p53 alteration in morphologically normal/benign breast luminal cells in BRCA carriers with or without history of breast cancer.

Germline mutations in BRCA genes have been shown to predispose patients to breast cancer. Studies have suggested that p53 alteration is a necessary step in tumorigenesis in BRCA carriers. Our previous study showed p53 alteration in morphologically normal/benign breast luminal cells in sporadic breast cancer patients, the so-called breast p53 signature. Here, we studied p53 status in 66 BRCA1/2 carriers' breasts: 29 patients with breast carcinoma (2 patients with bilateral breast carcinomas) and 37 without. Seven of the 12 (58%) triple-negative breast carcinomas in BRCA carriers were positive for p53 alteration (immunohistochemical stain and/or sequencing), the same frequency as in sporadic triple-negative breast carcinomas. Focal p53 positivity in adjacent normal/benign luminal cells was identified in 4 of the 7 cases with p53-positive carcinomas but not in breasts with p53-negative carcinomas, indicating that p53 positivity in normal/benign breast luminal cells is not a random event. Furthermore, in BRCA carriers' prophylactic mastectomies, 12 of the 94 (12.77%) breasts had focal p53 positivity in normal/benign luminal cells, with 2 cases in bilateral breasts, significantly higher than in previously studied mammoplasty specimens (0%). Our study suggests that germline BRCA gene mutations could result in genomic instability and an elevated gene mutation rate (such as the p53 gene) in breast luminal cells compared with the general population, predisposing BRCA carriers to develop p53-positive/triple-negative breast carcinomas.

Pigmented epithelioid melanocytoma (animal-type melanoma): An institutional experience.

BACKGROUND: Pigmented epithelioid melanocytoma (PEM) is an uncommon, recently described entity with unknown biologic behavior. There is a high rate of regional metastases, but limited evidence of distant metastases or disease-related death. OBJECTIVE: We sought to report our series of patients given a diagnosis of PEM at our institution and provide mutational analysis of genes commonly implicated in melanoma in 2 cases. METHODS: The pathology database was queried for cases of PEM diagnosed at the University of Rochester. Charts were reviewed for follow-up information. Mutational analysis of melanoma-associated genes was performed on 2 cases. RESULTS: Nine cases of PEM were retrieved in a 10-year retrospective review. Five patients underwent sentinel lymph node biopsy with 3 of 5 having a positive sentinel lymph node. All 9 patients are alive and disease-free with average follow-up of 38.75 months. Two tumors were tested for common melanoma-associated mutations, and were negative, except for a telomerase reverse transcriptase promoter deletion detected in 1 sample. The deletion has not been associated with melanoma, and therefore its biologic significance is unclear. LIMITATIONS: Small sample size, retrospective nature, and single institution experience are limitations. CONCLUSIONS: PEM appears to have an indolent behavior. However, currently the evidence is too limited to provide insight into its true biologic potential.

p53 alteration in morphologically normal/benign breast tissue in patients with triple-negative high-grade breast carcinomas: breast p53 signature?

p53 alterations have been identified in approximately 23% of breast carcinomas, particularly in hormone receptor-negative high-grade carcinomas. It is considered to be an early event in breast carcinogenesis. Nevertheless, the putative precursor lesion of high-grade breast carcinoma remains elusive. Breast excision specimens from 93 triple-negative high-grade invasive ductal carcinomas, 48 estrogen receptor (ER)-positive/progesterone receptor-positive/Her2-negative non-high-grade invasive ductal carcinomas, and 50 mammoplasty breasts were selected. At least 2 tissue blocks with tumor and adjacent benign tissue were sectioned and subjected to immunohistochemistry staining for p53. TP53 gene sequencing was performed on select tumors. Further immunohistochemistry staining for ER and Ki-67 was performed on consecutive sections of tissue with p53-positive normal/benign cells. Of the 93 high-grade carcinomas, 51 (55%) were positive for p53 alteration, whereas only 3 (6.25%) of the 48 non-high-grade carcinomas were p53 altered. Focal p53 positivity in adjacent normal/benign breast tissue was identified in 19 cases, and 18 of them also had p53 alteration in their carcinomas. Only 1 case had focal p53 staining in normal/benign tissue, but the tumor was negative for p53 alteration. No p53 staining positivity was identified in the mammoplasty specimens. The p53-stained normal/benign cells were ER negative and did not show an increase in the Ki-67 labeling index. These findings indicate that the p53 staining positivity in normal/benign breast tissue is not a random event. It could be considered as the "p53 signature" in breast and serve as an indicator for future potential risk of p53-positive high-grade breast carcinoma.

Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma.

