EM proves invaluable in the confirmation of chordoma in the sacral mass of a middle-aged man.

A case of a 47-year-old male with a sacral spine mass was investigated by histology, immunohistochemistry (IHC), and electron microscopy (EM). The light microscopy of the first core biopsy revealed scant cellularity with spindle and round cells with eosinophilic cytoplasm within a fibromyxoid background. Immunostaining with pancytokeratin, cytokeratin 19, and S100 was nonspecific. Another biopsy was attempted to obtain a more definitive diagnosis. Light microscopy of the second core had scant cellular material. However, the tissue was specifically requested for ultrastructural evaluation and revealed features diagnostic of chordoma. After definitive diagnosis, radical resection of the mass was performed. This case illustrates how EM was instrumental in the definitive diagnosis before radical resection in a case that was not clear by hematoxylin and eosin (H&E) and IHC alone.

Association between HLA-DQB1 and cervical dysplasia in Vietnamese women.

Host genetic background seems to play a key role in cervical carcinogenesis as only a small subset of women infected with high-risk human papillomaviruses (HPVs) develop cervical cancer. The rate of cervical cancer in Vietnamese women is notably high. To explore the association of human leukocyte antigen (HLA)-DQB1 alleles, HPV infection, and cervical dysplasia in this population, cervical smears were obtained from 101 women attending the obstetrics and gynecology clinic of Da Nang General Hospital in Vietnam. Besides the Papanicolaou test, HPV and HLA-DQB1 genotyping were performed using cervical smear DNA. Additionally, a subset of 30 blood samples was used as the gold standard for HLA genotyping. HLA-DQB1 alleles showed no association with HPV infection status. However, a positive association with cervical dysplasia was found for HLA-DQB1*0302 (P= 0.0229, relative risk (RR) = 4.737) and HLA-DQB1*0601 (P= 0.0370, RR = 4.038), whereas HLA-DQB1*0301 (P= 0.0152, RR = 0.221) was found negatively associated. The low diversity of HLA-DQB1 alleles, wide spectrum of HPV genotypes, and high prevalence of HPV 16 and HPV 18 observed in the study population suggest a permissive/susceptible genetic background that deserves further research. Total concordance of HLA-DQB1 genotyping results between blood and cervical cells confirms the potential value of cervical smears as an effective tool for the development of cervical cancer biomarkers.

The ultrastructural and immunohistochemical heterogeneity of CD-30-positive neoplasms: so-called anaplastic large cell Ki-1 lymphomas.

Anaplastic large cell lymphoma (ALCL), also referred to as Ki-1 lymphomas, was first recognized as an entity with characteristic light microscopic appearance in 1985. This tumor is composed of variably cohesive cells, often with large, markedly atypical, and multinucleated cellular forms. The recognition of ALCL resulted from the development of a monoclonal antibody in Kiel, Germany, named Ki-1, which was initially believed to be a putative marker for Reed-Sternberg cells. This antibody was later found to be specific against the epitope CD-30. Attempts to create strict criteria to preserve this neoplasm as a specific entity have undergone evolution. However, it is now clear that included in this group are a variety of pleomorphic neoplasms with CD-30 immunoreactivity. Some of these neoplasms are nonlymphoid and show marked heterogeneity in their immunohistochemical and ultrastructural profiles. This article aims to highlight the ultrastructural spectrum of neoplasms exhibiting CD-30 positivity that are within the spectrum of ALCL. It remains to be determined if there are subgroups of these CD-30-positive neoplasms that can be segregated on the basis of ultrastructural and immunohistochemical criteria with corresponding clinical correlates that may impact on their management, treatment, and prognosis. We review here the heterogeneity of CD-30-positive neoplasms (so-called anaplastic large cell Ki-1 lymphomas).

Comparison of fine-needle aspiration cytology and core biopsy for diagnosis of breast cancer.

Both fine-needle aspiration (FNA) cytology and core biopsy are useful in the diagnosis of breast cancer. In order to compare the sensitivities of these procedures, we reviewed 209 patients with breast cancer who had either FNA, core biopsy, or both, and also either mastectomy or lumpectomy. Sensitivities for FNA and core biopsies for diagnosing breast cancer were calculated and compared. Sensitivity for FNA or core biopsies interpreted as either atypical or malignant was 93.8% for FNA and 90.1% for core biopsy (P > 0.05). Sensitivity for FNA or core biopsies interpreted as malignant was 65.4% for FNA and 88.7% for core biopsy (P < 0.0001). Sensitivities of FNA interpreted as either atypical or malignant were 92.4% for FNA performed by pathologists and 100% for FNA by nonpathologists (P > 0.05). Sensitivities of FNA interpreted as malignant were 75.8% for FNA by pathologists and 20.0% for FNA by nonpathologists (P < 0.00001). Both FNA and core biopsies are sensitive procedures for the detection of breast cancer. There was no significant difference between sensitivity of FNA and core biopsies interpreted as either atypia or malignancy, although the sensitivity of core biopsies interpreted as unequivocal malignancy was greater than that of FNA. FNAs performed by pathologists were more sensitive than FNAs performed by nonpathologists in making an unequivocal diagnosis of breast cancer.

