Todd S. Laughlin

Identification and Characterization of Optimal Gene Expression Markers for Detection of Breast Cancer Metastasis

Sentinel lymph node (SLN) status is highly predictive of overall axillary lymph node involvement in breast cancer. Historically, SLN-positive patients have undergone axillary lymph node dissection in a second surgery. Intraoperative SLN analysis could reduce the cost and complications of a second surgery; however, existing histopathological methods lack standardization and exhibit poor sensitivity. Rapid molecular methods may lead to improved intraoperative diagnosis of SLN metastasis. In this study, we used a genome-wide gene expression analysis of breast and other tissues to identify seven putative markers for detecting breast cancer metastasis. We assessed the utility of these markers for identifying clinically actionable metastases in lymph nodes through reverse transcriptase-polymerase chain reaction analysis of SLNs from 254 breast cancer patients. Polymerase chain reaction signals were compared to pathology on a per-patient basis. The optimal two-gene combination, mammaglobin and cytokeratin 19, detected clinically actionable metastasis in breast SLNs with 90% sensitivity and 94% specificity. Application of stringent criteria for identifying presumptive hematoxylin- and eosin-positive samples increased sensitivity and specificity to 91 and 97%, respectively. This study represents the first comprehensive demonstration of the utility of gene expression markers for detecting clinically actionable breast metastases. An intraoperative molecular assay using these markers has the potential to significantly reduce second surgeries for patients undergoing SLN dissection.

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.

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.

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.

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.

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.

Clonal antigen receptor gene PCR products outside the expected size range

Polymerase chain reaction (PCR) of the antigen receptor genes has clinical utility in establishing clonality in lymphoproliferations, which is an important correlate of lymphoid neoplasia. The most frequently used procedures for this purpose were developed by the BIOMED-2 consortium. One of the criteria for establishing monoclonality using PCR of the antigen receptor genes is the finding of an abundant amplicon within a size range determined by the positions of the PCR primers and the known variability in size inherent in the recombination events that assemble a functional antigen receptor gene. However, several cases have been reported in which an amplicon outside this size range has been shown to be a valid indicator of clonality after DNA sequence analysis. In this paper, we will report and discuss several additional cases in which an amplicon outside the accepted size range was consistent with a monoclonal lymphoproliferation. We conclude that oversized and undersized amplicons may indeed represent evidence for a monoclonal lymphoproliferation, but that this interpretation should preferably be confirmed by sequence analysis to avoid a false-positive result.

Genotyping of Hepatitis C Virus by Sequence Analysis of the Amplicon from the Roche Cobas AmpliPrep/Cobas TaqMan Viral Load Assay

Hepatitis C virus (HCV), a positive single-stranded RNA virus, is one of the major causes of end-stage liver disease worldwide (5). The nucleotide sequence of the HCV genome is an important predictor of response to antiviral therapy (6). Genotypic analysis is used together with measurement of viral load (VL) to help in the management of patients with HCV infection (1). The 5′ untranslated region (UTR) is often used for monitoring VL because it is less variable than other regions of the genome and consequently less likely to suffer PCR failures due to sequence variation at the primer binding sites (3, 6, 8). Although this region is not ideal for determination of the genotype, it is often used for this purpose because of the availability of the template after a VL assay and also because the genotypic information needed for clinical use can be obtained from it (2, 4, 9, 10).