Immunohistochemistry Detection of Histone H3 K27M Mutation in Human Glioma Tissue.

The presence of the histone 3 (H3) K27M mutation in diffuse midline glioma has implications for diagnosis, prognosis, and treatment, making rapid and accurate H3 K27M characterization vital for optimal treatment. This study evaluated an immunohistochemical assay using a commercially available monoclonal anti-H3 K27M in human central nervous system tumors. H3 K27M-positive glioma specimens were obtained from clinical sites with prior H3 K27M testing using local methods; negative control glioblastoma tissue was obtained from a tissue library. Specimens were stained with a rabbit anti-H3 K27M monoclonal antibody; slides were evaluated for the proportion of H3 K27M-positive tumor cells and staining intensity by a board-certified pathologist. H-score was calculated for each sample. Sensitivity, specificity, accuracy, repeatability, and reproducibility were evaluated. Fifty-one central nervous system specimens were stained (H3 K27M, n=41; H3 wild type, n=10). All H3 K27M-mutant specimens had positive nuclear staining, and most specimens had an H-score ≥150 (31/40, 77.5%). No nuclear staining occurred in H3 wild-type specimens; all cores in the normal tissue microarray were negative. Results were 100% sensitive, specific, and accurate for H3 K27M detection relative to local methods. Repeatability and reproducibility analyses were 100%, with a high degree of concordance for staining intensity. H3 K27M antigen was stable for at least 12 months at ambient temperature. Immunohistochemistry using a commercially available anti-H3 K27M monoclonal antibody provides a highly sensitive, specific, and stable method of establishing H3 K27M status in human glioma; this method may facilitate diagnosis in cases where sequencing is not feasible or available.

Immunohistochemical detection of PD-L1 among diverse human neoplasms in a reference laboratory: observations based upon 62,896 cases.

Targeting of the PD1/PD-L1 immune checkpoint pathway has rapidly gained acceptance as a therapeutic strategy for a growing number of malignancies. Testing for expression of PD-L1 in tumor cells and immune cells has been used as a companion or complementary test for drugs targeting the PD1/PD-L1 pathway. We evaluated the results of PD-L1 testing in a large reference lab cohort. Using Food and Drug Administration-approved methods and interpretive instructions for each individual test, 62,896 cases were evaluated for PD-L1 using antibody clone 22C3, 28-8, SP142, or SP263. Case data analyzed included test results and information on tumor location and clinical history. No clinical outcome information was available and no attempt was made to correlate PD-L1 results with any other tests performed. The following numbers of cases were evaluated: 22C3 with tumor proportion score [n = 52585], 22C3 with combined positive score [n = 2631], 28-8 [n = 4191], SP142 [n = 850], and SP263 [n = 70]. In 22C3/tumor proportion score cases, the general results were as follows: negative 33.1% (n = 17,405), (low) expression 33.9% (n = 17,822), and high expression 29.5% (n = 15,486). In cases identified as metastatic, the results were as follows: negative 35.9% (n = 1411), (low) expression 30.8% (n = 1211), and high expression 30.7% (n = 1208). We found broad ranges of expression in tumor types with increasing positivity, as adenocarcinomas were reported as poorly differentiated, whereas squamous cell carcinomas showed more positivity as tumors were described as well-differentiated. The results of many individual tumor types were evaluated and showed, in general, high levels of positive expression. Practical challenges and observations of PD-L1 stain results and interpretation are also discussed.

Motility induction in breast carcinoma by mammary epithelial laminin 332 (laminin 5).

Host interactions with tumor cells contribute to tumor progression by several means. This study was done to determine whether mammary epithelium could interact with breast carcinoma by producing substances capable of inducing motility in the cancer cells. Conditioned medium of immortalized 184A1 mammary epithelium collected in serum-free conditions induced dose-dependent motility in the MCF-7 breast carcinoma cell line by both a semiquantitative scattering assay and a Boyden chamber assay. Purification of the motility factor revealed that it was laminin 332 (formerly laminin 5) by mass spectroscopy. A Western blot of the 184A1 conditioned medium using a polyclonal antibody confirmed the presence of laminin 332 in the conditioned medium. Blockage of the motility with antibodies to the laminin 332 and its receptor components, alpha(3) and beta(1) integrins, provided further evidence that tumor cell motility was caused by the laminin 332 in the conditioned medium. Invasion of MCF-7, BT-20, and MDA-MB-435 S was induced by purified laminin 332 and 184A1 conditioned medium and blocked by an anti-alpha(3) integrin antibody. Staining of carcinoma in situ from breast cancer specimens revealed that laminin 332 in the myoepithelium adjacent to the preinvasive cells provided a source of laminin 332 that could potentially encourage the earliest steps of stromal invasion. In metaplastic breast carcinomas, the presence of laminin 332-producing cells coexpressing alpha(3) integrin and the greater metastatic potential of tumors with higher laminin 332 levels suggest that laminin 332 expression is associated with aggressive features in these human breast cancers.

The Anal Pap Smear: Cytomorphology of squamous intraepithelial lesions.

BACKGROUND: Anal smears are increasingly being used as a screening test for anal squamous intraepithelial lesions (ASILs). This study was undertaken to assess the usefulness and limitations of anal smears in screening for ASILs. METHODS: The cytomorphological features of 200 consecutive anal smears collected in liquid medium from 198 patients were studied and findings were correlated with results of surgical biopsies and/or repeat smears that became available for 71 patients within six months. RESULTS: Adequate cellularity was defined as an average of 6 or more nucleated squamous cells/hpf. A glandular/transitional component was not required for adequacy. Dysplastic cells, atypical parakeratotic cells and bi/multinucleated cells were frequent findings in ASIL while koilocytes were infrequent. Smears from LSIL cases most frequently showed mildly dysplastic and bi/multinucleate squamous cells followed by parakeratotic cells (PK), atypical parakeratotic cells (APK), and koilocytes. HSIL smears contained squamous cells with features of moderate/severe dysplasia and many APKs. Features of LSIL were also found in most HSIL smears. CONCLUSIONS: In this study liquid based anal smears had a high sensitivity (98%) for detection of ASIL but a low specificity (50%) for predicting the severity of the abnormality in subsequent biopsy. Patients with cytologic diagnoses of ASC-US and LSIL had a significant risk (46-56%) of HSIL at biopsy. We suggest that all patients with a diagnosis of ASC-US and above be recommended for high resolution anoscopy with biopsy.

Nuclear morphometry and molecular biomarkers of actinic keratosis, sun-damaged, and nonexposed skin.

Computer-assisted image analysis is useful for quantifying the histologic and molecular changes of sun-induced squamous cell carcinoma progression. We used the CAS 200 image analysis system to measure nuclear morphometric parameters, p53 expression, and proliferation markers in actinic keratosis (AK), sun-exposed, and normal skin in 51 patients. Nuclear morphometry revealed significant increases in nuclear absorbance, irregularity of nuclear shape, and nuclear size in AK compared with normal and sun-damaged skin. These parameters showed significantly greater variability in AK nuclei. Argyrophyllic nucleolar organizer area and number were also significantly greater in AK compared with sun-damaged skin and normal skin. Ki67 and p53 expressions were both increased in sun-damaged skin relative to normal and greater still in AK. These data are evidence that sun damage induces proliferation and p53 abnormalities before the appearance of nuclear abnormalities and their associated DNA instability. Following these changes during a skin cancer chemopreventative trial can then help assess the efficacy of the agent and help determine where in the progression of neoplastic changes it exerts its biological effects.