Ozlen Saglam

Exploring chromosomal abnormalities and genetic changes in uterine smooth muscle tumors

Smooth muscle tumors of the uterus are a diagnostically challenging group of tumors. Molecular surrogate markers reliably distinguishing between benign and malignant tumors are not available. Therefore, the diagnosis is based on morphologic criteria. The aim was to investigate a well-characterized group of challenging uterine smooth muscle tumors consisting of 20 leiomyomas, 13 leiomyomas with bizarre nuclei, and 14 leiomyosarcomas for copy number alterations, MED12 mutations and FH deletions to search for potential diagnostically useful surrogate markers. MED12 mutations were detected in 47, 15, and 25% of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas, respectively. MED12 mutations in leiomyomas with bizarre nuclei were detected outside the hotspot region. FH-deletions were seen in 27, 30.8, and 25% of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas, respectively. By using copy number alteration profiling a clear separation of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas could not be observed. Copy number alterations revealed clear genetic similarities between leiomyomas with bizarre nuclei and leiomyosarcomas. Leiomyosarcomas showed a similar pattern of gains and losses as leiomyomas with bizarre nuclei, with additional copy number alterations and more homozygous losses and high-level amplifications compared to leiomyomas with bizarre nuclei. In conclusion, this study demonstrates that known FH-deletions, a recurrent molecular change in leiomyomas, occur in morphologically challenging variants of leiomyomas, leiomyomas with bizarre nuclei and leiomyosarcomas. Although MED12 mutations are common in leiomyomas, they infrequently occur in leiomyomas with bizarre nuclei and leiomyosarcomas. The genetic similarities between leiomyomas with bizarre nuclei and leiomyosarcomas raise the intriguing possibility that uterine leiomyomas with bizarre nuclei and leiomyosarcomas are closely related and challenge the traditional concept that leiomyoma with bizarre nuclei is a tumor with just marked ‘degenerative’ cellular changes. These findings support the hypothesis that tumor progression within uterine smooth muscle tumors might occur.

Influence of a 21-Gene Recurrence Score Assay on Chemotherapy Delivery in Breast Cancer

BACKGROUND We performed an analysis to determine the relative contribution of the Oncotype DX (ODX) recurrence score (RS) results in adjuvant therapy delivery compared with traditional pathologic factors. METHODS AND MATERIALS We performed a retrospective review of women with stage I-IIIA breast cancer treated at the Yale Comprehensive Cancer Center from 2006 to 2012 with available ODX results. We constructed separate logistic models with the clinicopathologic factors alone and also integrating RS and compared these models using the likelihood ratio test and c-statistic to determine whether integration of the RS will result in better prediction of chemotherapy (CTx) delivery. RESULTS We identified 431 women with a median age of 58 years. The RS was low (< 18), intermediate (18-30), and high (> 30) in 56%, 37%, and 7%, respectively. CTx was delivered to 30% of the patients. Age, differentiation, lymphovascular invasion, and progesterone receptor (PR) positivity < 50% were associated with CTx delivery in multivariable logistic regression of clinicopathologic factors alone (P < .05). In the model integrating the RS, an intermediate or a high RS was the most influential factor for CTx delivery (odds ratio, 7.87 vs. 265.35, respectively; P < .0001). The PR results and grade were no longer significant (P = .74 and P = .06, respectively). The integration of the RS resulted in improved model fit and precision, indicated by the likelihood ratio test (ΔG2, 100.782; df = 2; P < .0001) and an improved c-statistic (0.720 vs. 0.856). CONCLUSION Gene expression profiling appears to account for a substantial amount of variability in CTx delivery in current practice. Further work is needed to ensure appropriate test usage and cost-effectiveness.

Matricellular protein CCN1 (CYR61) expression is associated with high-grade ductal carcinoma in situ

Cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene (CCN) comprise a family of matricellular proteins that have multiple physiologic functions including development, tissue repair, cell adhesion, migration, and proliferation. The expression of CCN1, cyclin D1, β-catenin, and p53 was explored by immunohistochemistry in different grades of ductal carcinoma in situ (DCIS) cases. These cases did not contain any infiltrating carcinoma components. In addition, all cysteine-rich protein 61 gene exons (encoding the CCN1 protein) were sequenced in 30 samples. Allred and H-scores were calculated for expression in both DCIS and the surrounding benign breast tissue. All cases of DCIS showed degrees of cytoplasmic CCN1 staining with median H-scores of 170, 160, and 60 in grades 3, 2, and 1, respectively (P = .043). Twelve of 28 DCIS 3, 1 of 15 DCIS 2, and 0 of 18 DCIS 1 also showed nuclear staining for CCN1. The cytoplasmic staining difference was preserved when the cases were divided into estrogen receptor (ER)+/DCIS grade 1, ER+/DCIS 2 and 3, and ER-/DCIS 2 and 3 by the H-score (P = .037). Cyclin D1 expression was positively correlated with the CCN1 cytoplasmic H-score in all DCIS samples (P = .038). Membranous β-catenin expression correlated with the grade of intraepithelial carcinoma by both H-score (P = .047) and Allred score (P = .026). Our results suggest that CCN1 has a role in the development of intraepithelial carcinoma. CCN1 expression correlates with grade of DCIS independent of ER status. It can induce cell cycle progression through cyclin D1. It is warranted to study high expression of CCN1 in DCIS as an independent risk factor in a larger cohort.

AKT, EGFR, C-ErbB-2, and C-kit expression in uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) accounts for approximately 15% of uterine cancer-associated deaths in the United States. With lack of effective treatment modalities, identification of underlying molecular defects may allow the introduction of targeted treatments. The expression of AKT, epithelial growth factor receptor, C-Kit, and C-ErbB-2 were studied by immunohistochemistry and exons 9 and 20 of PIK3CA gene were sequences in a cohort of 37 UCS, including 23 early-stage (I and II) and 14 late-stage (III and IV) tumors. Twenty-three (62%) of the UCS were homologous; the reminder contained heterologous elements. The carcinomatous component was pure serous carcinoma in 13 (35%), endometrioid in 12 (32%) cases. An immunostaining score ranging from 0 to (6+) was calculated for AKT, epithelial growth factor receptor, and C-Kit. C-ErbB-2 staining was scored by American Society of Clinical Oncology/College of American Pathologists criteria. AKT staining was seen in 35/37 cases with an immunostaining score ranging from (2+) to (5+). AKT was expressed significantly more in the early-stage than late-stage disease (P=0.016). The expression of AKT in the epithelial component was associated with the survival (P=0.026). Epithelial growth factor receptor was positive in 21/37 cases. Only 8 cases showed (⩽3+) immunostaining score with C-Kit. C-ErbB-2 immunostain was (3+) in only 1 case. An H1047R mutation on PIK3CA gene was detected in both carcinomatous and sarcomatous components in a single case. These results indicate that AKT pathway may be important in pathogenesis of UCS. Further studies with larger cohorts are warranted to confirm the observed associations in this study.

Expression of Hormone Receptors, Cell Regulatory and Myoepithelial Cell Markers in Lactating Breast and Associated Carcinomas

To the Editor: Morphological changes in female breast during menstrual cycle, pregnancy, lactation, and menopause have been studied and documented. Alterations in hormone receptor expression in the epithelial cells and variations in the morphology of the myoepithelial cells (MEC) associated with pregnancy related physiological lactational changes have not been studied in detail, however. Understanding these normal variations is important to help distinguish physiological from pathological states. Furthermore, although 3% of mammary carcinomas manifest during pregnancy (1), the impact of hormonal surges of pregnancy on the immunoprofile of normal mammary cells or on the development and progression of breast carcinoma is not well understood. Although the prognosis of pregnancy-associated breast carcinomas (PABC) may appear to be worse than non-pregnancy associated carcinoma (NPABC) (2), the differences are not significant when corrected for age and stage (3). To provide some basic data on these aspects, the expression of various markers in epithelial and MEC in lactating breast was compared with that of a few malignant lesions detected during pregnancy and postpartum period. Thirty lactating breast specimens including 10 biopsies, 17 lumpectomies and 3 mastectomies were retrieved from the Surgical Pathology files of Yale University. Twenty-five of these were lactating adenomas (LA), five of which also had either pure ductal intraepithelial neoplasia (DIN; n = 2), infiltrative duct carcinoma (IDCA) with associated DIN (n = 2) and pure IDCA (n = 1) diagnosed during pregnancy. An additional five samples reflected age-matched benign breast tissue samples used as normal non-lactating controls. Each case was evaluated for the expression of ER, PR, AR, prolactin receptor (PRLR), MIB1, calponin, p63 and p53. Any staining of ‡1% was recorded as positive for all markers except calponin, p53 and p63. For MIB 1, the percentage of positivity was recorded by counting 100 cells in the most mitotically active area of the breast. Both cytoplasmic staining and nuclear staining were recorded for p63 since cytoplasmic positivity has been previously observed in secretory mammary epithelial cells (4). Immunostain for p53 was interpreted using a score of 0 to 3+ positive with variable intensity. At least 10% positivity with (2+) intensity was required for interpretation of a positive p53. The state of pregnancy was recorded for 16 of 25 women with LA, 11 of whom had their surgical excision during the third trimester. Two others were in their second trimester, while three women were postpartum. The mean age for the LA group was 28 years. A mass lesion with an average size of 2.7 cm was an initial presentation in 13 of 25 patients. The five women with either IDCA and ⁄ or DIN were 29, 33 (two patients), 37, and 38 years of age. The IDCA ranged from well to poorly differentiated. Of the two pure DIN cases, one was a grade II apocrine DIN, while the other was a comedo DIN3. Both the epithelial and MEC in all 25 LAs were negative for ER, PR, AR and PRLR. The well to moderately differentiated IDCAs were positive for ER and PR but negative for AR and PRLR. The lactating epithelium and the surrounding MEC were negative for all four markers. The poorly differentiated IDCA was negative for ER, PR, AR, and PRLR while the surrounding lactating lobules (SLL) displayed weak (1–5%) positivity for ER, PR and AR. The apocrine DIN2 was positive for AR (90%) but negative for the Address correspondence and reprint requests to: Ozlen Saglam, MD, Yale New Haven Hospital—Pathology, PO Box 208070, New Haven, CT 06510, USA, or e-mail: ozlen.saglam@yale.edu.

Metastatic leiomyosarcoma presenting as bilateral, multifocal breast masses

Here we describe a case of metastatic leiomyosarcoma presenting as bilateral, multifocal breast masses. This case represents the convergence of three rare entities: leiomyosarcoma of unknown primary origin, metastases to the breast and bilateral, multicentric breast disease.