Immunohistochemical studies of metastatic germ-cell tumors in retroperitoneal dissection specimens: a sensitive and specific panel.

Germ-cell tumors (GCTs) are the most common malignancies in adolescent and young men. These tumors are highly treatable, even at an advanced stage; therefore, accurate diagnosis is imperative. In this study, we evaluated immunohistochemical stains for SALL4, NANOG, glypican-3 (GPC3), D2-40, and CD30 with adequate control in retroperitoneal dissection specimens under the same laboratory conditions. The study groups included 31 cases of metastatic testicular GCTs with the following components: 11 seminomas, 14 embryonal carcinoma (ECs), 12 yolk sac tumor (YSTs), 8 teratomas, 10 cases of metastatic melanomas, 14 cases of malignant lymphomas, and 11 cases of metastatic, poorly differentiated carcinoma. SALL4 showed diffuse nuclear labeling for all seminomas, ECs, and YSTs. NANOG showed diffuse nuclear positivity in all seminomas and ECs. Metastatic carcinomas, melanomas, and malignant lymphomas were negative for these 2 markers. Gypican-3, D2-40, and CD30 showed sensitive staining for YSTs, seminomas, and ECs, respectively. In conclusion, SALL4 and NANOG are sensitive and specific markers for GCTs. GPC3, D2-40, and CD30 are sensitive but not specific for individual components of GCTs and may be useful in aiding in the differential diagnosis for the individual component of GCTs when the identity of GCT is established.

Sirtuin 1 (SIRT1): a potential immunohistochemical marker and therapeutic target in soft tissue neoplasms with myoid differentiation.

Sirtuin, silent mating-type information regulation 2 homolog Saccharomyces cerevisiae 1 (SIRT1), is a protein that has been implicated in multiple mammalian functions including cell aging, stress resistance, and differentiation. SIRT1 has also been shown to be involved in multiple tumors. In addition, new pharmacotherapies have recently been approved that target SIRT1. The purpose of this study was to use immunohistochemistry to characterize SIRT1 protein expression in human soft tissue neoplasms with the hopes of finding new diagnostic and therapeutic modalities. SIRT1 immunoreactivity was reviewed in a series of 164 soft tissue tumors including alveolar soft part sarcoma, angiomyolipoma, clear cell sarcoma, desmoid/fibromatosis, desmoplastic small round cell tumor, Ewing sarcoma, gastrointestinal stromal tumor, glomus tumor, leiomyoma, leiomyosarcoma, lipoma, liposarcoma, malignant peripheral nerve sheath tumor, nodular fasciitis, osteosarcoma, rhabdomyosarcoma, schwannoma, solitary fibrous tumor, synovial sarcoma, undifferentiated pleomorphic sarcoma, and Wilms tumor. In addition, numerous benign tissues were tested for SIRT1 reactivity. In nonneoplastic tissue, strong cytoplasmic SIRT1 reactivity was observed in all prostate stroma, smooth muscle, and striated muscle. A similar pattern of cytoplasmic SIRT1 expression was observed in soft tissue neoplasms with myoid differentiation, namely, angiomyolipoma (100%), glomus tumor (100%), leiomyoma (90%), leiomyosarcoma (76.5%), and rhabdomyosarcoma (87%). The other lesions examined were negative. Although the physiologic role of SIRT1 remains to be clarified in myoid tissues and neoplasms differentiating along these lines, this observation points to a potential role for this marker in diagnostic immunohistochemistry. Furthermore, the recent emergence of drugs capable of selectively inhibiting SIRT1 raises the possibility of a potential application for targeted therapy. Additional studies are necessary to further characterise the role of SIRT1 in myoid tissues and neoplasms.

Malignant mixed mullerian tumor: an immunohistochemical study.

Malignant mixed Mullerian tumor (MMMT) is an uncommon aggressive neoplasm composed of both malignant epithelial and mesenchymal components. In this study, immunohistochemical stains of germ cell markers, including SALL4, OCT3/4, glypican-3, and alpha-fetal protein (AFP), and CDX2 were performed in a series of MMMTs. SALL4 nuclear immunoreactivity was detected in 6 out of 19 cases (33%). The staining extent ranged from focal to extensive. The staining intensity was usually intermediate to strong (the score ranged from 1.5 to 3, and average score was 2.3 ± 0.5 in the positive cases). In addition, glypican-3 cytoplasmic reactivity was detected in 14 out of 16 cases (88%) with a mean score of 1.8 ± 0.7 (score ranging from 1 to 3). In contrast, OCT3/4 was only positive in 1 out of 19 cases and AFP in 2 out of 18 cases (11%). In summary, SALL4 and glypican-3 were frequently expressed in a subset of MMMTs. Their roles in the pathogenesis and biology of MMMT are yet to be determined. MMMT should be included in the differential diagnosis when a tumor is positive for SALL4 and/or glypican-3.

Aberrant connexin 43 and 26 expression in cervical dysplasia.

OBJECTIVE: To determine whether connexin (Cx) expression is altered in cervical dysplasia. STUDY DESIGN: Cx proteins form gap junctions and are expressed in squamous epithelia including ectocervix. We used multispectral imaging to perform a quantitative immunohistochemical survey of Cx43 Cx26 in 37 archival human cervical specimens. RESULTS: Cx43 expression was very low in normal cervix (100%), but was increased in low-grade squamous intraepithelial lesions (LSILs) (64%), primarily in a parabasal distribution. High-grade squamous intraepithelial lesions (HSILs) showed weak full-thickness Cx43 staining (53%) or lacked Cx43 (47%). An aberrant increase in Cx43 expression was often (62%) present in histologically normal areas of specimens that elsewhere harbored dysplasia. Cx26 was highly expressed in the basal layer of normal ectocervix (100%). In LSIL, 57% showed a decrease in Cx26 and the rest showed no change relative to the normal pattern. In HSIL, Cx26 was expressed in the full thickness of the epithelium, at a high level in 80% of cases and a low level in the rest. CONCLUSION: Cx alteration is moderately consistent in cervical dysplasia, and for Cx43 can precede histologic changes. The resulting changes in Cx signaling may be important in the pathogenesis of cervical intraepithelial neoplasia.

p53 in pure epithelioid PEComa: an immunohistochemistry study and gene mutation analysis.

Pure epithelioid PEComa (PEP; so-called epithelioid angiomyolipoma) is rare and is more often associated with aggressive behaviors. The pathogenesis of PEP has been poorly understood. The authors studied p53 expression and gene mutation in PEPs by immunohistochemistry, single-strand conformation polymorphism, and direct sequencing in paraffin material from 8 PEPs. A group of classic angiomyolipomas (AMLs) were also analyzed for comparison. Five PEPs were from kidneys and 1 each from the heart, the liver, and the uterus. PEPs showed much stronger p53 nuclear staining (Allred score 6.4 ± 2.5) than the classic AML (2.3 ± 2.9) (P < .01). There was no p53 single-strand conformation polymorphism identified in either the PEPs or the 8 classic AMLs. p53 mutation analyses by direct sequencing of exons 5 to 9 showed 4 mutations in 3 of 8 PEPs but none in any of the 8 classic AMLs. The mutations included 2 missense mutations in a hepatic PEComa and 2 silent mutations in 2 renal PEPs. Both the missense mutations in the hepatic PEComa involved the exon 5, one involving codon 165, with change from CAG to CAC (coding amino acid changed from glutamine to histidine), and the other involving codon 182, with change from TGC to TAC (coding amino acid changed from cysteine to tyrosine). The finding of stronger p53 expression and mutations in epithelioid angiomyolipomas might have contributed to their less predictable behavior. However, the abnormal p53 expression cannot be entirely explained by p53 mutations in the exons examined in the PEPs.

TFE3 expression in tumors of the microphthalmia-associated transcription factor (MiTF) family.

The DNA-binding factor TFE3 is closely related to microphthalmia-associated transcription factor (MiTF) and is over-expressed in alveolar soft part sarcoma (ASPS) and select renal cell carcinomas. Reports of TFE3 expression in PEComa prompted investigation into TFE3 expression among other members of the putative MiTF group of neoplasms. The authors examined cases of PEComa (n = 6), conventional angiomyolipoma (AML; n = 22), metastatic melanoma (n = 16), and clear cell sarcoma (CCS; n = 9) for TFE3 expression. Nuclear immunostaining was observed in 74% (39/53) of cases, as follows: 5/6 PEComas, 18/22 AMLs, 10/16 metastatic melanomas, and 6/9 CCSs. However, with the exception of PEComas, compared with ASPS controls, TFE3 staining was significantly less intense in the tumors examined. These results illustrate that TFE3 immunoreactivity is detectable in other members of the MiTF family of neoplasms. For this reason, such neoplasms warrant consideration in the differential diagnosis with nuclear TFE3 immunoreactivity, particularly when staining is focal and less intense.

Analysis of HIF-1a and its regulator, PHD2, in retroperitoneal sarcomas: clinico-pathologic implications.

Hypoxia is known to play important role in cancer biology.  In sarcomas, hypoxia-induced protein biomarkers such as Hypoxia Inducible Factor-1a (HIF-1a), vascular endothelial growth factor (VEGF), and Erythropoietin (Epo) have been previously reported in only a few studies.  Moreover, the biologic significance and relationship to tumorigenesis of these hypoxia-induced biomarkers is not well understood in the context of sarcoma. The HIF negative regulator, Prolyl Hydroxylase Domain protein 2 (PHD2) has not been evaluated in sarcomas.  We examined the expression of PHD2, HIF-1a, and several other hypoxia induced biomarkers in a series of clinically characterized, retroperitoneal sarcomas with immunohistochemical methods.  Expression of these proteins was analyzed and correlated with clinical outcome.  Increased HIF-1a expression was associated with shorter overall and disease free survival.  PHD2 expression was detected in the majority of sarcoma cases, with increased expression correlating with high tumor grade but not with survival.  Though changes in PHD2 expression alone did not correlate with overall and disease free survival, reduced/absent PHD2 expression in the presence of HIF-1a expression was associated with shorter overall and disease-free survival than that of other HIF-1a/PHD2 expression profiles.  These observations suggest that regulation and expression of both PHD2 and HIF-1a are important to the biology of sarcomas, and that loss of PHD2 function has an additional adverse effect in the prognosis of sarcomas in tumors expressing HIF-1a.  The biologic and therapeutic implications of HIF-1a and PHD2 expression in retroperitoneal sarcomas warrant further investigation.

Histologic predictors of fibrosis progression in liver allografts in patients with hepatitis C virus infection.

BACKGROUND & AIMS: Recurrent hepatitis C with ensuing fibrosis is the leading cause of liver allograft loss. We investigated whether histologic features in early posttransplant liver biopsies could predict the rate of fibrosis progression in this population. METHODS: From 1999 to 2007 there were 476 liver transplants performed for hepatitis C at our center. We reviewed all available posttransplant biopsies for these patients; patients were categorized as rapid, intermediate, or slow fibrosers based on their METAVIR fibrosis score at 24 months. Stage F0 biopsies for rapid and slow fibrosers were analyzed histologically and immunohistochemically. RESULTS: We identified 52 rapid fibrosers and 61 slow fibrosers in our cohort. There was a significant increase in the fibrosis progression rate in the group transplanted between 2003 and 2007 compared with between 1999 and 2002. The course of fibrosis progression was determined early in the posttransplant period and the rate was constant. Rapid fibrosers had more hepatocyte apoptosis than slow fibrosers (P = .001), but no difference in hepatitis activity on stage F0 biopsies. Rapid fibrosers also experienced more episodes of acute rejection after transplantation (P < .001). Cytokeratin 19 (CK19) and vimentin expression on F0 stage biopsies could distinguish rapid from slow fibrosers (CK19: area under the curve, 0.71; P = .0034; vimentin: P = .0219). CONCLUSIONS: CK19, vimentin, and hepatocellular apoptosis are promising early markers of rapid fibrosis progression in patients transplanted for hepatitis C. The rate of fibrosis progression is established early in the posttransplant period; this initial rate dictates long-term outcome.

Asymptomatic changes in cardiac function can occur in ductal carcinoma-in-situ patients following treatment with HER-2/neu-pulsed dendritic cell vaccines.

BACKGROUND: Targeting HER-2/neu with Trastuzumab has been associated with development of cardiac toxicity. METHODS: Twenty-seven patients with ductal carcinoma in situ (DCIS) of the breast completed an IRB approved clinical trial of a HER-2/neu targeted dendritic cell based vaccine. Four weekly vaccinations were administered prior to surgical resection. All subjects underwent pre- and post-vaccine cardiac monitoring by MUGA/ECHO scanning allowing for a comparison of cardiac function. RESULTS: In 3 of 27 vaccinated patients (11%) transient asymptomatic decrements in ejection fraction of greater than 15% were noted after vaccination. Notably, evidence of circulating anti-HER-2/neu antibody was found prior to vaccination in all three patients, but cardiac toxicity was not noted until induction of cellular mediated immune responses. CONCLUSIONS: This is the first description of HER-2/neu targeted vaccination associated with an incidence of cardiac changes, and the induction of cellular immune responses combined with antibody may contribute to changes in cardiac function.

IMP-3 is differentially expressed in normal and neoplastic lymphoid tissue.

IMP-3 is a member of the insulin-like growth factor II mRNA binding protein (IMP) family of proteins that play a role in RNA trafficking and stabilization and cell growth and migration during embryogenesis but which are down-regulated in adult tissue. However, IMP-3 has recently been shown to be overexpressed in several epithelial malignancies, with increased expression correlating with aggressive behavior. To our knowledge, there is no published literature evaluating IMP-3 in lymphoid tissue. Accordingly, we immunohistochemically evaluated IMP-3 expression in normal lymphoid tissue and 141 lymphoid neoplasms. Physiologically, IMP-3 expression was restricted to germinal center B cells. Among lymphoid neoplasms, Hodgkin lymphoma demonstrated the highest percentage of positive cases (26/26, 100%) often with bright staining. Burkitt lymphoma was positive in 10 (83%) of 12 cases with moderate to bright staining. Although follicular lymphoma was also positive in a high percentage of cases (12/15, 80%), the intensity was exclusively weak to moderate. Although 22 (85%) of 26 of diffuse large B-cell lymphomas were positive for IMP-3, there was wide variability in staining intensity, which did not correlate with classification into activated B cell versus germinal center B origin. By contrast, lower proportions (8%-20%) of other non-germinal center B lymphoma subtypes were IMP-3-positive. In conclusion, although IMP-3 expression is seemingly restricted to physiologic germinal center B cells, its expression in lymphomas of germinal center B origin is less robust. However, there does appear to be some association with the latter group of lymphomas, which may prove to have diagnostic or therapeutic relevance as the biologic role of IMP-3 is further elucidated.

CDX2 expression in yolk sac component of testicular germ cell tumors.

CDX2 has been detected in the majority of colorectal adenocarcinoma cases and may be useful in determining the sites of origin of tumors. In this study, the authors evaluated CDX2 expression in germ cell tumors (GCTs) by immunohistochemistry. Forty cases of testicular GCTs and 8 cases of metastatic GCTs were retrieved for study. In the 40 cases of testicular GCTs, 13 were pure seminomas and 27 mixed GCTs. Yolk sac tumor (YST) was identified by morphology and glypican 3 staining in 20 testicular mixed GCTs. Of these 20 cases, 8 cases showed 1+ positivity for CDX2. Other primitive components of GCTs were negative. For the 6 cases of metastatic mixed GCT with YST, 4 cases were positive, 2+ in 2 cases and 1+ in 2 cases. The positivity of CDX2 in GCTs warrants including YST in the differential diagnosis of adenocarcinoma of unknown origin.

Thyroid transcription factor-1 expression in normal gynecologic tissues and its potential significance.

SUMMARY: Thyroid transcription factor-1 (TTF-1) is a 38-kd nuclear protein, and a member of the NKx2 family of homeodomain transcription factors. It is highly expressed in normal and neoplastic thyroid and lung tissues, and is considered a reliable marker for lung adenocarcinoma and thyroid carcinoma. Recently, expression of TTF-1 has also been reported in ovarian, endometrial, and endocervical epithelial neoplasms. Little is known about TTF-1 immunoreactivity in normal gynecologic tissues. In this study, TTF-1 expression in various non-neoplastic gynecologic tissues was investigated by standard immunohistochemistry. One hundred and eight samples of benign gynecologic tissues from adult patients who had no known history of neoplastic condition were collected. Twenty-eight endometria (12 proliferative, 11 secretory, and 5 inactive), 26 fallopian tubes, 28 cervixes (14 endocervical and 14 ectocervical), 14 myometria, and 12 ovaries were studied. In addition, 4 normal fallopian tubes and 2 ovaries from 5 pediatric patients (aged from 3 mo to 11-yr old) were evaluated. Variable TTF-1 nuclear reactivity was identified in 25 of 26 (96%) fallopian tubes (extent of positivity ranged from 2% to 60%, median 25%), 15 of 28 (54%) endometria (1% to 10%, median 5%), and 6 of 14 (43%) endocervical samples (<5%). TTF-1 was also identified in 2 of 4 (50%) pediatric fallopian tubes with 5% and 20% of the tubal epithelium being positive, respectively. No TTF-1 expression was detected in ovarian tissue (neither epithelial nor stromal tissue; neither adult nor pediatric samples), ectocervical squamous epithelium or myometrium, nor in stromal tissue in endometrium, tube, or cervix. TTF-1 reactivity was detected in both proliferative and secretory endometrium, but not in 5 inactive endometria. TTF-1 is frequently expressed in normal/non-neoplastic tubal, and less frequently in functional endometrial and endocervical epithelia, but not in ovarian surface epithelium. TTF-1 might have a functional and developmental role in normal fallopian tube and endometrium, as it is highly expressed in tubal epithelium of both adults and young children, and in functional endometrium but not in inactive endometrium. The high TTF-1 expression in tubal epithelium but not in normal ovarian surface epithelium suggests that some TTF-1-positive ovarian tumors might be related to the tubal epithelium.

Value of P63 and CK5/6 in distinguishing squamous cell carcinoma from adenocarcinoma in lung fine-needle aspiration specimens.

The current FDA-approved standard of care for nonsmall cell lung cancer is Carboplastin/Taxol/Avastin based upon an impressive survival benefit; however, patients with squamous carcinoma (SQCC) cannot receive Avastin because of a 30% mortality rate due to fatal hemoptysis. In this study we evaluated the role of cytomorphology and immunohistochemistry in differentiating SQCC from adenocarcinoma (ADC) in lung FNA specimens. The case cohort included 53 FNA cases of nonsmall cell lung carcinoma with surgical pathology follow-up. All FNA specimens were reviewed independently by a panel of cytopathologists to differentiate between SQCC and ADC. The cell block material was available in 23 cases (11 ADC and 12 SQCC) to perform immunohistochemical stains for TTF-1, CK7, CK20, P63, and CK5/6. On surgical resection, 35/53 (66%) cases were diagnosed as ADC and 18/53 (34%) as SQCC. The number of cases classified correctly on the basis of cytomorphology was 66% for ADC and 53% for SQCC (combined accuracy 60%). By immunohistochemical staining, 14/23 (61%) cases expressed TTF-1. Nine cases were TTF-1 negative; eight of the TTF-1 negative cases (89%) were SQCC. Twenty-three cases expressed CK7 (87%); one ADC case (4%) showed focal CK20 positivity. Both P63 and CK5/6 expression was seen in 9/12 (75%) SQCC cases; none of the ADC cases showed this dual expression. Cytomorphology alone may not be able to stratify all cases of nonsmall cell lung carcinoma into ADC and SQCC in FNA specimens. The immune-panel of TTF-1, CK7, CK20, P63, and CK5/6 is useful in differentiating SQCC from ADC.

TTF-1 expression in ovarian and uterine epithelial neoplasia and its potential significance, an immunohistochemical assessment with multiple monoclonal antibodies and different secondary detection systems.

Thyroid transcription factor-1 (TTF-1) is a 38-kd homeodomain containing DNA-binding protein, identified in thyroid and lung as a regulator of thyroid-specific genes and surfactant and Clara cell secretory protein gene expression. TTF-1 has been used as a reliable lineage marker for lung adenocarcinoma and thyroid carcinoma in surgical pathology. However, TTF-1 expression has been recently reported in carcinomas of other origins including female genital tract. We evaluated TTF-1 expression with 3 primary different antibodies (8G7G3/1, SPT24, and BGX-397A) and 2 secondary automated detection systems (Envision+/Dako autostainer versus Refine/Bond Max) in 104 ovarian and endometrial tumors on routine surgical specimens and 108 ovarian tumors on tissue microarray (TMA) specimens. SPT24 and Refine/Bond Max autostainer was the most sensitive system among the primary antibodies and secondary detection/autostainers tested. By using SPT24/Refine/Bond Max, TTF-1 reactivity could be detected in all major histologic subtypes of gynecologic tumors and up to 26% of all cases tested on routine surgical specimens and 6.4% on TMA. TTF-1 was most frequently detected in uterine malignant mixed Müllerian tumor (82%), more common in uterine tumors than ovarian tumors, and more common in surgical specimen than TMA. When present, tumor cells can be rarely positive or diffusely positive for TTF-1 reactivity. In addition to malignant tumors, TTF-1 was also detected in benign tumors and benign tubal and endometrial epithelia. TTF 1 immunostaining has the potential to misguide a pathologist to conclude an ovarian or endometrial tumor being a lung metastasis. However, the role of TTF-1 in female genital tract and its tumors is unknown.

Expression of glial cell line-derived neurotropic factor receptor alpha-1 in immature teratomas.

The immaturity of teratomas is usually manifested as immature neuroepithelium. The amount of immature neuroepithelium has been correlated with the survival of adult patients with ovarian immature teratoma. To date, no immunohistochemical marker has been found to facilitate the identification of immature teratoma. In this study, we evaluated the expression of glial cell line-derived neurotropic factor receptor alpha-1 (GFRalpha-1) for this purpose. We retrieved 38 cases of germ cell tumors: 26 cases contained immature teratoma, of which 24 had immature neuroepithelium and showed strong membrane staining for GFRalpha-1. No significant staining was seen in other components including embryonal carcinoma, seminoma, yolk sac tumor, choriocarcinoma, immature mesenchyme, and intratubular germ cell neoplasia. Immunohistochemical staining for GFRalpha-1 in immature neuroepithelium may facilitate its identification.

Cytoplasmic overexpression of WT-1 in gastrointestinal stromal tumor and other soft tissue tumors.

The Wilms tumor 1 (WT-1) is a zinc finger transcription factor essential for the development of the kidneys and gonads. Alterations in the WT-1 gene were observed in several tumor types. Depending on the tumor types, WT-1 might function as a tumor suppressor or as a survival factor. WT-1 immunoreactivity in gastrointestinal stromal tumor (GIST) was currently not known. We, therefore, investigated the expression of WT-1 in GIST in comparison to other soft tissue tumors by immunohistochemistry and Western blot analysis. We found that all 28 cases (100%) of GIST are positive for WT-1, diffusely (>75%, 3+) in 13 (46.4%) cases, moderately (26% to 75%, 2+) in 13 (46.4%) cases, and focally (5% to 25%, 1+) in 2 (7.2%) cases. The staining intensity is usually strong. The staining pattern is predominantly cytoplasmic with rare scattered nuclear staining. Similar but less extensive cytoplasmic WT-1 immunoreactivity was detected in 16 of 25 (64%) uterine leiomyosarcoma and 14 of 24 (58.3%) soft tissue leiomyosarcoma. Rare scattered nuclear staining was also seen in uterine leiomyosarcoma and soft tissue leiomyosarcoma, which showed positive cytoplasmic WT-1 reactivity. Only 1 of the 10 solitary fibrous tumors showed weak cytoplasmic WT-1 positivity (10%). No WT-1 staining was detected in 6 cases of fibromatosis. The significance of cytoplasmic expression of WT-1 in GIST and some smooth muscle tumors is unclear and warrant further investigation. The potential roles of WT-1 in the diagnosis and treatment of GIST were discussed.

The selective expression of CD43 in adenoid cystic carcinoma.

CD43, a sialoglycoprotein expressed on hematopoietic cells, has only rarely been reported in nonhematopoietic tumors, mostly of colon. We describe CD43 expression by immunohistochemistry and mRNA in situ hybridization in adenoid cystic carcinomas (ACCs). CD43 immunoreactivity with 2 different antibodies was detected mainly in ACC but also 1 membranous type basal cell adenocarcinoma and 1 colonic adenocarcinoma. Nine of 20 (45%) ACC (8/17 salivary/mucoserous and 1/3 mammary) showed immunoreactivity with clone MT1 whereas 4 (23.5%) salivary ACC also showed immunoreactivity with clone DF-T1. The few high-grade, angioinvasive ACC in our study did not seem to express CD43. CD43 mRNA was detected by in situ hybridization in 2 of the 3 ACC that were CD43 positive on immunohistochemistry but not on any CD43 negative cases tested. Hence, CD43 seems to be selectively expressed in a subset of ACC and its significance in salivary tumors is discussed.

ZAP70 expression assessed by immunohistochemistry on peripheral blood: a simple prognostic assay for patients with chronic lymphocytic leukemia.

The somatic hypermutational (SHM) status of the immunoglobulin heavy-chain variable (IgVH) gene is a powerful prognostic factor in patients with chronic lymphocytic leukemia (CLL). However, IgVH SHM analysis is not well-suited to routine use in the clinical diagnostic laboratory. ZAP70 expression is a potential surrogate for the absence of SHM. Given the current problems with the standardization of ZAP70 assessment by flow cytometry, we sought an alternative approach, using immunohistochemistry (IHC). The utility of IHC is largely restricted to tissues, precluding its routine application to most patients with CLL who are typically diagnosed based upon peripheral blood (PB) findings. Accordingly, we developed an IHC assay that can be performed on PB. Enriched PB mononuclear cells from 29 patients with CLL were analyzed for ZAP70 expression by IHC on paraffin-embedded cell blocks, using standard techniques. IgVH SHM analysis was performed on all cases, and clinical features recorded. Seventeen specimens (59%) were negative for ZAP70 expression and 12 (41%) were positive for ZAP70 expression. SHM was evident in 20 specimens (69%), and absent in 9 (31%). Seventy-six percent of the specimens (22/29) displayed "concordant" ZAP70 and SHM results, in that 15 (52%) were SHM-positive/ZAP70 negative, whereas 7 (24%) were SHM-negative/ZAP70 positive. ZAP70 expression in this small cohort correlated with poor clinical outcome. Importantly, IHC analysis of ZAP70 in PB is a simple, reliable, robust assay that may have a valuable role in the routine clinical laboratory assessment of patients with CLL.

Immunoprofile of endocervical and endometrial stromal cells and its potential application in localization of tumor involvement.

To evaluate and compare the immunophenotype of endocervical and endometrial stromal cells and to asses its potential application in tumor localization. Paraffin sections of benign endocervix (n = 24), benign endometrium (n = 33), endocervical adenocarcinoma (n = 9), endometrial carcinoma (n = 13), and endometrial hyperplasia (n = 16) were stained with antibodies to CD10, Wilms Tumor-1, CD34, smooth muscle actin, and factor XIIIa by immunohistochemistry. In 16 cases, lower uterine segment was also available. Immunoreactivity of stromal cells was recorded as positive (>/=50% staining), focally positive (>/=5%-<50%) or negative (<5%). Endocervical stromal cells (ECSC) in either benign or malignant cervical epithelial lesions were predominantly CD34/CD10 (CD34 dominant immunophenotype). Endometrial stromal cells (EMSCs) in either benign or malignant epithelial lesions were primarily CD34/CD10 (CD10 dominant immunophenotype). Expression of Wilms Tumor-1 was decreased in EMSC of the EMCA when compared to their counterpart in endometrial hyperplasia. There was no differential expression of smooth muscle actin and factor XIIIa identified between ECSC and EMSC. The immunophenotypes of the ECSC and EMSC overlapped in the lower uterine segment. The functional status of the endometrium had no effect on the immunoprofile. The pattern of CD34 and CD10 immunostaining in stromal cells might be helpful in determining tumor involvement in uterine and cervical sites.

Extensive retraction artifact correlates with lymphatic invasion and nodal metastasis and predicts poor outcome in early stage breast carcinoma.

Retraction artifact resulting in clear spaces around tumor cell nests is frequently seen in histologic material and may present difficulty in their differentiation from lymphovascular invasion. We noticed that retraction artifact seemed to be more common around groups of breast cancer cells compared with benign acini, and when extensively present, metastasis to axillary lymph nodes was often seen. Thus, we performed a study of 304 cases of stage pT1 and pT2 breast carcinomas to test our hypothesis that extensive retraction artifact in tumors correlates with lymphatic spread and outcome. Tumors were evaluated to determine the presence and extent of retraction artifact around tumor cell nests and the presence of lymphatic invasion. Lymphatic invasion was confirmed by D2-40 immunostaining. The extent of retraction artifact in tumors was correlated with clinicopathologic tumor features and patient outcome. Variable degree of retraction artifact was present in 183 of 304 (60%) invasive carcinomas, with its extent ranging from 0% to 90% (median 5%). The extent of retraction artifact showed a significant correlation with tumor size, histologic type, histologic grade, presence of lymphovascular invasion, and nodal metastasis. Further, extensive retraction artifact was significantly associated with poor overall and disease-free survival in both univariate and multivariate analyses. We propose that the apparent retraction of the stroma from cells of invasive breast carcinoma on routine histologic sections is not a phenomenon merely due to inadequate fixation as currently believed. Rather, it likely signifies important biologic changes that alter tumor-stromal interactions and contribute to lymphatic spread and tumor progression.