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The cellular thermal shift assay (CETSA) provides a means of understanding the extent to which a small molecule ligand associates with a protein target of therapeutic interest, thereby inferring target engagement. Better analytical detection methods, including mass spectrometry, are being implemented to improve quantitation within these assays, providing both absolute quantitation and a very high analyte specificity. To understand the target engagement, and hence inhibition, of the protein dipeptidyl peptidase 9 (DPP9) in rat tissue, CETSA experiments, coupled with single-pot, solid-phase-enhanced sample preparation ("SP3") and absolute quantitation by high-resolution mass spectrometry, demonstrated a temperature-dependent "melting curve" by ex vivo incubation of compound with rat tissue and further demonstrated in vivo engagement by a dose-dependent response to treatment. These experiments illustrate the ability to extend the CETSA to in vivo dosed-animal samples using absolute quantitation of DPP9 by mass spectrometry and demonstrate a viable path for interrogating therapeutic molecules for drug discovery.

Disease modifying treatments for Alzheimer's disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.

The field of quantitative, label-free proteomics has evolved significantly over time, with most experiments performed "bottom-up" using proteolyzed protein mixtures. In these experiments, statistically significant peptide abundance differences between two or more experimental conditions are determined, and their corresponding proteins later identified. Recently, the rationale for extending this experimental design to mixtures of intact proteins has become clear, as analysis at the protein level allows for the independent detection of each protein form present, including those modified posttranslationally. This provides a level of specificity lost in bottom-up experiments. As such, the application of label-free top-down differential mass spectrometry has provided a means for understanding the subtle protein changes that define a particular phenotype. Described here is an approach for the top-down label-free quantitative analysis of the proteins which constitute human high-density lipoprotein particles. The methodology is conceptually very straightforward; however, it does require a level of rigor and consistency typically not addressed by more conventional proteomics experiments.

With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization. Described here is the implementation of existing advanced analytical technologies, including high-performance mass spectrometry (LTQ-Orbitrap XL-ETD) and a chip-based nanoelectrospray autosampling robot (TriVersa NanoMate), for the thorough identification and characterization of breakdown products derived from a force-degraded monoclonal antibody. Many anticipated breakdown products were identified, including Fab fragment (48,325 Da) and heavy chain polypeptide hydrolysis product (15,521 Da). Using high-resolution collisionally induced and electron transfer dissociation methods, additional identifications were made with specific localization of unpredicted modifications. As examples, a modified Fab fragment (N- and C-terminal cyclization, 47,902 Da) and a hydrolyzed free light chain impurity components (23,191 Da) were identified with a high degree of confidence (E value, <1e-5). This work describes the approach for top-down characterization of breakdown products and is readily applicable to additional monoclonal antibodies (mAb) characterization experiments, including charge isoform characterization and aggregate analysis, for a more thorough understanding of therapeutic mAb drug products.

Mass spectrometry based proteomic experiments have advanced considerably over the past decade with high-resolution and mass accuracy tandem mass spectrometry (MS/MS) capabilities now allowing routine interrogation of large peptides and proteins. Often a major bottleneck to 'top-down' proteomics, however, is the ability to identify and characterize the complex peptides or proteins based on the acquired high-resolution MS/MS spectra. For biological samples containing proteins with multiple unpredicted processing events, unsupervised identifications can be particularly challenging. Described here is a newly created search algorithm (MAR) designed for the identification of experimentally detected peptides or proteins. This algorithm relies only on predefined list of 'differential' modifications (e.g. phosphorylation) and a FASTA-formatted protein database, and is not constrained to full-length proteins for identification. The algorithm is further powered by the ability to leverage identified mass differences between chromatographically separated ions within full-scan MS spectra to automatically generate a list of likely 'differential' modifications to be searched. The utility of the algorithm is demonstrated with the identification of 54 unique polypeptides from human apolipoprotein enriched from the high-density lipoprotein particle (HDL), and searching time benchmarks demonstrate scalability (12 high-resolution MS/MS scans searched per minute with modifications considered). This parallelizable algorithm provides an additional solution for converting high-quality MS/MS data of multiply processed proteins into reliable identifications., (Copyright © 2011 John Wiley & Sons, Ltd.)

Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL(3). The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 --> 3)-Gal-beta(1 --> 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.

Eosinophilic syncytial change (ESC), also known as papillary syncytial change, occurs in association with endometrial breakdown and bleeding, especially in nonphysiological conditions. When prominent, this morphological alteration yields a pattern of eosinophilic epithelial cells, often in pseudopapillary arrangements that can mimic cellular changes seen in metaplastic and atypical endometrium. To determine if ESC represents a proliferative, regenerative process or a degenerative, retrogressive alteration, we assessed whether the cells of ESC were actively growing. Our methodology involved a retrospective immunohistochemical study on endometrial biopsies with proliferation markers Ki-67 (MIB-1 antibody) and phosphohistone H3 Ser 28 (pHH3) in 15 cases of multifocal ESC associated with benign endometrium, 5 cases of atypical hyperplasia, and 7 cases of endometrial carcinoma. The Ki-67 proliferative index and the pHH3 mitotic index were calculated per 100 cells for each case. On immunohistochemical analysis, the Ki-67 labeling index was 1.3% for cases of ESC (mean age, 53 yr), 15.8% in atypical hyperplasia (mean age, 51.6 yr), and 42.6% in endometrial carcinoma (mean age, 68.1 yr). In the endometrial cancers, the Ki-67 proliferative index was 10.6% for FIGO grade 1 tumors (n=3), 27.6% for grade 2 tumor (n=1), and 79.6% for serous carcinoma (n=3). The mitotic index calculated from pHH3 immunostaining was zero in all cases of ESC, whereas it was 2.3% in atypical hyperplasia and 4.8% in endometrial carcinomas (2.4% for grade 1, 3% for grade 2, and 7.8% for serous). Our results indicate that ESC is a regressive change. Furthermore, when there is a question of whether eosinophilic endometrial epithelium represents this change, a combination of Ki-67 and pHH3 immunostains can be helpful in distinguishing this entity from more significant processes including carcinoma.

Adenocarcinoma in situ rarely occurs in vulvar papillary hidradenoma. We encountered 2 cases of adenocarcinoma in situ arising in a papillary hidradenoma of the vulva. Both patients were asymptomatic women, aged 83 and 92 years, who presented with nodules (1.0 and 2.0 cm) on the vulva. Macroscopically, the lesions seemed tan-pink, fleshy, and well circumscribed. One tumor ulcerated the overlying epidermis. On microscopic examination, the tumors showed focal features of benign hidradenoma at the periphery with transitions into areas of increasing cytologic atypia that fulfilled criteria for adenocarcinoma in situ similar to that seen in the breast. One tumor showed a predominant cribriform pattern with moderate atypia and many mitoses; the other showed a mixture of cribriform and micropapillary patterns, mild atypia, and fewer mitotic figures. There was no evidence of destructive invasion or desmoplasia in either tumor. Both lesions show areas strongly immunoreactive for mammaglobin and gross cystic disease fluid protein 15 as well as estrogen and progesterone receptor protein in 1 case. There was no evidence of benign ectopic breast tissue within or adjacent to the neoplasms. Both patients underwent local excision with no evidence of tumor recurrence at 15 and 32 months of follow-up. The fact that these tumors displayed morphologic and immunohistochemical features that resembled ductal carcinoma in situ of the breast demonstrates the close homology between papillary hidradenoma and breast epithelium. In the absence of invasion, our experience suggests that these tumors can be cured by local excision.

Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.25:1 to 4:1 to make two sets of samples. The resulting mixtures were analyzed by microcapillary LC-FTMS and dMS. dMS successfully identified five out of the six peptides as statistically significant differences (p

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several nonnatural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of noncognate dipeptides on the second module.

Hyperplasia of the endometrium is a process characterized by an irregular, noninvasive proliferation of glands with a variable amount of stroma. Precise classification of endometrial hyperplasia in biopsy material is important in order to identify those hyperplasias that are likely to be precursors of endometrial adenocarcinoma. The current World Health Organization (WHO) classification provides a scheme that has become widely accepted, primarily dividing hyperplasias in to those with and those without cytologic atypia while the degree of glandular crowding (simple vs. complex) has secondary importance. A wide variety of other endometrial changes, ranging from artifacts, metaplasias and polyps to well-differentiated adenocarcinoma must be considered in the differential diagnosis. Well-differentiated adenocarcinoma is diagnosed when one of 3 essential criteria is found in biopsy specimens: (1) a confluent gland pattern; (2) an extensive papillary pattern; or (3) a desmoplastic stromal response. Using the WHO classification allows segregation of endometrial hyperplasia into clinically meaningful categories. Strict morphologic criteria also enable separation of hyperplasia from well-differentiated adenocarcinoma.

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.

Complex arrays of thioester bound intermediates are present on 100-700 kDa enzymes during the biogenesis of diverse types of pharmacophores and natural product drugs. These multidomain enzymes, known as nonribosomal peptide synthetases and polyketide synthases (NRPSs and PKSs, respectively), synthesize from simple, physiologically available substrates bioactive compounds that can be further tailored by a host of modifying domains (e.g., methylation, cyclization, and epimerization) to increase the complexity of the mature final product. Interrogation of such covalent intermediates using mass spectrometry (MS) presents an underutilized method for understanding the covalent catalysis executed by NRPS and PKS enzymes. For the PKS module (205 kDa) from the yersiniabactin (Ybt) gene cluster of Yersinia pestis, limited proteolysis afforded a key 11 kDa peptide from the acyl-carrier protein (ACP) domain upon which at least five covalent intermediates could be detected (42, 70, 86, 330, and 358 Da). The isotopic resolution achieved by Fourier transform mass spectrometry (FTMS) allowed for the incorporation of substrates with stable isotopes to confirm the structural assignments of three intermediates (86, 330, and 358 Da) on the Ybt biosynthetic pathway to within 1 Da. Approximately 75% of the enzyme capacity is lost to unproductive decarboxylation of malonyl-S-ACP partly constraining the 1.4 min(-)(1) rate of Ybt production in vitro. Acyl transfer to the ACP domain (on the Ybt pathway) was promoted by a factor of approximately 10 over unproductive CO(2) loss in the presence of the cosubstrate S-adenosylmethionine (SAM), with S-adenosylhomocysteine unable to restore the condensation yield observed with SAM. The data are consistent with Claisen condensation from KS to the ACP carrier site being reversible, with the absence of downstream methylation providing more opportunity for unproductive CO(2) loss. Extension of such FTMS-based studies will allow the direct visualization of multiple intermediates in determining the catalytic order of events and kinetics of NRPS and PKS systems.

For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine Lys residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of approximately 60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most of which were below 6 kDa); 15% sequence coverage was obtained. For the > 90% pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scale are considered.

Objective: To evaluate the prognostic significance of the Bethesda system's cytologic categories in patients with endometrial malignancy., Methods: Patients with biopsy or hysterectomy-proven endometrial malignancy and a Papanicolaou smear result reported using the Bethesda system within 1 year of diagnosis were identified through retrospective review of our computerized database., Results: After introduction of the Bethesda system in our laboratory on November 1, 1992, until January 1, 1997, 112 eligible patients were identified (108 with carcinomas and four with carcinosarcomas). Patients with cytologic diagnoses of malignancy (n = 17) were significantly more likely to have International Federation of Gynecology and Obstetrics (FIGO) grade 3 tumors and high-risk histology (serous, clear cell, and adenosquamous carcinoma and carcinosarcoma) than those with atypical glandular cells of uncertain significance (n = 33) or those with cytology not suspicious for malignancy (n = 63). Patients with malignant smears were also significantly more likely to have cervical extension, malignant peritoneal cytology, and FIGO stage II, III, or IV than those with atypical glandular cells of uncertain significance or those with cytology not suspicious for malignancy., Conclusion: Papanicolaou smears obtained within 1 year of histologic diagnosis of endometrial malignancy and interpreted using the Bethesda system were suspicious for (atypical glandular cells of uncertain significance) or diagnostic of malignancy in nearly half of all cases (29 and 15%, respectively). Patients having malignant glandular cells were more likely to have poor prognostic pathologic findings.

Objectives: Histologic findings in biopsy specimens obtained from patients with atypical glandular cells of uncertain significance were studied to define the utility and limitations of this category., Study Design: Computerized records over a 3-year period were retrospectively analyzed. The most significant histologic diagnosis from all biopsy specimens submitted was compared with the subcategory of the first Papanicolaou smear obtained showing atypical glandular cells of uncertain significance., Results: Biopsy results were available for 531 of 1117 patients with atypical glandular cells of uncertain significance (48%). Biopsy-proved preinvasive (83%) or invasive (17%) lesions were present in 191 patients (36%). Eighty-nine percent of the preinvasive lesions were squamous, whereas 97% of the invasive lesions were glandular. Glandular lesions were more likely to be invasive, whereas squamous lesions were more likely to be preinvasive (p < 0.001). Twenty-eight patients had endometrial carcinoma, which represents 88% of all invasive carcinomas detected., Conclusions: Almost three fourths of patients with atypical glandular cells of uncertain significance and with lesions have squamous lesions, not glandular as suggested by the name of the category. Unlike patients with atypical squamous cells of uncertain significance, patients with atypical glandular cells of uncertain significance have a significant risk of malignant lesions, which are nearly all glandular and predominantly arise from the endometrium.

Objective: To establish the frequency of the atypical glandular cells of uncertain significance (AGCUS) category, and its subcategories, as defined by the Bethesda System (TBS)., Methods: Our computerized records of cervical/vaginal cytology specimens submitted from January 1, 1993, through December 31, 1995, were retrospectively reviewed for specimens diagnosed as AGCUS. When appropriate, our subcategory of "AGCUS favor premalignant/malignant lesion" was further qualified as "favor endocervical adenocarcinoma in situ" or "suspicious for endometrial carcinoma." The number of specimens and patients diagnosed for each subcategory were grouped by calendar year. Differences in frequency between time periods were tested for statistical significance using chi 2 analysis., Results: AGCUS was diagnosed in 1181 of 177,715 submitted specimens (0.66%). The frequency of subcategories was as follows: "favor reactive" (65%), "unable to further classify" (30%), "favor premalignant/malignant" (2.9%), "suspicious for endometrial carcinoma" (1.9%), and "favor endocervical adenocarcinoma in situ" (0.4%). From 1993 to 1995 there was an increase in the rate of diagnosis of AGCUS (0.55 to 0.73%; P < 0.001) and a decrease in the percentage of specimens with AGCUS subclassified as "favor premalignant/malignant" (6.2 to 0.5%; P < 0.001). Other subcategories showed no significant change in frequency over this time period. The rate of biopsy-proven preinvasive or invasive lesions in AGCUS patients also showed no significant change from year to year over this time period., Conclusion: The AGCUS diagnosis can be anticipated at a low but consistent rate from a cytology laboratory using TBS. Any comparison of laboratories should take into consideration the change in reporting frequencies that occurs as part of the "learning curve" following introduction of TBS reporting. Uniform diagnostic criteria and additional reports with large numbers of cytologic specimens will be needed to establish the expected frequency of AGCUS and its subcategories.

A case of low-grade endometrial stromal sarcoma (endolymphatic stromal myosis) occurring in a patient who had received tamoxifen citrate for 3 years following surgical treatment for breast cancer is presented. Endometrial adenocarcinomas have been the most frequently reported tumor associated with tamoxifen use. More recently, uterine sarcomas have also been described in association with the use of tamoxifen. This report adds only the second case of a low-grade endometrial stromal sarcoma associated with tamoxifen use. As in the first report, the tumor demonstrated a sex-cord-like pattern of differentiation, an uncommon feature of endometrial stromal sarcomas. This suggests a possible association between tamoxifen use and the subsequent development of low-grade endometrial stromal sarcoma.

Well-differentiated endometrial adenocarcinoma of the secretory type (FIGO Grade 1) with minimal myometrial invasion occurred in a postmenopausal patient on tamoxifen therapy 5 years after mastectomy for breast carcinoma. We believe that this is the first report of secretory carcinoma of the endometrium associated with tamoxifen use. This unusual pattern of low-grade endometrial carcinoma adds to the spectrum of uterine neoplasia associated with tamoxifen therapy.

Objective: To report the finding of squamous metaplasia within endometrial glands occurring as a result of progestin therapy of hyperplasia., Design: Case series. Retrospective analysis of two cases from 9235 accessioned endometrial biopsies., Setting: Hospital-based pathology laboratory processing inpatient and outpatient specimens., Patients: Two women, ages 49 and 34 years, with endometrial hyperplasia without squamous metaplasia who were treated with progestin., Results: Extensive squamous metaplasia was found in endometrial glands following progestin therapy. The post-therapy biopsies also showed progestin-related alterations but no evidence of active hyperplasia., Conclusion: Squamous metaplasia in these cases appears to be a consequence of progestin therapy. The absence of hyperplasia in the progestin-treated endometrium suggests that the squamous metaplasia does not represent persistence or progression of the original hyperplastic lesion.

This study evaluated the microscopic changes in leiomyomata following the use of a gonadotropin releasing hormone (GnRH) agonist. Seventeen women with symptomatic leiomyomata were included. Nine were treated with a GnRH agonist for three to six months prior to surgery, and the remaining eight served as controls. Following myomectomy, paraffin sections were prepared from the tumors. These sections were examined microscopically by two gynecologic pathologists, who were blind to the patient groups. The results showed increased cellularity and hyalinization in leiomyomata following GnRH agonist treatment.

Papillary syncytial change (PSC) of endometrial epithelium, often regarded as a metaplastic change, shows syncytial to papillary aggregates of eosinophilic cells along the surface epithelium. To determine the cause and significance of PSC, 250 consecutive endometrial biopsy and curettage specimens and curettings in patients with suspected endometrial abnormalities were reviewed. Papillary syncytial change was found in 43 (17%) of the cases. Often PSC was focal, but in 12 cases it was multifocal and in 7 cases it was extensive. Patients with PSC ranged from 22 to 86 years of age. The primary pathologic findings in endometria with PSC included a variety of benign organic lesions and hyperplasia, as well as proliferative and secretory changes that suggested dysfunctional bleeding. One consistent finding in all cases was associated active endometrial bleeding with glandular and stromal breakdown, cell necrosis, and neutrophils in close proximity to PSC. Immunohistochemical study of 6 cases with extensive PSC showed no difference in reactivity to high and low molecular weight keratin, vimentin, and carcinoembryonic antigen compared with surrounding unaffected epithelium. The association of PSC with endometrial breakdown in a variety of conditions suggests that PSC is a benign retrogressive alteration rather than a metaplastic transformation to another cell type. Papillary syncytial change appears to be a useful histologic marker of acute endometrial breakdown and bleeding, and recognition of this phenomenon will prevent misclassification of this relatively common finding.

Carcinoma metastatic to the spleen is found at autopsy in 6% to 13% of patients who die of cancer, yet clinical symptoms referable to splenic metastases are unusual. Two cases of breast carcinoma metastatic to the spleen discovered incidentally at therapeutic splenectomy for idiopathic thrombocytopenic purpura are described. On gross examination, the spleens were mildly enlarged with a homogeneous congested cut surface; rare 0.2-cm white nodules were present in one case. Microscopic examination revealed large, poorly cohesive cells that diffusely involved both the red and white pulp. Histochemical, immunohistochemical, and ultrastructural analyses confirmed the epithelial nature of the cellular infiltrate. These cases show that idiopathic thrombocytopenic purpura may herald the presence of diffuse splenic metastases when metastatic disease is not otherwise clinically suspected. The lack of a discrete tumor mass in the spleen in such cases may make the diagnosis of metastatic carcinoma a challenge both clinically and pathologically. Immunohistochemical and electron microscopic examinations are useful to establish the appropriate diagnosis in such cases.

Strumal carcinoid is an unusual form of monodermal ovarian teratoma with thyroid-like follicles admixed with typical carcinoid tumor patterns. We encountered a case of this neoplasm in a patient with multiple endocrine neoplasia, type IIA (Sipple's syndrome), including a medullary thyroid carcinoma diagnosed 24 years previously. During evaluation of bilateral adrenal pheochromocytomas, a unilateral left ovarian strumal carcinoid was discovered. Subsequently, the patient had a parathyroid adenoma excised. The ovarian tumor was immunohistochemically reactive for neuron-specific enolase, chromogranin, synaptophysin, and serotonin, but did not stain for calcitonin. The follicular structures stained for thyroglobulin. This unusual case shows that ovarian strumal carcinoid, like carcinoid tumors at other sites, may arise in association with multiple endocrine neoplasia.

Primary sarcoma of the ovary is rare, and only 16 reported examples of leiomyosarcoma are known to us. We encountered a primary ovarian leiomyosarcoma in a 58-year-old woman. The tumor was characterized by interlacing bundles of plump spindle cells that showed immunoreactivity for desmin, vimentin, and muscle-specific actin. Ultrastructural features included abundant smooth-muscle-type filaments, notched nuclei, pinocytotic vesicles, and extracellular collagen. This case illustrates the utility of ancillary techniques in the classification of pure ovarian sarcomas and leads us to propose ultrastructural and immunohistochemical criteria for primary ovarian leiomyosarcoma.

The authors identified five leiomyosarcomas (LMS) in a review of 13 nonmatrix-producing spindle cell sarcomas of bone. Only two were initially recognized as LMS; the others had been diagnosed as malignant fibrous histiocytoma (two) and fibrosarcoma (one). The patients, four of whom were women, ranged in age from 32 to 70 years. Sites included proximal humerus (two), distal femur (two), and rib (one). All tumors presented with clinical and radiographic features consistent with a diagnosis of primary bone neoplasms, although one probably represented a solitary metastasis from a primary uterine LMS. Radiographs showed lytic bone destruction with a moth-eaten appearance, and three cases had soft tissue extension. Histologically, all tumors showed broad, interlacing fascicles of spindle cells with pleomorphic nuclei, frequent mitoses, and necrosis. Two cases had a focal storiform pattern and bizarre multinucleated cells, and two other cases had focally prominent osteoclast-like giant cells. Extensive immunoreactivity for muscle actin was seen in all cases and for desmin in three. In each case, electron microscopy showed definite smooth muscle differentiation including cytoplasmic filaments with densities. At this writing, two patients are free of disease (including the patient with a presumed metastasis), one is alive with locally recurrent disease, and two are dead of disease. Experience suggests that LMS of bone is a distinct clinicopathologic entity that may be more common than previously recognized. Application of immunohistochemistry and electron microscopy to nonmatrix-producing bone sarcomas should facilitate diagnosis of additional cases.

We examined the immunohistochemical profile of 21 granular cell tumors (GCTs) and a single clinically malignant GCT using a panel of commercially available antibodies. All cases showed diffuse cytoplasmic and nuclear staining for S100 protein. Fourteen cases stained for myelin basic protein, Leu-7, or both. Immunostains for neurofilament protein and glial fibrillary acidic protein were negative in all cases. Stains for cathepsin B and alpha 1-antichymotrypsin were positive in 21 and 15 cases, respectively. Cathepsin-B reactivity may reflect autodigestion of myelin, while the presence of alpha 1-antichymotrypsin is less specific and may be related to cellular production of this product or to nonspecific uptake of alpha 1-antichymotrypsin in serum during the formation of phagolysosomes. All tumors expressed vimentin, often in a distinctive peripheral cytoplasmic pattern. Focal desmin staining was seen in three separate specimens from the patients with the malignant GCT, but this tumor also expressed S100 protein, myelin basic protein, and Leu-7 and did not stain for muscle-specific actin. The desmin reactivity in this single case probably represents non-specific staining rather than myogenous differentiation, since the reactivity to other nerve sheath markers shows histogenetic similarity with the benign GCTs. These findings support a Schwann cell origin for nongingival GCTs and illustrate a useful panel of commercially available antibodies to diagnose these distinctive tumors.

The immunohistochemical and DNA profiles of two cases of florid papillomatosis of the nipple (FPN) were compared with the immunohistochemical and DNA profiles of mammary intraductal carcinomas (IC) to assess the relationship between these two proliferative neoplasms. Both examples of FPN were circumscribed papillary tumors in the subareolar breast that showed cytologic atypia, intraductal necrosis, and a distinct myoepithelial cell layer. An antibody to muscle-specific actin (MSA) decorated a continuous myoepithelial layer in one case that was confirmed by electron microscopy. MSA showed patchy, discontinuous staining of apparent myoepithelium in the ICs. Flow cytometric analysis showed that both FPN lesions were diploid, rapidly proliferating lesions with S-phase fractions of 10.9% and 34.4%. One IC was aneuploid, and the five diploid neoplasms showed S-phase fractions ranging from 6.4 to 15.8%. In FPN many epithelial cells stained intensely for S-100 protein, but each IC also showed at least focal expression of S-100 protein. One case of FPN was focally positive for gross cystic disease fluid protein 15 (GCDFP-15), but neither stained for tumor-associated glycoprotein-72 (TAG-72) nor for the product of the c-erbB-2 oncogene. In comparison, three ICs expressed focal GCDFP-15, four stained for TAG-72, and one was positive for the c-erbB-2 oncogene product. These preliminary observations suggest that the tandem proliferation of epithelial and myoepithelial cells and the preservation of a normal structural relationship between the two appears to separate FPN from intraductal carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody B72.3 reacts with a tumor-associated glycoprotein designated TAG-72 that is expressed in many adenocarcinomas but not in normal tissues. The authors evaluated the immunoreactivity of B72.3 to benign, hyperplastic prostate, and to primary adenocarcinoma of the prostate to determine the frequency of TAG-72 production by benign and malignant prostatic epithelium. Focal cytoplasmic staining of gland cells was seen in 19 of 20 cases of glandular hyperplasia, and weak, homogeneous staining of secretions was seen in five cases. In contrast, 27 of the 35 (77%) adenocarcinomas studied showed at least focal intense staining of secretions, and 30 (86%) of the tumors showed some cytoplasmic immunostaining with B72.3. Positive staining occurred in all of the well-differentiated adenocarcinomas (100%) but was seen less often in moderately differentiated (82%) and poorly differentiated adenocarcinomas (58%). Because benign gland cells may express the TAG-72 antigen, immunohistochemistry results must be interpreted with caution and with regard to the overall morphologic pattern. Nonetheless, a positive B72.3 immunostain may be useful in identifying adenocarcinoma of the prostate, especially when an intense luminal reaction is found. A negative stain does not exclude the presence of adenocarcinoma, however.

A case of angiosarcoma of the skin of the breast is described in a woman 7 years after a primary breast carcinoma was treated by means of lumpectomy and irradiation. On mammograms, the angiosarcoma showed redevelopment of skin thickening and increase in breast density. Clinically, the skin showed patchy discoloration. Although there is an established association of angiosarcoma with lymphedema and therapeutic irradiation, there have been few other reports of this rare complication of local therapy for breast carcinoma. Recognition of the mammographic and clinical manifestations may help in the earlier diagnosis of additional cases.

We encountered an unusual ovarian tumor consisting of a mixture of typical endodermal sinus tumor (EST) and mucinous cystadenofibroma that occurred in the ovary of an 82-year-old female patient. The EST component showed the classic histologic features of this tumor. Serum alpha-fetoprotein (AFP) level was not determined. Tumor stains were negative for AFP but positive for alpha-1-antitrypsin. The malignant germ cell component was intimately associated with the benign mucinous component. Focal production of epithelial mucin and carcinoembryonic antigen (CEA) in the EST component suggested a probable association between the two tumor types. The tumor was confined to one ovary, and the patient is disease-free 2 years after surgical therapy. This neoplasm is unique not only for the malignant germ cell component occurring in an 82-year-old woman, but for the unusual combination of tumor types. The pathogenesis is unknown.

Pelvic fibromatoses are only unusually encountered in gynecologic practice, but then consistently create a diagnostic and therapeutic challenge. The reported patient typifies the difficulties encountered in surgical excision. A discussion of theories of etiology and management options follows.

We describe a case of endolymphatic stromal myosis (ESM) in which the lesion presented uniquely as recurrent frond-like cervical polyps. The lesion behaved aggressively, spreading to the peritoneum. Ultrastructurally, this tumor closely resembled proliferative phase endometrial stroma, in accord with the theory that ESM is a low-grade stromal sarcoma. The ultrastructural correlations may aid in developing future therapy, including progestagen administration for these unusual tumors.

A case of an ovarian endometrioid cystadenofibroma with epithelial atypia and Meigs' syndrome is described. The patient had a large pleural effusion that resolved rapidly after extirpation of the tumor. The S-phase fraction, measured by in vitro incorporation of tritiated thymidine, was extremely low, suggesting that this unusual tumor has a limited growth potential despite its atypical features. Ultrastructurally, the epithelial component has few features described in other ovarian endometrioid tumors. The cells have deeply clefted nuclei and numerous secondary cytolysosomes, features more commonly seen in Brenner tumors; the morphologic similarities may reflect a low epithelial turnover rate. Despite a review of the literature and an analysis of the case, the authors were unable to determine the pathogenetic mechanism of Meigs' syndrome.

The fine structure of 26 metastatic melanomas, including 13 pigmented and 13 amelanotic tumors, as studied to define ultrastructural criteria for diagnosis. Several important features in addition to melanosome granules were identified. Dendritic cytoplasmic processes were seen in 25 of the 26 cases, cell clusters formed by concentric aggregates of several cells and their processes were present in 18, and microvilli were found on cell surfaces in all cases. The cytoplasm was complex, containing numerous mitochondria, Golgi systems, endoplasmic reticulum, nonspecific filaments, and microtubules. Nuclear morphology was variable. Basement membranes and cell junctions including desmosomes were often encountered, and some melanomas shared features with Schwann cells, including complex membrane interdigitations. All tumors contained melanosomes, although the classic forms were frequently difficult to identify and abnormal variant forms often predominated. Knowledge of the variant melanosome morphology and an understanding of the other fine structural features of malignant melanocytes can help identify those cases that lack classic premelanosome granules.

Examples of focal clearing of endometrial epithelial nuclei associated with the presence of trophoblastic tissue have been observed recently in our surgical pathology laboratories. These nuclear alterations were initially observed in endometria from first- and second-trimester spontaneous abortions, term pregnancies, and a uterus harboring choriocarcinoma. Subsequently, an identical change was seen in small foci of endometriosis removed during postpartum tubal ligation. The prominent vacuolated appearance to the nuclei resembled, in some cases, the inclusions seen in herpesvirus infection, but electron-microscopic study showed that the nuclear clearing was due to replacement of normal chromatin by a network of fine filamentous material rather than herpesvirus DNA. A subsequent review of 200 consecutive first-trimester abortions found less-striking degrees of this change in 7% of cases. The endometrial nuclear clearing was focal; often the endometrium also contained cells which demonstrated the Arias-Stella reaction. The etiology of this type of nuclear clearing is not known, but the fibrils may represent a thread-like substructure of normal chromatin which forms as a result of hormonal stimulation of endometrial epithelial cells.

The ultrastructure of optically clear endometrial nuclei is presented. These nuclear alterations have been found in spontaneous abortion, term pregnancy, endometriosis, and uterine choriocarcinoma.

A case of adenocarcinoma of the rete testis was encountered in a 36-year-old white man. The tumor fulfilled established criteria for determining origin in the rete and showed an unusual biphasic morphology with papillary adenocarcinoma mixed with a prominent component of cytologically malignant spindle cells. Immunohistochemical study demonstrated a positive reaction in the epithelium for cytokeratin and epithelial membrane antigen, and the cytoplasm of a few of the spindle cells also reacted with these antibodies. Electron microscopic study confirmed the biphasic pattern, showing epithelial gland formation and mesenchymal cells. The results indicate that this tumor is a metaplastic carcinoma of the rete testis. Recognition of this pattern of rete carcinoma may further enhance our knowledge of primary tumors at this unusual site.

Ten trophoblastic tumors, including seven classical choriocarcinomas, two choriocarcinomas with atypical histology, and one placental-site trophoblastic tumor (PSTT), were studied to compare their fine structural features. Ultrastructurally, the classical choriocarcinomas showed well-defined cytotrophoblasts and syncytiotrophoblasts. The cytotrophoblasts were primitive epithelial cells, while the syncytiotrophoblasts were complex cells with multiple nuclei and dense cytoplasm containing dilated endoplasmic reticulum, lysosomes, vesicles, and tonofilaments. The syncytiotrophoblast cell membranes often contained numerous microvilli. In the choriocarcinomas, scattered intermediate trophoblasts showed features transitional between the cytotrophoblasts and the syncytiotrophoblasts, with moderately complex cytoplasm containing some of the organelles found in the syncytiotrophoblasts. Histologically, the atypical choriocarcinomas showed a predominance of mononucleate and binucleate cells and indistinct syncytiotrophoblasts. Ultrastructurally, these atypical tumors were composed largely of intermediate trophoblasts, yet contained scattered syncytiotrophoblasts with microvilli in compressed aggregates. The PSTT was composed primarily of intermediate trophoblasts that contained prominent paranuclear filaments not seen in the intermediate trophoblasts of the choriocarcinomas. Rare cells resembling syncytiotrophoblasts were found in the PSTT, but no cytotrophoblasts were observed. Immunoreactivity for human chorionic gonadotropin and human placental lactogen was found in the intermediate trophoblasts and syncytiotrophoblasts of both the choriocarcinomas and the PSTT, demonstrating functional homology between these tumors despite some ultrastructural differences. These results demonstrate ultrastructural features of trophoblastic cells that correlate with the morphologic diversity seen in these tumors by light microscopy. Furthermore, the comparisons suggest that the PSTT is composed of a distinct form of intermediate trophoblast that appears to reflect its origin from the extravillous trophoblast.

A case of herpes simplex virus (HSV) infection was diagnosed by biopsy of the cervix and endometrium in a 28-year-old woman with abnormal uterine bleeding. The cervical biopsy demonstrated surface ulceration and underlying patchy necrosis of endocervical clefts and stroma. The endometrium was late secretory, with striking patchy necrosis of gland epithelium and stromal cells. Both sites contained occasional epithelial and stromal cells with nuclear inclusions consistent with HSV infection. Viral culture further confirmed the presence of HSV. Immunohistochemistry demonstrated the presence of HSV antigens in the tissue, and ultrastructural study of the endometrium revealed viral particles within epithelial and stromal cells. The results suggest endometrial involvement via an ascending infection from the cervix. Recognition of this unusual pattern of endometrial inflammation may facilitate diagnosis of additional cases.

A battery of nine lectins was applied to routinely processed endometrial biopsies to evaluate changes in glycoconjugate expression during the menstrual cycle and early gestation. Specific staining patterns emerged for glands, surface epithelium, predecidual and decidual cells, stromal granulocytes, and endothelium. Three lectins--wheat germ agglutinin (WGA), Ricinus communis-I (RCA-I), and Concanavalin ensiformis (Con-A)--showed consistent binding to epithelial and stromal cells in the menstrual cycle. WGA and RCA were bound to gland secretions whereas Con-A labeled epithelial cytoplasm. These three lectins stained occasional stromal cells early in the cycle, but WGA and RCA marked granulocytes whereas Con-A labeled predecidua in the late secretory phase. Succinylated WGA had a distribution similar to WGA but lacked its consistency and intensity. In the menstrual cycle peanut agglutinin reacted with ciliated cells. In addition, this lectin along with soybean agglutinin and Dolichos biflorus labeled gland secretions in pregnancy. The remaining lectins, Phaseolus vulgaris leucoagglutin and Ulex europaeus, marked some components, especially endothelium, but generally had less reactivity than the other lectins did. The results indicate that glycoconjugate production is correlated with the functional status of the endometrium. Presumably these glycoconjugates play a role in endometrial development necessary for nidation, and alterations in lectin distribution may reflect functional abnormalities not apparent by light microscopic study.

Twenty-eight gastric wall tumors classified by light microscopy as leiomyomas or leiomyosarcomas were reevaluated for histogenesis. Each case was analyzed for the presence of S-100 protein, a marker for cells derived from neuroectoderm, by the immunoperoxidase technique. Eight tumors contained cells with positive immunostaining for S-100 protein. Usually this staining was focal, but in one case staining was diffuse. In three additional cases the immunostaining outlined a nerve within the tumor. In contrast, two esophageal and 10 uterine leiomyomas, as well as normal gastric smooth muscle, gave negative reactions for S-100 protein. Twelve cases had tissue processed for electron microscopy. Only two of the tumors, one leiomyoma and one leiomyosarcoma, contained cytoplasmic myofilaments with densities expected in cells derived from smooth muscle; neither of these tumors stained for S-100 protein. In one case, the tumor with diffuse staining for S-100 protein, the cells resembled Schwann cells ultrastructurally. The remaining nine cases had neither smooth muscle nor Schwann cell features. They did contain interposed cell processes, primitive junctions, and large cytoplasmic vacuoles. The results of this study indicate that many gastric wall tumors are not derived from smooth muscle. The presence of S-100 protein suggests a nerve sheath origin in some cases. While the ultrastructure of many gastric tumors does not resemble nerve sheath cells in most peripheral nerves, the myenteric nervous system is a possible source for perineurial or mesenchymal nerve sheath cells with distinctive fine structure. Further study is needed to refine our knowledge of the histogenesis of gastric stromal tumors.

In order to better define the frequency and patterns of metastasis to the female genital tract, all cases of nonhematopoietic metastases to the adnexa, uterus, vagina, and vulva encountered in patients treated at Barnes Hospital between 1950 and 1981 were reviewed. Three hundred twenty-five metastatic cancers from 269 patients were recovered. One hundred forty-nine cases were from extragenital primaries; the remaining tumors were intragenital metastases. Ovary and vagina were the most frequent metastatic sites for both extragenital and genital primaries. The majority of the extragenital metastases were adenocarcinomas from the gastrointestinal tract, but a variety of other primaries did spread, on occasion, to the genital tract. Twenty-seven percent of the metastases presented as possible primary gynecologic lesions, and 75% of these tumors had an extragenital origin. It is shown that despite certain trends in the distribution of metastases, all sites in the female genital tract are at risk for the occurrence of metastases.

A cystic tumor composed of atypical glands in a cellular stroma arose in the pelvis of a 49-year-old man. Two years later an identical tumor was again excised from the pelvis. Morphologic, immunohistochemical and ultrastructural studies indicate that this neoplasm arose in the seminal vesicle, possibly from a seminal vesicle cyst. The tumor did not involve the prostate gland, and immunohistochemical stains for prostate-specific antigen and prostatic acid phosphatase were negative. Ultrastructural study showed that both the glandular and mesenchymal components of the tumor recapitulated features of normal seminal vesicle, further establishing origin from this site. This tumor resembles the rare cystadenoma of the seminal vesicle, yet the cytologic atypia suggests low grade malignant potential. Following the second excision, the patient has had a disease-free interval of 18 months. Long term follow-up and recognition of additional cases is necessary to define the biologic potential of this unusual tumor.

A 33-year-old woman had a metastasizing leiomyoma in which mitotic figures could not be found. The tumor was composed of well differentiated smooth muscle indistinguishable from uterine leiomyoma or normal myometrium by light and electron microscopy. The S-phase fraction of the tumor, measured by in vitro tritiated thymidine labeling, was near the upper limit of the range seen in seven ordinary uterine leiomyomas and was distinctly lower than that in a uterine leiomyosarcoma. The content of estrogen receptors in the cytosol was within the range seen in uterine leiomyomas. Metastasizing leiomyoma is an actively proliferating neoplasm of mature smooth muscle that appears to be hormonally responsive. The tumor in our patient showed no clear differences from ordinary leiomyomas in findings relating to the rate of cellular proliferation, morphology, or estrogen receptor content.

Four new cases of primary choriocarcinoma arising in the placenta during a seemingly normal gestation were studied at the Trophoblastic Disease Center of Northwestern University. In each case the patient presented with disseminated metastases while carrying an intrauterine gestation with a normally developing fetus. All four placental primaries were small; three of the tumors were microscopic and found only after extensive sectioning. Histologically, these tumors all appeared to arise from the cytotrophoblastic cells covering the stromal portion of villi, and in some areas the involved villi retained a portion of normal investing trophoblast. This study shows that gestational choriocarcinoma unassociated with hydatidiform mole can have an early stage in which chorionic villi are present. The consistently small size of the lesions studied suggests that primary placental choriocarcinoma may frequently be overlooked or missed, and that choriocarcinoma possibly has its origin in the placenta more often than in retained or persistent trophoblast following pregnancy.