Kristina Fields

Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma

The cytodiagnosis of melanoma in fine‐needle aspiration (FNA) specimens can be challenging, often requiring the use of immunocytochemistry. As constitutively activating mutations in the BRAF oncogene are present in at least 40% of melanomas, the use of FNA material to interrogate the BRAF mutational status is likely to increase. Because cell blocks, traditionally used for these studies, can occasionally exhibit insufficient tumor cellularity, the authors investigated the utility of direct smears for immunocytochemistry and BRAF mutational analysis.

Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using 35S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry.

Frequent PD-L1 expression in primary and metastatic penile squamous cell carcinoma: potential opportunities for immunotherapeutic approaches

BACKGROUND Despite aggressive multimodal therapy, locally advanced and/or metastatic penile squamous cell carcinoma (SqCC) is associated with significant morbidity and mortality, indicating a need for new therapeutic options. Given the emerging clinical utility of immunotherapeutics, we sought to assess the incidence and potential clinical significance of PD-L1 expression in penile SqCC. PATIENTS AND METHODS Using an anti-PD-L1 primary antibody (clone 5H1), immunohistochemistry was carried out on whole tumor sections from 37 patients with penile SqCC treated at our institution between 2005 and 2013. PD-L1-positive tumors were defined as those with membranous staining in ≥5% of tumor cells. Association between PD-L1 expression and clinicopathologic parameters was examined using Fishers exact test. Correlation between PD-L1 expression in primary tumors and matched metastases was assessed using the Spearman rank correlation coefficient (ρ). The difference in cancer-specific mortality between PD-L1-positive and -negative groups was examined using the log-rank test. RESULTS Twenty-three (62.2%) of 37 primary tumors were positive for PD-L1 expression, and there was strong positive correlation of PD-L1 expression in primary and metastatic samples (ρ = 0.72; 0.032 < P < 0.036). Primary tumor PD-L1 expression was significantly associated with usual type histology (P = 0.040) and regional lymph node metastasis (P = 0.024), as well as decreased cancer-specific survival (P = 0.011). CONCLUSIONS The majority of primary penile SqCC tumors express PD-L1, which is associated with high-risk clinicopathologic features and poor clinical outcome. These data provide a rational basis for further investigation of anti-PD-1 and anti-PD-L1 immunotherapeutics in patients with advanced penile SqCC.

Molecular approaches for the analysis of chromogranins and secretogranins.

Recent molecular analyses have contributed to our knowledge about the chromogranin/secretogranin (Cg/Sg) family and their utility in diagnostic pathology. The genes for five of these proteins have been cloned, and the deduced amino acid sequences have provided insights into the structure and possible functions of the Cgs/Sgs, including their role as prohormones. Northern hybridization and in situ hybridization histochemistry have provided a great deal of information about the tissue distribution of the Cg/Sg gene products. Some neoplasms such as small cell lung carcinomas, which have little stored Cg/Sg protein, have abundant cytoplasmic mRNAs that can be readily detected by hybridization studies. Some other neoplasms such as neuroblastomas have decreased CgA and increased Sgll mRNAs during maturation to ganglioneuromas. There is also a differential expression of Cgs/Sgs in some endocrine neoplasms such as parathyroid adenomas, which express abundant CgA mRNA and little CgB mRNA, and in pituitary prolactinomas, which express CgB mRNA but not CgA mRNA. The mRNA for CgA has been found unexpectedly in some neoplasms such as 15% of colonic adenocarcinomas. Thus, molecular approaches in the analysis of Cgs/Sgs should contribute to the diagnosis of endocrine neoplasms and may provide support for a molecular classification of neoplasms in diagnostic pathology.

Effects of Estrogens on Pituitary Cell and Pituitary Tumor Growth

Estrogens have been known to induce PRL cell hyperplasia in the anterior pituitary of some species for many decades. Recent studies have shown variable susceptibility to estrogen-induced hyperplasia in different strains of rats. The distinction between hyperplastic pituitaries and adenomas is usually not made by most investigators in this field, although true neoplasms can usually be propagated by serial transplantation. The growth of transplantable tumors is usually inhibited by estrogen in vivo. Estrogens have a biphasic effect on pituitary cell proliferation in vitro with higher concentrations of estradiol inhibit cell growth, and lower concentrations stimulating PRL secretion. Estrogens can regulate PRL gene methylation in vivo thus affecting PRL mRNA expression. Recent studies have suggested that estrogen regulates signal transduction by stimulating protein kinase C. Estrogens also regulate specific proto-oncogenes such as c-myc and c-fos. These observations may help to explain some of the regulatory effects of estrogens on cell proliferation and tumor development.

The application of immunocytochemistry to cytologic direct smears of metastatic merkel cell carcinoma

Merkel cell carcinoma represents a highly aggressive cutaneous malignancy characterized by regional recurrences, lymph node metastases, distant metastases, and high mortality. As the cytomorphology of Merkel cell carcinoma can be mimicked by other malignancies, especially lymphoma and pulmonary small cell carcinoma, immunocytochemistry is often useful in confirming the diagnosis. Cell blocks, which are traditionally utilized for immunocytochemistry, occasionally exhibit insufficient cellularity. Hence, we prospectively investigated the application of CK20 immunocytochemistry to air‐dried, unstained direct smears in the diagnosis of Merkel cell carcinoma fine needle aspirates (FNAs). Eight consecutive FNAs of Merkel cell carcinoma were prospectively examined in this series; seven (88%) cases exhibited immunoreactivity for CK20 in the tumor cells. The one CK20‐negative Merkel cell carcinoma was immunoreactive for synaptophysin and CD56. This immunophenotype was identical to that of the original primary tumor. For comparison, air‐dried direct smears prepared from three pulmonary small cell carcinoma FNAs were examined by CK20 immunocytochemistry. In all cases, no CK20 immunoreactivity was seen in any of the tumor cells. In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose Merkel cell carcinoma. This methodology allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where insufficient cell block cellularity is anticipated or encountered. Diagn. Cytopathol. 2013;41:729–733.

The application of immunocytochemistry to direct smears in the diagnosis of effusions

Metastatic malignancy represents a common cause of effusions. Immunocytochemistry (ICC) is useful in confirming malignancy and gaining insight into the site of origin. Cell blocks are commonly utilized for this purpose; nonetheless, when the malignant cells are sparse, they may not be represented in cell blocks thereby precluding immunophenotypic characterization. Thus, we sought to investigate the utility of direct smear preparations as a platform for ICC in the diagnosis of effusions. Air‐dried, unstained direct smears were prepared from 49 malignant effusions and 17 reactive effusions for comparison. ICC for EMA and MOC‐31 highlighted the tumor cells in 91 and 98% of the malignant effusions tested, respectively. EMA immunoreactivity was focally observed within the calretinin‐positive mesothelial cell population in 1 (6%) of the 17 reactive effusions. ICC for MOC‐31 was negative in all reactive effusions. Site‐specific immunomarkers were also evaluated. Immunoreactivity for Napsin‐A and TTF‐1 were observed in 78 and 67% of metastatic lung adenocarcinomas, respectively. ICC for PAX8 highlighted metastatic Müllerian and thyroid carcinomas in 100% of cases tested. CDX‐2 immunoreactivity was observed in 25, 60, and 100% of metastatic gastric, pancreatic, and colorectal adenocarcinomas, respectively. Positivity for p63 was observed in 75% of metastatic urothelial cell carcinomas and the one case of pulmonary squamous cell carcinoma examined. Calretinin ICC highlighted the tumor cells in both malignant mesothelioma cases tested as well as the benign mesothelial cells in the reactive effusions. In conclusion, direct smears represent an effective platform for the performance of ICC in the diagnosis of malignant effusions. Diagn. Cytopathol. 2013.

Young investigator challenge: The utility of GATA3 immunohistochemistry in the evaluation of metastatic breast carcinomas in malignant effusions.

It is not uncommon to encounter challenges in the immunohistochemical confirmation of metastatic breast cancer given the limited sensitivities of mammaglobin and gross cystic disease fluid protein 15 (GCDFP‐15/BRST‐2) and the significant proportion of triple‐negative breast carcinomas (ie, tumors that are negative for estrogen receptor [ER], and progesterone receptor [PgR], and human epidermal growth factor 2 [HER2]). GATA binding protein 3 (GATA3) has emerged as a potentially useful immunohistochemical adjunct during the evaluation of metastatic breast carcinomas in cytology specimens. The objective of the current study was to examine GATA3 expression in the context of malignant effusions secondary to both mammary and extramammary malignancies.

Pit-1/ghf-1 transcription factor expression in rodent pituitaries

The Pit-1/GHF-1 (Pit-1) transcription factor is important for the development of anterior pituitary cells that produce GH and PRL. We examined the expression of Pit-1 mRNA in pituitary tissues from rats and mice. Analysis of pituitaries from normal and GHRH transgenic mice showed that Pit-1 transcripts were readily detected in normal, hyperplastic, and neoplastic pituitaries. A cell line (GHRH-CL1) established from a GhRH transgenic mouse pituitary tumor in our laboratory also expressed Pit-1 mRNA. Normal rat pituitaries and those with estrogen-induced PRL cell hyperplasia expressed Pit-1 mRNA. There was a decrease in Pit-1 mRNA in hyperplastic rat pituitaries concomitant with a decrease In GH mRNA amounts and an increase in PRL mRNA amounts after estrogen treatment. Similarly, analysis of GH3 cells in vitro showed that estrogen and bFGF modulated PRL but not Pit-1 mRNA levels. Pit-1 mRNA was localized by combined in situ hybridization and immunohistochemistry to predominantly GH and PRL cells, although some TSH and LH cells in the rat pituitary also expressed Pit-1 mRNA, indicating wide distribution of the mRNA for this transcription factor in various anterior pituitary cell types. Analysis of cell proliferation in normal rat pituitary and GH3 cells revealed that estrogen and bFGF stimulated cell proliferation in normal pituitaries but inhibited proliferation in GH3 cells, whereas Pit-1 transcripts remained unchanged in both groups of cultured cells. These results indicate that Pit-1 mRNA is readily detected in normal, hyperplastic, and neoplastic rodent pituitaries. Changes in Pit-1 mRNA amounts appear to correlate more closely with changes in GH than PRL mRNA levels in cultured pituitary cells.Endocr Pathol 4:146–154, 1993.

Regulation of dopamine receptors in the MtT/W15 transplantable pituitary tumor by estrogen.

We investigated the effects of estrogens on the regulation of dopamine receptors in the MtT/W15 transplantable rat pituitary tumor. Diethylstilbestrol (DES) and 17beta-estradiol treatment in female rats significantly decreased the number of dopamine binding sites (B max) from 85 +/- 3.9 fmol/mg protein in untreated rats to 9.2 +/- 1.2 and 8.2 +/- 2.8 fmol/mg protein in DES and 17beta-estradiol-treated rats, respectively, while the binding affinities (Kd) did not change significantly. Testosterone treatment did not change the B max, while ovariectomy resulted in a significant increase in the B max (146.3 +/- 6.7 fmol/mg protein). The effects of DES on the B max were reversible, since removal of the DES for one week before sacrificing the animals led to a marked increase in the B max (54.9 +/- 3.1 fmol/mg protein). Pituitaries from normal female rats treated with DES for 6 and 9 weeks had a significant decrease in the B max. These results show that the number of dopamine binding sites in the membranes of MtT/W15 tumors is decreased by estrogen treatment and that this effect is reversible after removal of the estrogenic stimulus.