Differentiated PeIN was p16INK4a negative and Ki-67 positive, with variable p53 positivity. Basaloid and warty PeINs were consistently p16INK4a and Ki-67 positive, with variable p53 positivity. The use of a triple p16INK4a/p53/Ki-67 immunohistochemical panel was found to be helpful in the classification, differential diagnosis, and morphologic standardization of penile intraepithelial lesions.

Distribution and characterization of subtypes of penile intraepithelial neoplasia and their association with invasive carcinomas: a pathological study of 139 lesions in 121 patients

We are presenting the morphological features of 121 cases of atypical penile intraepithelial lesions. The term penile intraepithelial neoplasia (PeIN) was used to encompass all of them, and lesions were classified into 2 major groups, differentiated and undifferentiated. The latter was further divided in warty, basaloid, and warty-basaloid subtypes. Ninety-five cases were associated with invasive squamous cell carcinomas. Differentiated lesions predominated (68%), followed by warty-basaloid (14%), basaloid (11%), and warty (7%) subtypes. Multifocality was found in 15% of the cases. Differentiated lesions were preferentially located in foreskin, whereas warty and/or basaloid subtypes were more prevalent in the glans. The former lesions were preferentially seen in association with keratinizing variants of squamous carcinoma, whereas the latter subtypes were found mostly in conjunction with invasive warty, basaloid, and warty-basaloid carcinomas. Lichen sclerosus was present in 51% of cases of differentiated lesions and absent in warty and/or basaloid subtypes. In summary, PeIN can be classified into 4 distinctive morphological subtypes. The proper pathological characterization of these lesions may provide important clues to the understanding of the pathogenesis and natural history of penile cancer.

Electron Microscopic Findings in Skin Biopsies from Patients with Infantile Osteopetrosis and Neuronal Storage Disease

Infantile osteopetrosis with neuronal storage disease is a rare lysosomal storage disorder. It is an autosomal recessive disease that is associated with mutations in the OSTM1 and chloride channel ClCN-7genes. So far mutations in the OSTM1 gene have been identified in only 8 patients. To date, the clinical and morphological features of nine patients with infantile osteopetrosis with neuronal storage have been reported, but no ultrastructural findings of skin have been described in these patients. Skin biopsy is a cost-effective tool for the diagnosis of lysosomal storage disease. The purpose of this report is to define the ultrastructure of affected cells seen in skin biopsies of 2 boys whose mutation of OSTM1 has been characterized. The children presented in infancy with severe osteopetrosis and neurological deficiencies whose predominant symptoms were marked cerebral atrophy, decreased myelinization, and severe central nervous system involvement. Because of the difficulties in distinguishing this disorder from some lysosomal storage diseases such as mucopolysaccharidosis that have both neurological and skeletal abnormalities, the authors elected to examine skin biopsies from these children. Ultrastructural examination revealed the presence of swollen unmyelinated axons containing spheroids, reduced numbers of myelinated axons, and the presence of secondary lysosomes in Schwann cells containing lipofuscin. This study demonstrates that electron microscopy of skin biopsy is a useful diagnostic method to identify patients with clinical features of osteopetrosis with neuronal storage disease.

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.

Convexity dural cavernous malformation with intradural and extradural extension mimicking a meningioma: a case report.

BackgroundDural-based cavernous malformations are rare and have been more commonly described in the middle fossa. Fewer than 20 cases outside of the middle fossa have been reported and they often mimic more commonly found lesions such as meningiomas or hemangiopericytomas.Case descriptionWe describe the unusual case of a right frontal convexity dural cavernous malformation with intradural and extradural components as well as erosion through the calvarium. The patient underwent a right frontal craniotomy and en-bloc resection of the mass. Final pathologic interpretation confirmed a cavernous malformation that had eroded through the calvarium.ConclusionDural-based cavernous malformations are a rare entity, but should be considered in the differential diagnosis of atypical appearing dural-based lesions and soft subgaleal masses. If atypical features are present, further radiographic investigations should be undertaken. To our knowledge, this is the only reported case of a dural-based cavernous malformation eroding through the calvarium and presenting initially as a soft scalp mass.