Expression of the translation initiation factor eIF4E in the polyp-cancer sequence in the colon.

The eukaryotic translation initiation factor 4E (eIF4E) has been shown to play a key role in cell growth, and several studies have documented an increased expression of eIF4E in a number of solid tumors, including breast, bladder, cervical, and head and neck cancers. This study was done to evaluate the potential role of eIF4E in the polyp-cancer sequence in the colorectum. Eighty-seven cases with lesions in the colorectum with a variety of histopathological diagnoses were randomly selected from the archives of the Pathology Department at Louisiana State University Health Sciences Center-Shreveport. Appropriate sections were selected for immunostaining with eIF4E. The medical records of the patients were reviewed, and demographic information was collected. All statistical analyses were performed using SAS software. A statistically significant relationship was found between the level of eIF4E expression and histological type of lesion: the lowest level of eIF4E expression was found in normal colon tissue, whereas the highest level of eIF4E expression was found in colorectal adenocarcinomas. Carcinomatous lesions were found to have a 43 times higher chance of having a high level of eIF4E expression compared with normal tissue (95% confidence interval, 8.0-213.6, P < 0.0001). In a multivariate analysis, histological type was the only variable that showed a significant relationship with eIF4E expression; no effect was found due to age, gender, race, history of polyps, and family history. The results from this study are consistent with other data from the literature and support the suggestion that eIF4E is strongly involved in colon tumorigenesis. eIF4E might be a useful intermediate biomarker for use in chemoprevention intervention studies in patients with colorectal polyps.

Placebo-controlled trial of indole-3-carbinol in the treatment of CIN.

OBJECTIVE: Most precancerous lesions of the cervix are treated with surgery or ablative therapy. Chemoprevention, using natural and synthetic compounds, may intervene in the early precancerous stages of carcinogenesis and prevent the development of invasive disease. Our trial used indole-3-carbinol (I-3-C) administered orally to treat women with CIN as a therapeutic for cervical CIN. METHODS: Thirty patients with biopsy proven CIN II-III were randomized to receive placebo or 200, or 400 mg/day I-3-C administered orally for 12 weeks. If persistent CIN was diagnosed by cervical biopsy at the end of the trial, loop electrocautery excision procedure of the transformation zone was performed. HPV status was assessed in all patients. RESULTS: None (0 of 10) of the patients in the placebo group had complete regression of CIN. In contrast 4 of 8 patients in the 200 mg/day arm and 4 of 9 patients in the 400 mg/day arm had complete regression based on their 12-week biopsy. This protective effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0. 25 to 0.99) P = 0.023) for the 200 mg/day group and a RR of 0.55 ((95% CI, 0.31 to 0.99) P = 0.032) for the 400 mg/day group. HPV was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day group, and in 8 of 9 in the 400 mg/day group. CONCLUSIONS: There was a statistically significant regression of CIN in patients treated with I-3-C orally compared with placebo. The 2/16 alpha-hydroxyestrone ratio changed in a dose-dependent fashion.

Immunoelectron microscopy in the age of molecular pathology.

The introduction of molecular biology-based diagnostic procedures in pathology has created substantial expectations in regard to screening, characterization, monitoring, and detection of predisposition to a variety of diseases, most notably malignant neoplasms. It should be emphasized, however, that molecular studies are only one component of the diagnostic process and that more traditional methods are still required in the evaluation of tumors and management of patients. The data obtained from the molecular biology-based studies must be always interpreted in conjunction with the clinical history, immunomorphologic findings, and other pertinent ancillary data. Routine evaluation of tissues using traditional light microscopy remains the backbone of pathologic evaluation. The additive role of molecular diagnostics often depends on how accurate the initial evaluation has been. Ancillary techniques such as immunohistochemistry and electron microscopy remain essential in properly characterizing diseased tissues and in speciation of tumors. Ultrastructural immunolabeling capitalizes on combining these two techniques and providing exquisite immunomorphologic evaluation. The extra time and effort required are more than compensated by the degree of sophistication that can be achieved when this diagnostic technique is utilized and the added expense is rather reasonable. The value of molecular biology-based diagnostics is potentially questionable if the tissue samples are not initially accurately characterized. The question that molecular diagnostics may be trying to answer may be the wrong one or the answer obtained may be interpreted incorrectly if the context of the clinicopathologic situation has not been clearly defined using traditional diagnostic techniques.

HIV+ patients have increased lymphocyte infiltrates in CIN lesions.

OBJECTIVE: The objective of our study was to analyze immunocyte infiltrates in CIN lesions from HIV+ patients to assess whether local immunosuppression, defined as a decrease in T cell infiltrates, could explain the aggressive nature of CIN in HIV-infected patients. MATERIALS AND METHODS: Cervical tissue was obtained from 6 HIV+ CIN patients, 6 HIV- CIN patients, who underwent LLETZ (large loop excision of the transformation zone) for CIN, and 17 normal patients who underwent hysterectomy for benign indications. The following cell surface markers were analyzed: CD20 (B cells), CD4 (T helper cells), and CD8 (T suppressor/cytotoxic cells). Each tissue section was visualized with a Leica microscope at 400x and the image was captured for analysis by Harmony Group image analysis software. RESULTS: A significantly higher number of lymphocytes (both B and T cells) was detected in the stroma of HIV+/CIN tissue compared to either HIV-/CIN or normal tissue. There was also a significant increase in CD8+ cells in the HIV+/CIN group compared to HIV-/CIN or normal tissue. There was a trend toward a decreased CD4+/CD8+ ratio in the HIV+/CIN compared to the other two groups; however, this did not reach statistical significance. CONCLUSIONS: This study indicates that HIV+/CIN cervical tissue has a greater number of tissue lymphocytes recruited to the neoplastic site compared to HIV- individuals. In addition, HIV+ patients may have a decreased CD4/CD8 ratio in locally infiltrating immunocytes in CIN lesions. The local immunomodulatory effects of HIV may be detectable early in infection and therefore may explain the aggressive nature of CIN in the HIV+ patient.

Glomerulopathic light chain-mesangial cell interactions modulate in vitro extracellular matrix remodeling and reproduce mesangiopathic findings documented in vivo.

Glomerulopathic light chains (LCs) are associated with two distinct mesangiopathies: AL (light-chain-related) amyloidosis and light-chain deposition disease (LCDD) with immunomorphologic features that are well documented in the literature. Even though both conditions are caused by monoclonal LCs, these entities differ dramatically in their morphologic expressions. In AL amyloidosis the mesangial matrix is replaced by amyloid fibrils, while in LCDD the matrix increases as a consequence of deposition of excess extracellular matrix (ECM). The immunomorphologic mesangial alterations observed in biopsy material are closely reproduced in vitro when mesangial cells grown on an artificial matrix are incubated with monoclonal light chains obtained from the urine of patients with either condition. This article summarizes previously reported data, reports new findings, and focuses on integrating all the available information on the subject. When mesangial cells are incubated with LCDD-LCs, production of ECM proteins (collagen IV, laminin, fibronectin, and tenascin) is increased, with maximum effect at 72 hours post LC treatment. A concomitant decrease in collagenase IV activity further accentuates the accumulation of mesangial matrix. These effects are mediated through transforming growth factor-beta (TGF-beta) activation. In contrast, when mesangial cells are incubated with Am-LCs, a decrease in ECM protein production and a stimulatory effect on collagenase IV is observed, which results in matrix degradation and facilitates amyloid deposition. The decreased TGF-beta documented in the literature in this setting precludes adequate matrix repair. These findings substantiate the morphologic alterations observed in renal biopsy specimens and in the in vitro model. Using this in vitro model, it is then possible to delineate the LC interactions with putative receptors at the mesangial cell surface that regulate mesangial cell pathobiologic responses and mesangial matrix homeostasis.

Elevated expression of eIF4E in confined early breast cancer lesions: possible role of hypoxia.

The translation-initiation factor eIF4E is rate-limiting for protein synthesis, and its over-expression results in oncogenic transformation of mammalian cells. eIF4E facilitates the synthesis of several powerful tumor angiogenic factors (FGF-2 and VEGF) by selectively enhancing their translation. In breast carcinomas, eIF4E is commonly over-expressed, but the pathology where this elevation is initially manifested is presently unknown. To probe whether the elevation of eIF4E marks an early stage of cancer development, we focused our research on early cancerous lesions. We have analyzed 70 invasive ductal carcinomas (IDCs), 78 ductal carcinomas in situ (DCIS), 51 benign lesions and 4 model cell lines for elevated expression of eIF4E by several different methods: Northern/Western blots, immuno-histochemistry and in situ RT-PCR. eIF4E expression was markedly increased in IDC and in islets of viable cells in the center of poorly vascularized DCIS, which are not easily identifiable by standard histological stains. We also show that expression of eIF4E is increased by hypoxia and, presumably, in hypoxic areas of these lesions. We propose that clonal expansion of cancer cells, permanently over-expressing eIF4E, gives them a critical advantage to survive hypoxia and marks the transition toward the vascular phase of cancer progression. Hence, eIF4E may be useful in stratifying DCIS lesions according to their malignant stage.