Telomerase is frequently expressed in cancer and contributes to carcinogenesis. Two recent publications report the identification of a set of recurrent mutations in melanoma in the promoter of the telomerase reverse transcriptase gene (TERT) that appears to be the result of mutagenesis from ultraviolet (UV) radiation. Both groups reported that the mutations increase the transcription of TERT. This prompted our search for similar mutations in two other UV-related skin cancers, basal cell carcinoma, and squamous cell carcinoma. We found that the activating TERT promoter mutations reported in melanoma are also frequent in squamous cell carcinoma (50%) and basal cell carcinoma, the latter including both sporadic tumors (78%) and tumors from patients with nevoid basal cell carcinoma syndrome (68%). These mutations were found in only 1 of 11 Bowen's disease (squamous cell carcinoma in situ) specimens, and in none of 15 non-malignant skin specimens and 57 blood specimens. The mutations were frequently homozygous or hemizygous, with little or no normal signal at the mutated positions. These data suggest that TERT promoter mutations are the most frequent putative oncogenic mutations in cutaneous cancer.

Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies.

BACKGROUND: The recent development of antibodies specific for the major hotspot mutations in the epidermal growth factor receptor (EGFR), L858R and E746_A750del, may provide an opportunity to use immunohistochemistry (IHC) as a screening test for EGFR gene mutations. This study was designed to optimize the IHC protocol and the criteria for interpretation of the results using DNA sequencing as the gold-standard. METHODS: Tumor sections from fifty lung adenocarcinoma specimens from Chinese patients were immunostained using L858R and E746_A750del-specific antibodies using three different antigen retrieval solutions, and the results were evaluated using three different sets of criteria. The same specimens were used for DNA purification and analysis of EGFR gene mutations. RESULTS: In this study the optimal buffer for antigen retrieval was EDTA (pH 8.0), and the optimal scoring method was to call positive results when there was moderate to strong staining of membrane and/or cytoplasm in >10% of the tumor cells. Using the optimized protocol, L858R-specific IHC showed a sensitivity of 81% and a specificity of 97%, and E746_A750del-specific IHC showed a sensitivity of 59% and a specificity of 100%, both compared with direct DNA analysis. Additionally, the mutant proteins as assessed by IHC showed a more homogeneous than heterogeneous pattern of expression. CONCLUSIONS: Our data demonstrate that mutation-specific IHC, using optimized procedures, is a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2059012601872392.

Is the association of "cup-like" nuclei with mutation of the NPM1 gene in acute myeloid leukemia clinically useful?

Cup-like nuclear invaginations (NIs) in acute myeloid leukemia (AML) blasts have been associated with NPM1 mutations. Precision for enumeration of NI blasts has not been previously studied. Furthermore, the sensitivity and specificity for the morphologic prediction of NPM1 mutations have been variously reported. By using 66 AML specimens (17 with NPM1 mutations and 49 without), we found that interobserver reproducibility for enumeration of NI blasts was high (r = 0.98) and that identification of this feature was teachable (r = 0.96). No NPM1 mutation-negative case had greater than 7% NI blasts. The fraction of NI blasts was highly variable among 17 NPM1 mutation-positive cases, ranging from 0% to greater than 40%. These data indicate that an NI blast fraction of more than 10% is highly specific for NPM1 mutation-positive cases but with a sensitivity of about 30%. Therefore, although NI blasts can be reliably identified in routine smears and although they are a specific marker of NPM1 mutation-positive cases, the majority of NPM1 mutation-positive cases lack this distinctive finding.

PCR assays detect B-lymphocyte clonality in formalin-fixed, paraffin-embedded specimens of classical hodgkin lymphoma without microdissection.

Hodgkin lymphoma (HL) was shown to be a B-cell malignancy using polymerase chain reaction (PCR) clonality studies of microdissected Reed-Sternberg cells. While methods for the detection of B-cell clonality could aid in the diagnosis of HL, microdissection is not practical in most clinical settings. We assessed the standardized BIOMED-2 IGH and IGK PCR primers for the detection of clonality using 50 consecutively diagnosed formalin-fixed, paraffin-embedded (FFPE) classic HL specimens. Without microdissection, clonality was detected in 23 of 47 assessable cases. The IGK assay was significantly more sensitive than the IGH assay (18 vs 10 positive results). These data and 2 representative cases demonstrate that PCR-based B-cell clonality assays have usefulness when the histologic differential diagnosis of an FFPE specimen includes classic HL.

Detection of exon 12 Mutations in the JAK2 gene: enhanced analytical sensitivity using clamped PCR and nucleotide sequencing.

JAK2 V617F is the most frequently found somatic mutation in polycythemia vera (PV). Among the cases negative for V617F, a significant fraction have a mutation in exon 12 of the JAK2 gene. Several groups have reported that the exon 12 mutations are present in only a small fraction of the blood cells in some patients. We have developed an assay to detect these mutations with an analytical sensitivity of 0.1% by using a "PCR clamp" to inhibit amplification of the normal sequence and enhance amplification of DNA containing a mutation in the clamp target sequence. The products of this reaction were analyzed by capillary electrophoresis to detect deletions, which are the most frequent type of exon 12 mutations, or by nucleotide sequencing to detect all of the mutations. In a survey of 34 specimens from patients with PV or idiopathic erythrocytosis who did not have a JAK2 V617F mutation, we found four with a mutation in exon 12, 3 of 10 with PV, and 1 of 24 with idiopathic erythrocytosis. In two cases the mutation was present in a small fraction of the cells and difficult to detect without the use of the clamp. The use of an assay with increased analytical sensitivity enhances the ability to identify patients with mutations in exon 12 of the JAK2 gene.

A 46 XY phenotypic female adolescent with bilateral gonadal tumors consisting of five different components.

46 XY gonadal dysgenesis patients often develop gonadal tumors, including gonadoblastoma and other types of germ cell tumors. We report a case of a 16-year-old female adolescent with primary amenorrhea and a right adnexal mass. Subsequent study revealed that she is a 46 XY phenotypic female adolescent with complete gonadal dysgenesis and with no alterations of the sex-determining region Y gene. Microscopic examination of the gonads revealed bilateral gonadoblastoma mixed with dysgerminoma and mature teratoma. The tumor in the right gonad was also mixed with yolk sac tumor and immature teratoma with rhabdomyoblastic components, mimicking adult rhabdomyoma and rhabdomyosarcoma. No metastasis in the regional lymph nodes was identified and the patient is disease free 15 months postsurgery. The complexity of the tumorigenesis in this case indicates that the gonadal cells in gonadal dysgenesis are extremely unstable and highly tumorigenic. This tumorigenesis is not necessarily associated with sex-determining region Y gene alterations; therefore, it reinforces the recommendation of gonadectomy for gonadal dysgenesis patients, regardless of the sex-determining region Y gene status.

Rapid method for detection of mutations in the nucleophosmin gene in acute myeloid leukemia.

Mutations in exon 12 of the nucleophosmin gene (NPM1) that cause the encoded protein to abnormally relocate to the cytoplasm are found at diagnosis in about 50% of karyotypically normal acute myeloid leukemias and are associated with a more favorable outcome. We have devised a PCR-based assay for NPM1 exon 12 mutations using differential melting of an oligo probe labeled with a fluorescent dye. The nucleobase quenching (NBQ) phenomenon was used to detect probe hybridization, and an oligonucleotide containing locked nucleic acid (LNA) nucleotides was used as a PCR clamp to suppress amplification of the normal sequence and enhance the analytical sensitivity of the assay. After the NBQ assay, the specimens with a mutation were removed from the capillary and sequenced to identify the mutation. The use of the LNA clamp facilitates interpretation of the mutant sequence because of the lower intensity of the overlapping normal sequence. Analysis of a series of 70 patient specimens revealed 17 positive for an NPM1 mutation and 53 negatives. All of the NBQ results (positives and negatives) were confirmed with other methods. The analytical sensitivity of the NBQ assay is variable depending on the concentration of the PCR clamp and other parameters. Using a 100 nmol/L concentration of the LNA clamp, NPM1 mutations were detectable in a 10-fold excess of wild-type DNA. This assay may be valuable for screening disease specimens.

Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in the respiratory tracts of human infants following paramyxovirus infection.

Respiratory syncytial (RSV) and parainfluenza (PIV) viruses are primary causes of acute bronchiolitis and wheezing illnesses in infants and young children. To further understand inflammation in the airways following infection, we tested for the presence of matrix metalloproteinases (MMP) and natural tissue inhibitors of MMP (TIMP) in primary and established human cell lines, and in the nasopharyngeal secretions (NPS) of human infants infected with RSV or PIV. Using ELISA and multiplex-based assays, MMP-9 and TIMP-1 proteins were, respectively, detected in 66/67 and 67/67 NPS. During PIV or RSV infection TIMP-1 concentrations were associated with hypoxic bronchiolitis. TIMP-1 amounts were also negatively correlated with O2 saturation, and positively correlated with IL-6, MIP-1alpha, and G-CSF amounts following RSV infection. IL-6, MIP-1alpha, and G-CSF were negatively correlated with O2 saturation during RSV infection. Acute respiratory tract disease was not associated with MMP-9 protein/protease activity. Additional studies using real-time quantitative PCR suggested that MMP-9 mRNA copy numbers were elevated in normal human bronchial epithelial (NHBE) cells infected with RSV, while TIMP-1 and TIMP-2 were not increased. However, ELISA did not reveal MMP-9 protein in the NHBE cell culture supernatants. Hence, the data implied that airway epithelial cells were not the primary source of MMP or TIMP following paramyxovirus infection. Taken together, the data suggested that paramyxovirus infection perturbs MMP-9/TIMP-1 homeostasis that in turn may contribute to the severity of respiratory tract disease.

Characterization of recombinant respiratory syncytial viruses with the region responsible for type 2 T-cell responses and pulmonary eosinophilia deleted from the attachment (G) protein.

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpDeltaG150-222 or rA2cpDeltaG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37 degrees C, but pinpoint morphology was observed at 39 degrees C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log(10)-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpDeltaG150-222 or rA2cpDeltaG177-220. These findings are important for the development of attenuated RSV vaccines.

Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro.

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

Inhibition of respiratory syncytial virus infection with the CC chemokine RANTES (CCL5).

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1delta/CCL15, stromal cell derived factor (SDF)-1alpha/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein-epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4 degrees C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV.