New Technologies to Image Tumors.

The premise of this book is the importance of the tumor microenvironment (TME). Until recently, most research on and clinical attention to cancer biology, diagnosis, and prognosis were focused on the malignant (or premalignant) cellular compartment that could be readily appreciated using standard morphology-based imaging.

Association of quantitative assessment of the intrafollicular proliferation index with outcome in follicular lymphoma.

The role of the proliferation index (PI) as an outcome predictor in follicular lymphoma (FL) isn't clear. We have previously demonstrated that quantitative image analysis (QIA) is a robust tool for PI determination and the present study aimed to determine the significance of the PI for outcome in low-grade FL. One hundred and twenty-nine patients with grade 1-2 FL were retrospectively analysed. Slides were scanned digitally and follicle/tumour-involved areas were annotated. The intrafollicular PI was estimated by analysing a median of 10 follicles per case. Patients were divided into two groups: PI < 30%, PI ≥ 30% and clinical outcome was analysed. Among the 129 patients analysed, intrafollicular PI ranged from 0·6 to 63·2% with a median of 23·3%. Overall survival was not influenced by PI group. Among those patients initially observed, intrafollicular PI < 30% was associated with longer time to first therapy compared to patients with a PI ≥ 30%. In the group of patients that were treated at diagnosis, PI was not predictive of time to treatment failure (TTTF). Intrafollicular PI is an important predicator of TTFT for patients who are candidates for observation. Further confirmation in an independent cohort of patients is necessary to determine the clinical validity of the results.

Genetic analysis of patients with leukemic transformation of myeloproliferative neoplasms shows recurrent SRSF2 mutations that are associated with adverse outcome.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P=.05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P=.03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P<.05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation.

We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Effect of selumetinib vs chemotherapy on progression-free survival in uveal melanoma: a randomized clinical trial.

IMPORTANCE: Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation. OBJECTIVE: To assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma. DESIGN, SETTING, AND PARTICIPANTS: Randomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada. INTERVENTIONS: One hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (n = 50), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression. MAIN OUTCOMES AND MEASURES: Progression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013. RESULTS: Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < .001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction. CONCLUSIONS AND RELEVANCE: In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01143402.

Regulatory T cell infiltration predicts outcome following resection of colorectal cancer liver metastases.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) counts in colorectal cancer liver metastases (CRCLM) predict survival following resection. While CD4 and CD8 T cells have been correlated with outcome following CRCLM resection, the role of regulatory T cells (Treg) is not well defined. METHODS: TIL in 188 patients who underwent CRCLM resection between 1998 and 2000 were analyzed by immunohistochemistry using tissue microarrays. Correlation between TIL composition and outcome was determined while controlling for established prognostic factors. Total T cells (CD3), helper T cells (CD4), cytotoxic T cells (CD8), and Treg (FoxP3) were analyzed. RESULTS: Median follow-up time was 40 months for all patients and 95 months for survivors. Overall survival (OS) at 5 and 10 years was 40 and 25%, respectively. The CD4 T cell count correlated with OS (p = .02) and recurrence-free survival (p = .04). A high number of CD8 T cells relative to total T cells (CD8:CD3 ratio) predicted longer OS times (p = .05). Analysis of Treg revealed that high FoxP3:CD4 (p = .03) and FoxP3:CD8 (p = .05) ratios were independent predictors of shorter OS. Patients with a high clinical risk score (CRS) were more likely to have a high number of intratumoral Treg, and patients ≥65 years old had a less robust CRCLM T cell infiltration. CONCLUSIONS: A high number of Treg relative to CD4 or CD8 T cells predicted poor outcome, suggesting an immunosuppressive role for FoxP3 + TIL. The intratumoral immune response was an independent predictor of outcome in patients with colorectal liver metastases.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

Development of a LAG-3 immunohistochemistry assay for melanoma.

AIMS: A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumour tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardised, well-characterised assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision and reproducibility of this assay. METHODS: The specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a ≥1% cut-off to determine LAG-3 positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. RESULTS: The LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator and inter-reagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). CONCLUSIONS: We have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.

T cell infiltrate predicts long-term survival following resection of colorectal cancer liver metastases.

