Diffusion-collision model study of misfolding in a four-helix bundle protein.

Proteins with complex folding kinetics will be susceptible to misfolding at some stage in the folding process. We simulate this problem by using the diffusion-collision model to study non-native kinetic intermediate misfolding in a four-helix bundle protein. We find a limit on the size of the pairwise hydrophobic area loss in non-native intermediates, such that burying above this limit creates long-lasting non-native kinetic intermediates that would disrupt folding and prevent formation of the native state. Our study of misfolding suggests a method for limiting the production of misfolded kinetic intermediates for helical proteins and could, perhaps, lead to more efficient production of proteins in bulk.

Gamma probe guided biopsy of the sentinel node in malignant melanoma: a multicentre study.

Sentinel lymph node biopsy was attempted in 336 patients with clinically node-negative cutaneous melanoma. All patients were injected with technetium-99m labelled radiocolloid, with 108 patients simultaneously receiving vital blue dye for sentinel node identification. Sentinel lymph nodes were identified in 329 patients, giving a technical success rate of 97.9%. Metastatic disease was identified in 39 (11.9%) of the patients in whom sentinel nodes were found. Patients with negative sentinel nodes were observed and patients with positive sentinel nodes underwent comprehensive lymph node dissection. The presence of metastatic disease in the sentinel nodes and primary tumour depth by Breslow or Clark levels were joint predictors of survival based on Cox proportional hazards modelling. Disease recurrences occurred in 26 (8.8%) patients with negative sentinel lymph nodes, with isolated regional recurrences as the first site in 10 (3.4%). No patients with Clark level II primary tumours were found to have positive sentinel nodes or disease recurrences. One patient with a thin (<0.75 mm) Clark level III primary had metastatic disease in a sentinel node. Patients with metastases confined to the sentinel nodes had similar survival rates regardless of the number of nodes involved.

Sentinel lymph node biopsy in breast cancer: creating controversy and defining new standards.

Sentinel lymph node (SLN) biopsy is an exciting and promising new addition to surgical management and pathologic evaluation of breast cancer. Sentinel nodes are more likely to contain metastases than nonsentinel nodes providing an opportunity to more accurately stage breast cancer patients. In the future, surgical management of the axilla may be approached as a staged procedure, perhaps eliminating axillary dissection in SLN-negative patients, should clinical trials demonstrate the safety of this approach. The advent of SLN biopsy has propelled an old question back to the forefront of clinical investigation: do occult metastases in lymph nodes have prognostic, predictive, and clinical significance? This article provides one opinion and a review on the pathology and future of SLN biopsy in breast cancer. Practicing pathologists will need to stay abreast of the issues and facts in order to participate in the debate and shape the future of pathologic management of sentinel nodes.

Immunizations: recommendations and resources for active patients.

Although childhood vaccination rates are at an all-time high, those for adolescents and adults are suboptimal. All adolescents and adults should be immunized against measles, mumps, rubella, varicella, tetanus, and diphtheria, and many should also receive hepatitis A, hepatitis B, influenza, and pneumococcal vaccines. In addition, active patients who engage in outdoor activities may benefit from vaccination against Lyme and meningococcal disease. Regular, strenuous exercise and foreign travel may increase the risk of some infectious diseases. Athletes often see a physician only for sports physical exams and injuries, so it is important for providers to take the opportunity to vaccinate patients during these visits.

The National Health Service Corps for the 21st century.

The National Health Service Corps (NHSC) was created in 1970 to provide primary health care clinicians for the underserved. The article includes a review of the peer-reviewed and intragovernmental literature on the NHSC program from 1971 to 1998 and also presents a current profile of the program. Despite significant increases in NHSC field strength since 1991, the 2,439 clinicians meet only 12% of the need for primary health care providers in underserved areas. While the NHSC has successfully addressed clinician diversity and retention issues, community and site development remain barriers to increasing access. Most communities in need are not ready to recruit and support clinicians. The NHSC of the next millennium must work with the neediest communities to reach the appropriate stage of readiness. Only after completing the necessary "preplacement" activities can the NHSC assist in the recruitment and placement of clinicians to increase access.

Pathologic analysis of sentinel and nonsentinel lymph nodes in breast carcinoma: a multicenter study.

BACKGROUND: Axillary lymph node status is a powerful prognostic factor in breast carcinoma; however, complications after axillary lymph node dissection are common. Sentinel lymph node biopsy is an alternative staging procedure. The sentinel lymph node postulate is that tumor cells migrating from the primary tumor colonize one or a few lymph nodes before colonizing subsequent lymph nodes. To validate this hypothesis, the distribution of occult and nonoccult metastases in sentinel and nonsentinel lymph nodes was evaluated. METHODS: Original pathology material was reviewed from 431 patients enrolled on a multicenter validation study of sentinel lymph node biopsy in breast carcinoma patients. Paraffin embedded tissue blocks of sentinel and nonsentinel lymph nodes were obtained for 214 lymph node negative patients. Additional sections from 100 and 200 microm deeper into the paraffin block were examined for the presence of occult metastatic carcinoma. Both routine and cytokeratin immunohistochemical stains were employed. RESULTS: Metastases were identified in 15.9% of sentinel lymph nodes and 4.2% of nonsentinel lymph nodes (odds ratio [OR] 4.3[ P < 0.001]; 95% confidence interval [95% CI], 3.5-5.4). Occult metastases were identified in 4. 09% of sentinel lymph nodes and 0.35% of nonsentinel lymph nodes (OR 12.3 [P < 0.001]; 95% CI, 5.6-28.6). The overall case conversion rate was 10.3%. All the occult metastases identified were < or = 1 mm in greatest individual dimension. The likelihood (OR) of metastases in nonsentinel lymph nodes was 13.4 times higher for sentinel lymph node positive than for sentinel lymph node negative patients (P < 0. 001; 95% CI, 6.7-28.1). CONCLUSIONS: The distribution of occult and nonoccult metastases in axillary lymph nodes validates the sentinel lymph node hypothesis. In addition, pathology review of cases confirmed the authors' previously reported finding that the sentinel lymph nodes are predictive of the final axillary lymph node status. Occult metastatic disease is more likely to be identified in sentinel lymph nodes, allowing future studies to focus attention on one or a few sentinel lymph nodes. However, the relation between occult metastatic disease in sentinel lymph nodes, disease free survival, and overall survival must be evaluated prior to endorsing the intensive analysis of sentinel lymph nodes in routine practice. [See editorial on pages 971-7, this issue.]

Intraoperative ultrasound localization to guide surgical excision of nonpalpable breast carcinoma.

BACKGROUND: This report describes a technique of intraoperative tumor localization by ultrasound without the use of a needle or wire to guide the excision of nonpalpable breast cancers. The results of our experience with pathologic margin status are reviewed. STUDY DESIGN: From 1994 to 1998, 65 breast cancers in 62 patients with biopsy-proved nonpalpable breast cancer were excised using intraoperative ultrasound localization. The pathologic status of the margins from the initial surgical excision specimen and any further excisions, either at the first operation or later procedures, was recorded. The distance from the tumor to the closest margin of excision was also determined. RESULTS: The overall success in achieving pathologically negative excision margins at first operation was 97% (63 of 65 cancers). Three patients underwent a second operative procedure, two for positive margins and one for a margin less than 1 mm (second operation = 4.8% of patients). After completion of the first operative procedure, the mean distance to the closest margin of excision was 0.8 cm. CONCLUSIONS: Intraoperative ultrasound localization for excision of nonpalpable breast cancers is feasible and gives results, in terms of pathologic margins, that are comparable with those achieved by standard needle-wire-guided excisions.

Submission of lymph node tissue for ancillary studies decreases the accuracy of conventional breast cancer axillary node staging.

Pathologists are under increasing pressure to submit fresh tissue for ancillary studies and research protocols. In several tumor types (breast, lung, melanoma, colorectal, prostate), increased interest in detecting submicroscopic nodal metastases through reverse transcriptase polymerase chain reaction analysis of mRNA from portions of lymph nodes has precluded histologic analysis of the entire node for metastases. A retrospective review was undertaken of 227 breast cancer patients prospectively entered on a research protocol examining the usefulness of sentinel lymph node surgery. All of the patients ultimately underwent complete lymph node dissection. The research protocol required that all nodes greater than 8 mm in size be bisected and submitted separately. Positive lymph nodes were evaluated for unilateral or bilateral involvement in the node sections. Sixty node-positive patients were identified, yielding 230 positive nodes. One hundred seven of these nodes were confirmed to have been bisected. Carcinoma was identified in both lymph node sections in 64 (59.8%) nodes and in only one-half of the bisected lymph node in 43 (40.2%) nodes. Involvement of both sections was more likely when patients had multiple nodes positive. In 12 patients, involvement of one-half of the bisected nodes was the only evidence of metastatic disease (20.0% of node-positive patients). This evidence suggests that submission of less than the complete lymph node for histologic evaluation of metastatic disease decreases the accuracy of lymph node staging. Furthermore, a significant proportion of patients may be erroneously classified as histologically node negative.

Limitation in gamma probe localization of the sentinel node in breast cancer patients with large excisional biopsy.

BACKGROUND: Radiolocalization and selective biopsy of the sentinel node to correctly predict the status of remaining lymph nodes may provide an alternative to axillary dissection in selected breast cancer patients with clinically negative lymph nodes. STUDY DESIGN: In a nonrandomized, multicenter clinical trial, gamma probe localization for lymphatic mapping and sentinel node biopsy along with axillary dissection was performed on 75 patients with invasive breast cancer and clinically negative lymph nodes. The accuracy of the sentinel node biopsy to correctly predict the status of the remaining axillary lymph nodes was established through standard pathologic investigation. RESULTS: A sentinel node was identified in 70 of 75 patients with a technical success rate of 93%. Of these 70 patients, 21 (30%) had axillary nodal metastases identified pathologically. Four of these 21 (19%) had sentinel nodes negative for metastases. All 4 false-negative patients had prior excisional biopsies. The false-negative group had a larger mean maximal biopsy dimension than the true-positive group. Eleven of the 21 patients with axillary metastases had a diagnosis made by core needle biopsy with no false negatives. CONCLUSIONS: The accuracy of the sentinel node biopsy in correctly predicting the status of remaining axillary lymph nodes may be limited in patients with large excision before radiolocalization of the sentinel node. Our findings suggest that excisional biopsy should be avoided prior to lymphatic mapping for sentinel node biopsy.

Breast Cancer Cells in the Blood: A Pilot Study.

The goal of this pilot study was to determine in patients with operable breast cancer the incidence of breast cancer cells present in the blood, the clearance rate after surgical resection of the primary tumor, and the incidence of patients with persistent cancer cells in the blood after the primary tumor was removed. Twenty-one patients with operable breast cancer had 15 ml venous blood obtained twice prior to surgery and after surgery at 2, 4, 8, 12, 24, and 48 hours and also on days 7 and 14. Immunomagnetic selection of malignant cells was performed on each sample. Cells were then fixed on slides and immunocytochemistry performed on the collected cells. Cells that had a rosette of magnetic beads, cytoplasmic staining for keratin, and malignant morphology were counted as breast cancer cells. Eighteen of 19 of patients had cancer cells detected in at least one of the two blood samples preceding surgical removal of the primary tumor. The incidence of cancer cells in the blood of patients rapidly declined during the 48 hours postsurgery. The incidence of cancer cells in the blood remained stable in approximately 30% of patients to 14 days. The majority of breast cancer patients in this pilot study (even with small tumors and negative nodes) had detectable cancer cells in the blood prior to resection of the primary tumor. These findings justify further investigation. Successful application of this methodology may serve as a powerful indicator of which patients need systemic adjuvant therapy, the effectiveness of systemic adjuvant therapy, tumor recurrence, and early detection of breast cancer.

The early folding kinetics of apomyoglobin.

The folding pathway of apomyoglobin has been experimentally shown to have early kinetic intermediates involving the A, B, G, and H helices. The earliest detected kinetic events occur on a ns to micros time scale. We show that the early folding kinetics of apomyoglobin may be understood as the association of nascent helices through a network of diffusion-collision-coalescence steps G + H <--> GH + A <--> AGH + B <--> ABGH obtained by solving the diffusion-collision model in a chemical kinetics approximation. Our reproduction of the experimental results indicates that the model is a useful way to analyze folding data. One prediction from our fit is that the nascent A and H helices should be relatively more helix-like before coalescence than the other apomyoglobin helices.

Estrogen-receptor-related protein p29 in primary nonsmall cell lung carcinoma: pathologic and prognostic correlations.

BACKGROUND: Estrogen-dependent intracellular processes are important in the growth regulation of normal tissue and may play a role in the regulation of malignancies. Utilization of estrogen receptor assays in breast carcinoma is well established, but the role of such evaluation in other cancers largely is unknown. In this study, immunohistochemical expression of estrogen receptor (ER) and the ER-related protein p29 was correlated with survival of patients with nonsmall cell carcinoma of the lung. METHODS: All patients with a tissue diagnosis of primary nonsmall cell bronchogenic carcinoma diagnosed over a 6-year period at the Medical Center Hospital of Vermont were reviewed. Assays for p29 and ER using a streptavidin-biotin immunoperoxidase method were performed on each tumor. Results were correlated with clinical data, including survival. RESULTS: Of 111 tumors examined, 109 (98%) were positive for p29 whereas none of the tumors reacted with ER (ER1D5). The relation between p29 expression and survival time was different for men and women. A statistically significant negative relation for women was observed; this relation was most pronounced in patients with Stage I and II tumors. A positive but not statistically significant relation was observed for men. CONCLUSIONS: The ER-related protein p29 commonly is expressed in nonsmall cell carcinomas of the lung. The relation between p29 and survival time is different for males and females, suggesting the presence of gender specific factors that may influence tumor growth and overall patient survival, especially in patients with early stage lung carcinoma.

Localization of regional lymph nodes in melanomas of the head and neck.

OBJECTIVES: To study the efficacy of gamma-probe radiolocalization of the first draining (sentinel) lymph node (SLN) in stage N0 melanoma of the head and neck and to evaluate its potential role in the staging and treatment of this disease. DESIGN: Gamma-probe radiolocalization, a new alternative to blue-dye lymphatic mapping, uses a scintillation (gamma) probe to identify radiolabeled SLNs. In a consecutive sample clinical trial, gamma-probe radiolocalization of the SLN is compared with lymphoscintigraphy and blue-dye lymphatic mapping. Follow-ups ranged from 1.7 years to 4 years, with a mean follow-up of 2.5 years. SETTING: Tertiary and private care teaching hospital. PATIENTS: Between June 1993 and November 1995, 23 patients with stage N0 intermediate-thickness melanoma of the head and neck were enrolled in this volunteer sample. INTERVENTIONS: Twenty-four hours prior to surgery, a radioactive tracer was intradermally injected around the circumference of a primary melanoma. Twelve patients also had blue dye injected just prior to surgical resection. Using a handheld gamma probe, radiolabeled lymph nodes were identified and selectively removed with minimal dissection. In patients with nodes with histologic evidence of metastases, a regional lymphadenectomy was performed. MAIN OUTCOME MEASURES: The successful identification of radiolabeled SLNs, the correlation of SLN radiolabeling to lymphoscintigraphy and blue-dye mapping, and the long-term development of regional metastases. RESULTS: Surgeons successfully resected the radiolabeled SLNs in 22 (96%) of 23 patients. The success rate of blue-dye lymphatic mapping was 8 (75%) of 12 patients and lymphoscintigraphy was 20 (91%) of 22 patients. One hundred percent of blue-stained lymph nodes were radiolabeled. The one patient in whom no SLN could be identified developed regional disease at 17 months. CONCLUSIONS: Gamma-probe radiolocalization and resection of the radiolabeled SLN is a simple and reliable method of staging regional lymph nodes and determining the need for elective lymphadenectomy.

Desmoplastic small round cell tumour of unknown primary origin with lymph node and lung metastases: histological, cytological, ultrastructural, cytogenetic and molecular findings.

Desmoplastic small round cell tumour (DSRCT) is an extremely aggressive neoplasm belonging to the family of "small round blue cell tumours", which includes primitive neuroectodermal tumour (PNET), Wilms' tumour and Ewing's sarcoma. DSRCT is considered to be a neoplasm derived from a primitive cell. It is immunohistochemically reactive with epithelial, neuronal and mesenchymal cell markers, demonstrating divergent differentiation along three cell lines. Originally thought to arise from serosal surfaces, the tumour has recently been reported in the central nervous system and ovary. The tumour in this case is a neoplasm that meets the histological, immunohistochemical, cytological and cytogenetic criteria of DSRCT; it is not associated with serosal membranes, and it has supraclavicular and axillary lymph node deposits and multiple pulmonary and brain metastases. Tumour cells from our case show cytogenetic similarities with Ewing's sarcoma and PNET. Electron microscopic findings suggest similarities between DSRCT and Wilms' tumour. Cloning and sequencing of PCR products showed in-frame fusion of EWS exon 7 to WT1 exon 8.

Osteocalcin and osteonectin immunoreactivity in the diagnosis of osteosarcoma.

Osteosarcomas (OSAs) can be difficult to distinguish histologically from tumors with significantly different biologic potentials and treatment protocols. The correct diagnosis of OSA relies on identification of malignant osteoblasts that are capable of producing neoplastic bone. To determine the use of immunohistochemistry for the diagnosis of OSA, 106 tumors from the Massachusetts General Hospital and the University of Vermont were immunostained with monoclonal antiosteocalcin (OC) and antiosteonectin (ON) antibodies. They included 42 OSAs, 25 non-bone-forming sarcomas, 24 other malignant tumors including lymphomas, carcinomas, and melanomas, and 15 benign bone tumors. Cytoplasmic staining with OC showed 70% sensitivity and 100% specificity, while staining with ON showed 90% sensitivity and 54% specificity for bone-forming tumors, consistently staining cell types other than osteoblasts. Of the OSAs, 83% demonstrated matrix staining with one or both antibodies, whereas dense collagen was negative for both antibodies in all tumors. We conclude that tumor cell cytoplasmic staining with monoclonal OC may be helpful in distinguishing OSAs from other malignancies, and staining of extracellular matrix for OC and ON antibodies concurrently may help distinguish bone matrix from dense collagen.

Diagnostic accuracy of a rural live video telepathology system.

