Toward efficient analysis of >70 kDa proteins with 100% sequence coverage.

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.

Papanicolaou smears by the Bethesda system in endometrial malignancy: utility and prognostic importance.

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.

Biopsy findings in five hundred thirty-one patients with atypical glandular cells of uncertain significance as defined by the Bethesda system.

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.

Incidence of atypical glandular cells of uncertain significance in cervical cytology following introduction of the Bethesda System.

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.

Endolymphatic stromal myosis associated with tamoxifen use.

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.

Secretory endometrial adenocarcinoma in a patient on tamoxifen for breast cancer: a report of a case.

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.

Endometrial squamous metaplasia. An unusual response to progestin therapy of hyperplasia.

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.

Hyalinization and cellular changes in uterine leiomyomata after gonadotropin releasing hormone agonist therapy.

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.

Endometrial papillary syncytial change. A nonspecific alteration associated with active breakdown.

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.

Breast carcinoma diffusely metastatic to the spleen. A report of two cases presenting as idiopathic thrombocytopenic purpura.

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.

Ovarian strumal carcinoid in association with multiple endocrine neoplasia, type IIA.

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 ovarian leiomyosarcoma. An immunohistochemical and ultrastructural study.

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.

Leiomyosarcoma of bone. A clinicopathologic, immunohistochemical, and ultrastructural study of five cases.

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.

Granular cell tumor. Immunohistochemical analysis of 21 benign tumors and one malignant tumor.

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.

Florid papillomatosis of the nipple: immunohistochemical and flow cytometric analysis of two cases.

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)

Prostatic adenocarcinoma. Evaluation of immunoreactivity to monoclonal antibody B72.3.

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.

Cutaneous angiosarcoma of the breast 7 years after lumpectomy and radiation therapy.

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.

Uterine herpes virus infection with multifocal necrotizing endometritis.

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.

Adenocarcinoma of the rete testis with a spindle cell component. A possible metaplastic carcinoma.

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.