BACKGROUND: While tumor infiltrating lymphocytes (TIL) have been shown to independently predict survival in primary colorectal cancer, the prognostic implications of TIL in resectable colorectal cancer liver metastases (CRCLM) have not been previously defined. This study examines the correlation between TIL numbers and survival following hepatic resection. METHODS: We studied patients who survived or=10 years following CRCLM resection. Immunohistochemistry was performed on tissue microarrays (TMAs) to determine the number of T cells within CRCLM. Correlation between TIL frequency and or=10 year survival was determined while controlling for established prognostic factors. RESULTS: Of 162 patients, 104 survived or=10 years. Independent correlates of 10-year survival following CRCLM resection included a high number of CD8 T cells, a low number of CD4 T cells, and a clinical risk score of

The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2.

MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor alpha and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G(1) phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G(1) phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF(Skp2), which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G(1)/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.

Patterns of expression of cell cycle/apoptosis genes along the spectrum of thyroid carcinoma progression.

BACKGROUND: Genetic screening studies suggest that genetic changes underlie progression from well differentiated to anaplastic thyroid cancers. The aim of this study is to determine to what extent cell cycle/apoptosis regulators contribute to cancer progression. METHODS: Tissue microarrarys (TMAs) were constructed from well-differentiated papillary thyroid carcinoma (WDPTC; n = 41), poorly differentiated thyroid carcinoma (PDTC; n = 43), and anaplastic thyroid carcinoma (ATC; n = 22). TMAs were immunostained for 7 different cell cycle/apoptosis-related genes (p53, Ki-67, bcl-2, mdm-2, cyclin D1, p21, and p27). RESULTS: p53 (0%, 12%, 32%) and Ki-67 (5%, 49%, 82%) were expressed with increasing frequency, and bcl-2 (68%, 42%, 0%) and p21 (40%, 7%, 0%) with decreasing frequency in WDPTC to PDTC and ATC, respectively (P < .001). Interestingly, mdm-2 (54%, 5%, 0%) showed decreased expression along the progression axis (P < .001). p27 and cyclin D1 were expressed in <15% of cases, with a trend toward decreasing expression from WDPTC to PDTC to ATC. CONCLUSIONS: These data confirm the presence of increasing genetic complexity with progressive dedifferentiation in thyroid cancer, with aberrant tumor suppressor activity and increased proliferative activity being most prevalent in ATC. The data also confirm the intermediate position of PDTC in the classification scheme of thyroid carcinomas.

Resistance to imatinib in patients with chronic myelogenous leukemia and the splice variant BCR-ABL1(35INS).

PURPOSE: In patients with chronic myelogenous leukemia (CML), point mutations in the BCR-ABL1 kinase domain are the most common cause of treatment failure with a tyrosine kinase inhibitor (TKI). It is not clear whether the splice variant BCR-ABL1(35INS) is also associated with treatment failure. PATIENTS AND METHODS: We reviewed all CML patients who had BCR-ABL1 kinase mutation analysis performed between August 1, 2007, and January 15, 2014. Patients who had BCR-ABL1(35INS) detected had their medical records reviewed to determine response to TKI therapy. RESULTS: Two hundred and eighty four patients had kinase mutation testing performed; of these, 64 patients (23%) had BCR-ABL1(35INS) detected. Forty-five patients were in chronic phase (70%), 10 were in accelerated phase (16%), 6 were in blastic phase (9%), and 3 were in other settings (5%). Of the 34 chronic phase patients who began therapy with imatinib, 23 patients (68%) failed therapy: 8 patients (24%) had primary refractory disease, 11 patients (32%) progressed, and 4 patients (12%) had disease progression after dose interruption. In contrast to the patients with disease progression or lack of response, none of 23 patients who were responding to imatinib had BCR-ABL1(35INS) detected. DNA sequencing of commonly mutated spliceosomal genes SF3B1, U2AF1, SRSF2, ZRSR2, SFA31, PRPF408, U2A565, and SF1 did not reveal mutations in seven BCR-ABL1(35INS) -positive patients tested. CONCLUSIONS: The splice variant BCR-ABL1(35INS) is frequently found in patients who are resistant to imatinib. Mutations in the commonly mutated spliceosomal proteins do not contribute to this association.

KRAS Mutation Is a Significant Prognostic Factor in Early-stage Lung Adenocarcinoma.

The potential clinical impact of KRAS and epidermal growth factor receptor (EGFR) mutations has been investigated in lung adenocarcinomas; however, their prognostic value remains controversial. In our study, we sought to investigate the prognostic significance of driver mutations using a large cohort of early-stage lung adenocarcinomas. We reviewed patients with pathologic early-stage, lymph node-negative, solitary lung adenocarcinoma who had undergone surgical resection (1995 to 2005; stage I/II=463/19). Tumors were classified according to the IASLC/ATS/ERS classification and genotyped by Sequenom MassARRAY system and polymerase chain reaction-based assays. In stage I disease, the Kaplan-Meier method and cumulative incidence of recurrence analyses were used to estimate the probability of overall survival (OS) and recurrence, respectively. Of all, 129 (27%) patients had mutations in KRAS, 86 (18%) in EGFR, 8 (2%) in BRAF, 8 (2%) in PIK3CA, 4 (1%) in NRAS, and 1 (0.2%) in AKT1. EGFR L858R mutation correlated with lepidic predominant histology (P=0.006), whereas exon 19 deletion correlated with acinar predominant histology (P<0.001). EGFR mutations were not detected in invasive mucinous adenocarcinomas (P=0.033). The 5-year OS of patients with KRAS-mutant tumors was significantly worse (n=124; 5-year OS, 63%) than those with KRAS wild-type (n=339; 77%; P<0.001). In solid predominant tumors, KRAS mutations correlated with worse OS (P=0.008) and increased risk of recurrence (P=0.005). On multivariate analysis, KRAS mutation was an independent prognosticator of OS in all patients (hazard ratio, 1.87; P<0.001) and recurrence in solid predominant tumors (hazard ratio, 4.73; P=0.012). In patients with resected stage I lung adenocarcinomas, KRAS mutation was an independent prognostic factor for OS and recurrence, especially in solid predominant tumors.

Expression of p63 in diffuse large B-cell lymphoma.

The p63 gene, a homolog of the tumor suppressor gene TP53, maps to chromosome 3q27-28, a region frequently displaying genomic amplification in squamous cell carcinomas. p63 is expressed in a variety of epithelial tissues and has been reported to be critical for the normal development of stratified epithelia, including skin epidermis. In a previous study, the authors reported the expression of p63 in occasional cells in the germinal center of lymph nodes and also observed p63 expression in B-cell lymphomas, among other tumor types surveyed in that analysis. The present study was conducted to further analyze the potential clinical significance of identifying p63 expression, assessing a larger cohort of well-characterized patients with diffuse large B-cell lymphoma (DLBCL) (n = 172 cases) and a panel of established lymphoma cell lines. p63 expression at the microanatomic detail was examined by immunohistochemistry using a monoclonal antibody (clone 4A4), while distinction of p63 isoforms was analyzed by Western blotting and reverse transcription-polymerase chain reaction using isoform-specific primers. The authors found that a subset of DLBCL (32% of cases) expressed p63 in the nuclei of neoplastic lymphocytes. Examination of the different p63 isoforms revealed that the DeltaNp63 species was expressed by only one cell line, while the other p63 isoforms were found in most cell lines analyzed. The authors also observed that p63 expression correlated with high proliferative index, as assessed by Ki-67 immunostaining. Even though in univariate analysis p63 expression did not correlate with overall survival, the association of p63 with increased proliferative index suggests its involvement in DLBCL tumor progression.

p63 expression profiles in human normal and tumor tissues.

PURPOSE: The p63 gene, located on chromosome 3q27-28, is a member of the p53 gene family. The product encoded by the p63 gene has been reported to be essential for normal development. EXPERIMENTAL DESIGN: In this study, we examined the expression pattern of p63 in human normal and tumor tissues by immunohistochemistry using a monoclonal antibody (clone 4A4) that recognizes all p63 splice variants, and by reverse transcription-PCR using isoform-specific primers. RESULTS: We found that p63 expression was restricted to the nucleus, with a nucleoplasmic pattern. We also observed that the expression was restricted to epithelial cells of stratified epithelia, such as skin, esophagus, exocervix, tonsil, and bladder, and to certain subpopulations of basal cells in glandular structures of prostate and breast, as well as in bronchi. Consistent with the phenotype observed in normal tissues, we found that p63 is expressed predominantly in basal cell and squamous cell carcinomas, as well as transitional cell carcinomas, but not in adenocarcinomas, including those of breast and prostate. Interestingly, thymomas expressed high levels of p63. Moreover, a subset of non-Hodgkin's lymphoma was also found to express p63. Using isoform-specific reverse transcription-PCR, we found that thymomas express all isoforms of p63, whereas the non-Hodgkin's lymphoma tended to express the transactivation-competent isoforms. We did not detect p63 expression in a variety of endocrine tumors, germ cell neoplasms, or melanomas. Additionally, soft tissue sarcomas were also found to have undetectable p63 levels. CONCLUSIONS: Our data support a role for p63 in squamous and transitional cell carcinomas, as well as certain lymphomas and thymomas.