Accuracy of diagnoses rendered using a live video telepathology network was assessed for permanent sections of surgical pathology specimens. To determine accuracy, telepathology diagnoses were compared with those obtained by directly viewing the glass slide using a standard microscope. A total of 294 cases were read via both telepathology and glass slide by attending pathologists at a tertiary care medical center. Overall accuracy was defined as exact concordance between diagnoses. Clinically insignificant differences in diagnoses were excluded to determine clinically significant accuracy. For the 285 cases with complete data, the overall accuracy for telepathology was 0.912 (95% confidence interval [CI], 0.872-0.941), whereas the overall accuracy for glass slide readings was 0.968 (95% CI, 0.939-0.985). This difference is statistically significant (p = 0.009). When focusing on clinically significant discrepancies, where the difference in diagnosis might affect therapeutic decisions, the video accuracy was only slightly less than the glass slide accuracy (0.965 [95% CI, 0.934-0.982] vs. 0.982 [95% CI, 0.957-0.994], respectively), but this difference is not statistically significant (p = 0.302). Most of the cases with clinically significant differences involved lesions with inherently high interobserver variation. Certainty of diagnosis did not differ between video and glass slide readings (p = 0.911), but there was an association between certainty of diagnosis and diagnostic accuracy for video (p = 0.003 for clinically significant accuracies). Based on these findings, we recommend when using this telepathology system that only preliminary diagnoses should be given in the following situations: for diagnostic areas with known high interobserver variability; when the consultant has any degree of uncertainty about the presence or absence of the lesion in question; and when there is insufficient experience using telepathology as a diagnostic medium.

Three-dimensional reconstruction by confocal laser scanning microscopy in routine pathologic specimens of benign and malignant lesions of the human breast.

Confocal laser scanning microscopy (CLSM) has become an exciting new instrument because of its increased resolution over conventional wide-field microscopy and its high performance three-dimensional (3D) optical sectioning. Although CLSM has been used extensively in cell biology, few applications have been reported in routine clinical pathology. In this study, 3D reconstruction was performed on routine formalin-fixed, paraffin-embedded tissues of normal mammary duct, simple ductal hyperplasia, intraductal papillary hyperplasia, ductal carcinoma in situ, invasive carcinoma, and lymph node metastatic carcinomas of the human breast by using computer-assisted CLSM in conjunction with a 3D reconstruction software package (microVoxel). The selected specimens were sectioned at 30 microns, mounted on glass slides, and stained with the DNA fluorescent probe, YOYO-1 iodide. The nuclear DNA and chromatin texture were clearly demonstrated after pretreatment with RNAase and hydrolysis with 2 N HCl. High quality 3D images were obtained by processing the optical section stacks with volume render and surface display parameters in microVoxel. 3D morphologic characteristics of different breast lesions were examined in various orientations by angular image rotation. The clearly benign lesions (simple ductal hyperplasia and intraductal papillary hyperplasia) revealed similar 3D morphologic features, including: (1) smooth nuclear surface and homogeneous chromatin fluorescence intensity; (2) hyperplastic cell nuclei showing similar shape and volume; and (3) clearcut margin of basement membrane defined by spindle-shaped myocytes of the ductal outer layer. In contrast, carcinomas displayed remarkably different features in 3D morphology, including: (1) irregular nuclear surface: (2) marked nuclear pleomorphism (irregular, angulated and indented shape of nuclear volume); (3) irregular and coarse chromatin texture; (4) chaotic arrangement of tumor cell nuclei; and (5) absence of myocytes, indicating no clear margin at the site of infiltration of cancer cells. In conclusion, nuclear structure, specifically demonstrated by CLSM of YOYO-1 iodide fluorescently stained cells, used in tandem with 3D volume morphologic reconstruction, may provide a useful research diagnostic tool in pathology.

A positive, systematic approach to improve inpatient medical record completion in a family practice residency program.

BACKGROUND AND OBJECTIVES: Timely completion of medical records is a concern for many reasons. For physicians, incomplete records may result in loss of hospital privileges and income and increased legal liability after entering practice. Traditionally, medical record delinquencies have been dealt with punitively. We developed a multifaceted approach to inpatient medical record completion and implemented it at our hospital. METHODS: The intervention involved posting weekly lists of physicians who were in compliance or noncompliance with medical records completion requirements, giving positive rewards (candy bars) for those who were in compliance, and increasing faculty contact with residents over issues of medical records completion. RESULTS: This approach reversed an increasing hospital record delinquency rate that had exceeded 50% and reduced the rate to 10%-30%. Residents' views of the various components of the intervention were positive. CONCLUSION: Medical records completion rates can be increased with positive, rather than punitive, measures.