Mirka W. Jones

Carcinosarcomas (malignant mixed mullerian tumors) of the female genital tract: comparative molecular analysis of epithelial and mesenchymal components.

Female genital tract carcinosarcomas (FGTCS) are biphasic neoplasms composed of an admixture of malignant epithelial and mesenchymal elements. Histogenesis of FGTCS centers on two theories: (1) simultaneous formation of independent tumors (biclonal theory), (2) multidirectional differentiation of a single neoplasm (monoclonal theory). In an attempt to resolve this histogenetic controversy, we determined the presence, specific genotype, and timing of p53 mutational change in each component of FGTCS using a topographic genotyping (TG) approach. We selected 43 FGTCS from the files of Magee-Womens Hospital, Pittsburgh, and initially immunostained them for p53 protein. Strong p53 immunopositivity was detected in 35 (82%) of 43 tumors. Subsequently, topographic genotyping (TG) was performed on a subset of nine immunopositive tumors with sufficiently distinct malignant components to enable effective sampling. All nine tumors showed point mutations in p53 exons 5 through 8. In each case, the identical point mutational genotype was present in both components. Furthermore, in all nine cases mutations were present with loss of the wild-type allele. P53 gene mutation is a frequent event in progression of FGTCS. Of importance, both p53 mutation and allelic loss occur before the differentiation into separate epithelial and mesenchymal malignant components. These molecular findings strongly support monoclonal, multidirectional histogenesis of FGTCS.

Immunohistochemical profile of endometrial adenocarcinoma: a study of 61 cases and review of the literature.

The differences in immunohistochemical expression of p53, bcl-2, bax, estrogen receptor (ER), and progesterone receptor (PR) were evaluated in 40 endometrioid and 21 papillary serous carcinomas of endometrium and correlated with known predictors of survival, such as grade and stage. Uterine papillary serous adenocarcinomas (UPSA) showed significantly higher p53 expression than did uterine endometrioid adenocarcinomas (UEA) (76.2% versus 35%), whereas both ER and PR were more often positive in endometrioid than in serous tumors (p =.005 and.0005). No significant difference was found in bcl-2 and bax expression between both histologic types. However, there was definite decrease in intensity of bcl-2 in UPSA compared with UEA. In endometrioid carcinoma, p53 overexpression was associated with high-grade and advanced-stage tumors (p =.0006 and.006), whereas ER and PR expression was associated with low-grade and early-stage tumors (p =.0006 and.0001; p =.003 and.0006). Bcl-2 immunopositivity was more common in low-grade, early-stage rather than in high-grade, advanced-stage adenocarcinomas, but the difference was not statistically significant (p =.24 and.07). Bax immunopositivity was associated with well-differentiated (p =.04) and early-stage tumors. Furthermore, a significant inverse relationship between bax and p53 reactivity was defined (p =.05), especially in tumors of endometrioid type. Bax and PR immunoexpression correlated near the limit of statistical significance (p =.08), whereas no relationship was found among bax, bcl-2, and ER immunopositivity. Our results indicate that the differences in immunohistochemical profiles of endometrioid and serous carcinomas support the existence of different molecular pathways of their development. The correlation of immunohistochemical findings with histologic grade and clinical stage could help in predicting biologic behavior and planning treatment in patients who are diagnosed as having these tumors.

Whole Exome Sequencing in a Random Sample of North American Women with Leiomyomas Identifies MED12 Mutations in Majority of Uterine Leiomyomas

Uterine leiomyomas (uterine fibroids) arise from smooth muscle tissue in the majority of women by age 45. It is common for these clonal tumors to develop from multiple locations within the uterus, leading to a variety of symptoms such as pelvic pain, abnormal uterine bleeding, and infertility. We performed whole exome sequencing on genomic DNA from five pairs of leiomyomas and corresponding normal myometrium to determine genetic variations unique to leiomyomas. Whole exome sequencing revealed that the gene encoding transcription factor MED12 (Mediator complex subunit 12) harbored heterozygous missense mutations caused by single nucleotide variants in highly conserved codon 44 of exon 2 in two of five leiomyomas. Sanger re-sequencing of MED12 among these five leiomyomas confirmed the two single nucleotide variants and detected a 42 base-pair deletion within exon 2 of MED12 in a third leiomyoma. MED12 was sequenced in an additional 143 leiomyomas and 73 normal myometrial tissues. Overall, MED12 was mutated in 100/148 (67%) of the genotyped leiomyomas: 79/148 (53%) leiomyomas exhibited heterozygous missense single nucleotide variants, 17/148 (11%) leiomyomas exhibited heterozygous in-frame deletions/insertion-deletions, 2/148 (1%) leiomyomas exhibited intronic heterozygous single nucleotide variants affecting splicing, and 2/148 (1%) leiomyomas exhibited heterozygous deletions/insertion-deletions spanning the intron 1-exon 2 boundary which affected the splice acceptor site. Mutations were not detected in MED12 in normal myometrial tissue. MED12 mutations were equally distributed among karyotypically normal and abnormal uterine leiomyomas and were identified in leiomyomas from both black and white American women. Our studies show an association between MED12 mutations and leiomyomas in ethnically and racially diverse American women.

Adjuvant radiation for vulvar carcinoma: Improved local control

PURPOSE Local recurrence is a significant problem following primary surgery for advanced vulva carcinoma. The objectives of this study were to evaluate the impact of adjuvant vulvar radiation on local control in high risk patients and the impact of local recurrence on overall survival. METHODS AND MATERIALS From 1980-1994, 62 patients with invasive vulva carcinoma and either positive or close (less 8 mm) margins of excision were retrospectively studied. Thirty-one patients were treated with adjuvant radiation therapy to the vulva and 31 patients were observed after surgery. Kaplan-Meier estimates and the Cox proportional hazard regression model were used to evaluate the effect of adjuvant radiation therapy on local recurrence and overall survival. Independent prognostic factors for local recurrence and survival were also assessed. RESULTS Local recurrence occurred in 58% of observed patients and 16% in patients treated with adjuvant radiation therapy. Adjuvant radiation therapy significantly reduced local recurrence rates in both the close margin and positive margin groups (p = 0.036, p = 0.0048). On both univariate and multivariate analysis adjuvant radiation and margins of excision were significant prognostic predictors for local control. Significant determinants of actuarial survival included International Federation of Gynecologists and Obstetricians (FIGO) stage, percentage of pathologically positive inguinal nodes and margins of excision. The positive margin observed group had a significantly poorer actuarial 5 year survival than the other groups (p = 0.0016) and adjuvant radiation significantly improved survival for this group. The 2 year actuarial survival after developing local recurrence was 25%. Local recurrence was a significant predictor for death from vulva carcinoma (risk ratio 3.54). CONCLUSION Local recurrence is a common occurrence in high risk patients. In this study adjuvant radiation therapy significantly reduced local recurrence rates and may improve overall survival in certain subgroups. As salvage rates after developing local recurrence are poor adjuvant vulvar radiation should be considered for patients at risk after primary surgery.

Clinicopathologic study of 28 uterine leiomyosarcomas with metastasis

Twenty-eight smooth muscle tumors of the uterine corpus that exhibited metastasis were studied to determine gross and microscopic features associated with metastasis and potential for metastasis. Seventeen of 28 patients were advanced stage at initial diagnosis, and the other 11 patients developed metastases 3 months to 9 years later. Seventy-nine percent of tumors occurred in women 50 years or older. Size was a feature of metastatic capability, as 22 tumors were larger than 5 cm. Of the six neoplasms smaller than 5 cm, five had increased mitotic activity and cytologic atypia to qualify them as sarcomas. The remaining one with four mitotic figures (MF) per 10 high-power fields (HPF) and 1+ atypia had infiltrative margins and vascular invasion that probably accounted for its aggressive behavior. Sixteen cases were composed predominantly of spindle cells, 10 were epithelioid, and two were myxoid. Cytologic atypia was a prerequisite for metastasis as it was present in all. On a scale of 3, 12 were nuclear grade 3, 15 were grade 2, and one was grade 1. The average mitotic activity was 14 MF/10 HPF, and 86% had > 4 MF/10 HPF. Of the five leiomyosarcomas with low mitotic activity (fewer than 5 MF/10 HPF), three were the spindle cell variety, one was epithelioid, and one was myxoid. An epithelioid cell type occurred in 39% of tumors and is a feature that identifies a neoplasm capable of metastasis, even with low mitotic activity.

Adenosarcoma of the uterine cervix: a clinicopathological study of 12 cases.

The clinical and pathologic features of 12 cervical adenosarcomas from the files of the Armed Forces Institute of Pathology are described. The patients ranged in age from 13 to 67 years (mean 37). The majority (58%) presented with abnormal bleeding. All tumors were located in the cervix and consisted of soft, tan, polypoid or papillary masses ranging in size from 1.5 to 4.5 cm. Microscopically, they showed a biphasic pattern with mesenchymal and epithelial components. There was a characteristic stromal condensation below the epithelial surface and around glandular structures. The cytologic atypia of stromal cells was 1+ in three, 2+ in five, and 3+ in four. The mitotic activity ranged from four to 28 (mean 7.0) mitotic figures per 10 high-power fields. One neoplasm contained cartilage and one striated muscle. Myometrial invasion was present in three. Treatment consisted of hysterectomy in nine patients and excisional biopsy in three. Two patients received radiotherapy; one before surgery and the other after hysterectomy. Two were treated with chemotherapy. Follow-up ranged from 9 months to 18.8 years. Nine patients were alive and well with no evidence of recurrent tumor at postoperative intervals of 0.8-18.8 years. One patient died 1 year after diagnosis with intraabdominal metastasis. One developed a recurrent tumor. This study demonstrates a favorable prognosis for patients with cervical adenosarcoma. Similar to patients with uterine adenosarcoma, prognosis is mostly affected by the presence of deep myometrial invasion.

Prognostic significance of p53, bcl-2, vimentin, and S 100 protein-positive langerhans cells in endometrial carcinoma

Immunohistochemical expression of p53, Bc12, vimentin, and S100 protein-positive Langerhans cell was evaluated in 50 endometrial carcinomas (6 stage I, 14 stage II, 20 stage III, and 10 stage IV), in an attempt to use these markers as predictors of survival. Monoclonal antibodies to p53, Bcl-2, vimentin, and S100 proteins were applied to paraffin-embedded sections of endometrial adenocarcinoma, using the avidin-biotin peroxidase complex technique (ABC). All 20 patients with stage I and II carcinomas were alive with a mean follow-up of 3 years. Of 30 patients with stage III and IV carcinomas, 13 died of tumor (3-year survival, 57%; SE, 10%), eight were alive with tumor, and nine were alive with no evidence of tumor (mean follow-up, 46 months). Strong p53 positivity was present in 11 carcinomas (22%), including nine high-stage and two low-stage tumors. Bcl-2 positivity was identified in 33 tumors (66%). These tumors were mostly low stage; however, no correlation was found between Bcl-2 expression and prognosis. Vimentin positivity (P < .001), and tumor infiltration by a large number of S100 protein-positive Langerhans cells (P < .05) were associated with low-stage tumors. Vimentin was expressed in 23 carcinomas, including 70% of low-stage tumors and 20% of high-stage tumors. Most high-grade carcinomas were Langerhans cell depleted; most low-grade carcinomas showed >50 S100 protein-positive Langerhans cells/10 high-power fields. Our results indicate that Langerhans cell infiltration and vimentin positivity of tumor cells are favorable prognostic factors in endometrial carcinomas.

Genomic alterations in uterine leiomyosarcomas: Potential markers for clinical diagnosis and prognosis

Genomic alterations were analyzed in 21 uterine leiomyosarcomas (ULMSs) by comparative genomic hybridization. DNA copy number changes were detected in all 21 tumors. The most frequent losses were 13q (16/21 = 76%), 10q (13/21 = 62%), 16q (8/21 = 38%), 12p (7/21 = 33%), and 2p (9/21 = 43%). The most common gains were 17p (8/21 = 38%), Xp (7/21 = 33%), and 1q (7/21 = 33%). High‐copy‐number gains (ratio > 1.5) were identified in Xp, 1q, and 17p. Loss of 13q was identified in both low‐grade and high‐grade tumors. Inactivation of a tumor suppressor gene in 13q may be an early event in the development of leiomyosarcomas. Loss of 10q, 2p, and 12p and gains of 1q as well as 17p were frequently found in high‐grade tumors and recurrent tumors. Inactivation of tumor suppressor genes and activation of oncogenes in these regions may be associated with a more aggressive behavior of ULMS. Patients with only loss of 13q and without the other alterations listed above had longer survival times. Gains of Xp, 17p, and 1q and losses of 13q, 10q, 16q, 12p, and 2p have been reported in extra‐uterine leiomyosarcomas. Our findings indicate that the pathogenesis of uterine leiomyosarcomas and extra‐uterine leiomyosarcomas follows the same genetic pathways.

Fibrosarcoma-malignant fibrous histiocytoma of the breast. A clinicopathological study of 32 cases.

&NA; We report the clinical and pathologic features of 32 sarcomas of the breast with features spanning the spectrum of fibrosarcomas‐malignant fibrous histiocytomas. Neoplasms were categorized as high‐ or low‐grade lesions depending on a combination of the degrees of atypia and mitotic activity. The majority of high‐grade lesions had marked (3 +) nuclear atypia and at least five mitotic figures per 10 hpf. High‐grade lesions with moderate (2 +) nuclear atypia had a mitotic activity of six or more mitotic figures per 10 hpf. All low‐grade lesions had five or fewer mitotic figures per 10 hpf, and none had a score of the nuclear grade times mitotic figures of more than 10. The average mitotic activity in low‐grade lesions was two mitotic figures per 10 hpf; the high‐grade lesions had 12 mitotic figures per 10 hpf. Sixty‐nine percent of the lowgrade fibrosarcomas‐malignant fibrous histiocytomas showed mild (1 +) cytologic atypia, and 69% of the highgrade lesions showed severe (3 +) cytologic atypia. The herringbone pattern was associated with a more favorable prognosis than the malignant fibrous histiocytoma pattern. Compared to the high‐grade lesions, low‐grade fibrosarcomas‐malignant fibrous histiocytomas were slowgrowing, produced fewer recurrences, and did not metastasize. Of the 16 women with low‐grade lesions, all were free of tumor at last contact, despite recurrence in more than half of the patients. In contrast, 31% of the patients with high‐grade lesions died of tumor, and 13% were alive with disease. Twenty‐five percent of women with highgrade lesions developed distant metastases.

Well-differentiated villoglandular adenocarcinoma of the uterine cervix: oncogene/tumor suppressor gene alterations and human papillomavirus genotyping.

Twelve well-differentiated villoglandular adenocarcinomas (WDVAs) of the uterine cervix were retrospectively analyzed for the presence and specific genotype of human papillomavirus (HPV), tumor suppressor loss (p53, MCC, APC, BRCA1), cancer gene mutation (K-ras-2, exons 1 and 2, p53 exons 5 to 8), and oncogene amplification (c-erbB-2/HER-2/neu, int-2). Tissue for genetic evaluation was obtained by microdissection, using 4-&mgr;m-thick histology sections of archival, formalin-fixed, paraffin-embedded specimens. Genotyping involved nucleic acid amplification and DNA sequencing with gene-specific oligonucleotides and L1 region consensus primers for common strains of HPV. Point mutation and HPV strain determination were accomplished by DNA sequence analysis. Tumor suppressor gene loss and oncogene amplification were performed by allelic imbalance analysis in informative subjects based on DNA sequence and microsatellite-length polymorphisms. HPV was present in all tumors and consisted of type 16 (n = 5, 42%) and type 18 (n = 7, 58%) strains, which have been closely associated with cervical neoplasia. K-ras-2 and p53 genes did not manifest point mutational damage. There was no evidence of oncogene amplification or tumor suppressor gene loss. The presence of HPV in all 12 tumors supports the role of HPV infection in the molecular pathogenesis of this uncommon neoplasm. The absence of associated oncogene or tumor suppressor gene damage is consistent with indolent biological behavior and the favorable prognosis of this unusual tumor.