Cytology: screening or diagnostic tool?

Previously used only as a screening tool, cytology now emerges as a powerful diagnostic technique, especially since the advent of the fine needle aspiration (FNA) biopsy. This article highlights the use of ancillary techniques, primarily electron microscopy (EM), and immunohistochemistry (IHC). When coupled with routine cytological examination such as FNA and body cavity fluid cytology, EM and IHC can refine the diagnosis and make it more precise. The authors discuss how to solve common diagnostic dilemmas by the use of cytology along with IHC and EM. The following common diagnostic problems are addressed: mesothelioma versus adenocarcinoma, neuroendocrine neoplasms and their mimickers, melanoma versus carcinoma versus sarcoma, hepatocellular carcinoma versus adenocarcinoma and adenocarcinomas of unknown primary.

Role of electron microscopy in transplant renal pathology.

The crucial role that electron microscopy plays in diagnostic renal pathology is undisputed. By allowing recognition of findings not identifiable by light microscopic evaluation, electron microscopy has contributed significantly to the understanding of renal diseases and has proven to be of unquestionable value in many diagnostic situations. However, the percentage of cases in which electron microscopic examination adds important information that is either key for establishing or confirming a diagnosis or provides valuable data that influence patient's management remains controversial. This figure depends on the renal biopsy service that is surveyed, but it is reported that on the average ultrastructural evaluation is of value in approximately 30 to 45% of the cases. Correct interpretation of a renal biopsy depends on the ability to correlate light, immunofluorescence, and ultrastructural findings. In contrast, the role of electron microscopy in the examination of renal transplant specimens remains controversial. Many centers do not use routine electron microscopy to examine these specimens and insist that there are only a few specific indications that require ultrastructural evaluation. There is general agreement among renal pathologists that electron microscopy is of importance in the evaluation of renal specimens from patients with proteinuria to distinguish between transplant glomerulopathy, recurrent or de novo glomerulonephritis in order to correctly manage these patients and predict survival of the graft. The other possible indications are much more controversial. This paper summarizes and critically reviews the literature available on this subject and defines recommendations based on the information available at the current time.

Integrated expression of glomerular extracellular matrix proteins and beta 1 integrins in monoclonal light chain-related renal diseases.

Light-chain deposition disease (LCDD) and amyloid light-chain amyloidosis (AL-Am) represent the two classical diseases associated with glomerular alterations in monoclonal light chain-related renal diseases. LCDD is characterized by deposition of extracellular matrix proteins in the mesangium, thus creating the morphologic appearance recognized as nodular glomerulosclerosis. In AL-Am, the mesangial matrix is replaced by polymerized light chains in the form of amyloid fibrils. Integrins are responsible for cell-to-cell and cell-to-matrix communication and, therefore, are expected to play a key role in the alterations encountered in these two diseases. The present article addresses the expression of selected extracellular matrix proteins (collagen IV, laminin, fibronectin, and tenascin) and their respective receptor beta 1 integrins (alpha 2, alpha 3, alpha 5, and alpha 9) in glomeruli with LCDD and AL-Am by immunohistochemical methods. The corresponding integrin (alpha 9 beta 1) co-localized with tenascin in the center of the mesangial nodules in LCDD. In AL-Am, tenascin is found primarily at the periphery of replaced mesangial areas and in the remaining mesangium not replaced by the amyloid. Tenascin co-localized with alpha 9 beta 1 integrin in mesangial areas in the earlier phases of the process. Fibronectin, laminin, and collagen IV, although increased in absolute amounts, are pushed toward the periphery of mesangial areas, in which correlated expression of their corresponding beta 1 integrins (alpha 2, alpha 3, and alpha 5, respectively) is documented in both LCDD and AL-Am. Deposition of tenascin might be at least partially responsible for the perpetuation and irreversibility of the glomerular lesion in LCDD.

The role of ultrastructural pathology in the diagnosis of epithelial and unusual renal tumors.

The electron microscopic and immunohistochemical features of epithelial and unusual renal tumors are reviewed. Ultrastructural and immunohistochemical features of diagnostic value are highlighted. An attempt is made to address histogenesis/differentiation issues that would help in understanding morphologic findings, with an emphasis placed on the role that ultrastructural evaluation has played in demonstrating these.

Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion.

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca2+] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca2+, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca2+]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 microM and 90% at 7 microM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 microM trifluoperazine. Quantitative immunofluorescence using fluorescein-labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3-day bone-attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9 nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP-dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent Km for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated.