Comparative Use of Napsin A and Glypican 3 to Distinguish Endometrial Clear Cell from Serous and Endometrioid Carcinomas.

OBJECTIVE: Diagnosis of endometrial clear cell carcinomas is difficult owing to the low reproducibility of histological cell type in high-grade endometrial cancers. Recently, immunoreactivity for napsin A and glypican 3 has been reported in clear cell cancers. We sought to evaluate the use of napsin A and glypican 3 staining to distinguish clear cell carcinoma from other high-grade endometrial cancers. METHODS/MATERIALS: Twenty cases of pure and mixed endometrial clear cell carcinoma were extracted from the 2000-2014 archival material in the Departments of Obstetrics & Gynecology and Pathology at Montefiore Medical Center and compared to serous and grade 3 endometrioid controls. Representative sections were stained with monoclonal antibodies to napsin A and glypican 3. Immunostains were independently reviewed by 2 pathologists to assess frequency and pattern of staining. Charts were reviewed for clinicopathologic and treatment data. RESULTS: Granular cytoplasmic positivity for napsin A was observed in 70% of endometrial clear cell carcinomas; only 25% showed cytoplasmic or membranous glypican 3 positivity. No serous or high-grade endometrioid tumors stained for either marker. No cases of clear cell carcinoma that stained negative for napsin A stained positive for glypican 3. No difference in the immunohistochemical profile was found between pure and mixed clear cell carcinomas and between early- and advanced-stage clear cell carcinomas. CONCLUSIONS: Napsin A is a more sensitive marker for endometrial clear cell carcinoma than glypican 3. In histologically ambiguous cases, napsin A and glypican 3 may help distinguish clear cell carcinoma from other high-grade histologies. Further investigation of endometrial clear cell carcinoma is needed to identify additional diagnostic tools for this rare histology. Correlation of a unique immunohistochemical profile and clinical outcomes is necessary.

Differential expression of various clones of estrogen receptor in cell block preparation of lung adenocarcinoma.

BACKGROUND: Women treated for breast cancer are at increased risk of developing pulmonary nodules which could represent new primary lung carcinomas or metastatic breast carcinoma. The FNA biopsy is frequently the first diagnostic choice in determining the primary site of the tumor. Estrogen receptor (ER) positivity in diagnostic tissue is generally used to favor breast over lung primary. However, the recent studies have shown a wide range of ER antibody cross reactivity with lung adenocarcinoma. We studied the frequency of ER expression in cytology samples of lung adenocarcinoma using antibodies to three different ER clones. METHODS: Cytology cell block preparations, including 22 lung FNAs and 19 malignant pleural effusions (PE) from 41 patients, with clinically documented primary lung adenocarcinomas were selected for this study. All cases were stained with monoclonal antibodies to ER clones 6F11 and 1D5. Twenty nine cases with remaining available material (15 FNA and 14 PE) were stained with a third antibody to ER clone SP1. The extent of ER nuclear staining was scored as 3+ (50%-100% of tumor cells), 2+ (11%-49%), and 1+ (≤10%). RESULTS: Positivity for ER-6F11 clone was present in 4 of 22 lung FNAs (18.2%, 2+ staining). Two of the four 6F11 positive FNAs also co-expressed ER-1D5 (9.1%, 2+ staining). No immunoreactivity was observed for ER clones 6F11 and 1D5 in all 19 malignant PEs. In addition, none of the remaining 15 FNAs and 14 PEs showed immunoreactivity for ER-SP1 clone. CONCLUSIONS: A small subset of pulmonary adenocarcinomas shows immunoreactivity for ER clones 6F11 and 1D5 in FNA samples (18.2% and 9.1%, respectively). The absence of immunoreactivity for ER-SP1 clone indicates higher specificity of this clone in non-breast tissue. The differential diagnostic value of all ER clones in malignant PEs appears to be secure. Larger studies are necessary to validate this observation.

Hypopigmented mycosis fungoides in childhood and adolescence: a long-term retrospective study.

Patients with hypopigmented mycosis fungoides (HMF) present at a younger age than those with classic MF. Our goal was to describe the clinical presentation, histopathologic features and long-term outcome in patients who developed HMF before the age of 21. It was observed that among 69 pediatric patients diagnosed with MF between 1992 and 2010, 50 had HMF. Thirty-five patients had clinical follow-up. There were 37 males and 32 females with a mean age of 13.6 years. Most patients were African American or Hispanic and presented with multiple hypopigmented patches. All biopsies showed epidermotropism of T-lymphocytes, whereas fibroplasia and lichenoid infiltrate were variable. All specimens tested were CD8+. Treatment modalities included topical steroids, narrow band ultraviolet B and psoralen and ultraviolet A. HMF patients were followed for <1-12 years. Most children responded to treatment, but recurrence rates were high. One patient progressed to plaque/tumor stage. Others did not progress; however, many were lost to follow-up. We present a large cohort of children with HMF and report on the features of disease and progression. A major difference in histology of HMF was lack of fibroplasia and lichenoid infiltrate, probably because of presentation in the early patch stage. Most patients have a waxing-and-waning course and relapse after discontinuation of therapy, requiring repetitive treatment.

Estrogen, progesterone, and HER-2 receptor immunostaining in cytology: the effect of varied fixation on human breast cancer cells.

The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER-2 expression. The guidelines for testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. We evaluated various fixation protocols for optimal receptor testing by immunohistochemistry and immunocytochemistry (ICC) of human breast cancer cell lines MCF-7 (ER/PR positive) and SKBR-3 (overexpressing HER-2). The cells were fixed in 10% neutral buffered formalin or Saccomanno Fixative (SF) for various time points, and either embedded in paraffin as cell blocks or prepared as cytospins. ER and PR slides were assigned a proportion score (PS; 0-5), an intensity score (IS; 0-3) and a total score (TS = PS + IS). Standard DAKO scoring system ranging from 0 to 3+ was used for the evaluation of HER-2 staining. Human breast cancer cells stained successfully for ER, PR and HER-2 when fixed in formalin and prepared as cell blocks. The optimal fixation time for formalin-fixed cells ranged from 2 to 96 hours. Cells fixed in SF from 2 to 96 hours also stained well for ER and PR. However, SF produced variable results for HER-2 staining; particularly, SF fixation beyond 24 hours caused false negative results. The interpretation of HER-2 staining on cytospins was not feasible irrespective of the fixative and fixation time. In summary, formalin fixation from 2 to 96 hours and preparation of cells as cell blocks produces optimal results for ER, PR, and HER-2 testing in human breast cancer cells.

High expression of class III ß-tubulin predicts good response to neoadjuvant taxane and doxorubicin/cyclophosphamide-based chemotherapy in estrogen receptor-negative breast cancer.

BACKGROUND: Expression of class ΙΙΙ ß-tubulin (ßΙΙΙ-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether ßΙΙΙ-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy. PATIENTS AND METHODS: We determined ßΙΙΙ-tubulin expression in 85 breast cancers, including 41 localized breast cancers treated with primary surgery and 44 treated with neoadjuvant chemotherapy before surgery. ßΙΙΙ-tubulin expression was evaluated by immunohistochemical methods and was correlated with pathologic characteristics and response to neoadjuvant chemotherapy using residual cancer burden (RCB) score. RESULTS: High ßΙΙΙ-tubulin expression was significantly associated with poorly differentiated high-grade breast cancers (P = .003) but not with tumor size, estrogen receptor (ER) status, or human epidermal growth factor receptor 2 (HER2)/neu overexpression. In ER(-) tumors treated with neoadjuvant chemotherapy, high ßΙΙΙ-tubulin expression was associated with a significantly greater likelihood of achieving a good pathologic response to chemotherapy as reflected by lower RCB scores (P = .021). CONCLUSION: This study reveals differential ßΙΙΙ-tubulin expression in breast cancers of different histologic grades, hormone receptors, and HER2/neu status. It also suggests a potential role for ßΙΙΙ-tubulin as a predictive biomarker for response in neoadjuvant chemotherapy for ER(-) breast cancer, which has not been previously reported. These data provide a strong rationale for considering ßΙΙΙ-tubulin status and further validation of this marker in a large study.

Predictors of disease progression in ductal carcinoma in situ of the breast and vascular patterns.

Breast carcinoma-induced angiogenesis helps meet growing metabolic needs of tumors and progressively increases with malignant transformation of benign ducts to ductal carcinoma in situ (DCIS) and ductal carcinoma in situ to invasive carcinoma. There are conflicting data regarding the difference in angiogenesis in low-, intermediate-, and high-grade ductal carcinoma in situ. If angiogenesis is related to ductal carcinoma in situ progression, the types of ductal carcinoma in situ with more aggressive biologic potential would have different vascular patterns than the less aggressive ones. In this study, we classified 51 cases of ductal carcinoma in situ as low (10-20 years to progression to invasive carcinoma), moderate, or high aggressive (2-5 years to progression to invasive carcinoma), based on criteria outlined by Tsikitis and Chung (Am J Clin Oncol 2006; 29:305), which takes into account nuclear grade, mitotic rate, Ki-67, Her2Neu, P53, estrogen, and progesterone receptor expression. We correlated these 3 groups of ductal carcinoma in situ with the extent of periductal and stromal vascularity and the presence and type of vascular breaks. No association of aggressive biologic behavior of ductal carcinoma in situ with any vascular pattern was found. Moreover, no correlation was found between vascular patterns and classifiers of aggressiveness, microvascular density, or outcome (local recurrence, invasive carcinoma, or metastatic disease). To validate our cohort, we confirmed expected correlations of all measured parameters of aggressiveness by correlating them with each other. In summary, vascular patterns in ductal carcinoma in situ do not correlate with the predictors of aggressive behavior, suggesting that the biologic potential of ductal carcinoma in situ is independent of angiogenesis.

Spectrin isoforms: differential expression in normal hematopoiesis and alterations in neoplastic bone marrow disorders.

Spectrins are large, rod-like, multifunctional molecules that participate in maintaining cell structure, signal transmission, and DNA repair. Because little is known about the role of spectrins in normal hematopoiesis and leukemogenesis, we immunohistochemically stained bone marrow biopsy specimens from 81 patients for αI, αII, ßI, and ßII spectrin isoforms in normal reactive marrow (NRM), myelodysplastic syndrome, myeloproliferative neoplasm, acute myeloid leukemia (AML) with well-characterized cytogenetic abnormalities, acute erythroid leukemia (EryL), and acute megakaryoblastic leukemia (MegL). In NRM, spectrin isoforms were differentially expressed according to cell lineage: αI and ßI in erythroid precursors; αII and ßII in granulocytes; and ßI and ßII in megakaryocytes. In contrast, 18 (44%) of 41 AMLs lacked αII spectrin and/or aberrantly expressed ßI spectrin (P = .0398; Fisher exact test) and 5 (100%) of 5 EryLs expressed ßII spectrin but lacked ßI spectrin. The frequent loss and/or gain of spectrin isoforms in AMLs suggests a possible role for spectrin in leukemogenesis.

Alpha- and beta-synucleins are new diagnostic tools for acute erythroid leukemia and acute megakaryoblastic leukemia.

α-Synuclein is a key component of the Lewy body, a large globular protein complex that forms in the nervous system of patients with Parkinson disease and other dementias [1-3]. Since α-synuclein also occurs in megakaryocytic and erythroid lineages [4-7], we wondered what role synucleins had in the hematopoietic system. Therefore, we studied the expression of α-, ß-, and γ-synucleins in a comprehensive panel of patient bone marrows and leukemic cell lines. We observed under expression of α-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow (NRM) or myelodysplastic syndrome (MDS). Conversely, we observed over expression of ß-synuclein in the blasts of megakaryoblastic leukemias (MegL), but not acute myeloid leukemia (AML) or erythroleukemia (EryL), suggesting that α- and ß-synucleins could be useful adjunct markers for the early detection of MDS and the differential diagnosis of EryL and MegL from other AMLs.

Podoplanin (d2-40): a new immunohistochemical marker for reactive follicular dendritic cells and follicular dendritic cell sarcomas.

The diagnosis of follicular dendritic cell (FDC) sarcoma can be challenging because of its morphologic overlaps with many other spindle cell neoplasms and, therefore, new phenotypic markers will be helpful in its differential diagnosis. Podoplanin is a mucin-type transmembrane glycoprotein that has recently been detected in reactive FDCs. In this study, we investigated the expression patterns of podoplanin using a new mouse monoclonal antibody D2-40, and compared them with CD21, a well-established FDC marker, in a comprehensive panel of cases. The panel included 4 FDC sarcomas, 38 spindle cell neoplasms of other types, 25 reactive lymphoid hyperplasia, and 117 lymphoid and 5 myeloid malignant hematopoietic neoplasms. Our study revealed that D2-40 strongly stained 3 of 4 FDC sarcomas. In contrast, D2-40 stained only 2/38 other spindle cell neoplasms tested. Furthermore, we observed that D2-40 highlighted more FDC meshworks than CD21 in Castleman's disease, follicular lymphoma, nodular lymphocyte predominance Hodgkin lymphoma, and residual reactive germinal centers in a variety of lymphoma types. D2-40 and CD21 stained an equal number of cases of reactive lymphoid hyperplasia, progressively transformed germinal centers and angioimmunoblastic T-cell lymphoma. No expression of podoplanin was detected in normal or neoplastic lymphoid and myeloid cells. We conclude that podoplanin (D2-40) is a sensitive and specific FDC marker, which is superior or equal to CD21 in evaluating both reactive and neoplastic FDCs. In addition, our results suggest that podoplanin (D2-40) can be used to support the diagnosis of FDC sarcoma.

Mechanisms of progression of ductal carcinoma in situ of the breast to invasive cancer. A hypothesis.

Ductal carcinoma in situ (DCIS), a known precursor lesion of invasive cancer of the female breast, is surrounded by a thick basement membrane and a layer of myoepithelial cells. For DCIS to become invasive, both these barriers must be breached by cancer cells. It has been repeatedly suggested that proteolytic enzymes are somehow involved in this process but a direct proof of this event has never been provided. It is our hypothesis that invasion of the DCIS by capillary vessels derived from the periductal necklace of vessels is the most likely mechanism of breaching the basement membrane, providing an escape hatch for cancer cells. This hypothesis was initially tested on ten randomly selected cases of DCIS, with or without invasion. Capillary vessels were visualized by staining histologic sections with an antibody to CD 34 and, in three cases, by combined stain for CD 34 and collagen IV. In five of the 10 cases of DCIS, the presence of discrete capillary vessels invading DCIS could be documented. In two of these five cases, the vessels subdivided the cancerous ducts into territories of unequal sizes. Vascular invasion of DCIS is a plausible mechanism of breaching the basement membrane in DCIS as a prelude to invasion. This hypothesis must be further tested on a much larger number of cases. The hypothesis, if confirmed, may suggest that invasive cancer derived from DCIS may be prevented by antiangiogenic therapy.