Evaluation of inadequate, indeterminate, falsenegative and false-positive cases in cytological examination for breast cancer according to histological type

ABSTRACT: BACKGROUND: We previously investigated the current status of breast cytology cancer screening at seven institutes in our area of southern Fukuoka Prefecture, and found some differences in diagnostic accuracy among the institutions. In the present study, we evaluated the cases involved and noted possible reasons for their original cytological classification as inadequate, indeterminate, false-negative and false-positive according to histological type. METHODS: We evaluated the histological findings in 5693 individuals who underwent cytological examination for breast cancer (including inadequate, indeterminate, false-negative and falsepositive cases), to determine the most common histological types and/or features in these settings and the usefulness/limitations of cytological examination for the diagnosis of breast cancer. RESULTS: Among 1152 cytologically inadequate cases, histology revealed that 75/173 (43.6%) cases were benign, including mastopathy (fibrocystic disease) in 38.6%, fibroadenoma in 24.0% and papilloma in 5.3%. Ninety-five of 173 (54.9%) cases were histologically malignant, with scirrhous growing type, invasive ductal carcinoma (SIDC) being significantly more frequent (49.5%) than papillotubular growing type (Papi-tub) (P < 0.0001), solid-tubular growing type (P = 0.0001) and ductal carcinoma in situ (DCIS) (P = 0.0001). Among 458 indeterminate cases, 54/139 (38.8%) were histologically benign (mastopathy, 30.0%; fibroadenoma, 27.8%; papilloma, 26.0%) and 73/139 (52.5%) were malignant, with SIDC being the most frequent malignant tumor (37.0%). Among 52 false-negative cases, SIDC was significantly more frequent (42.3%) than DCIS (P = 0.0049) and Papi-tub (P = 0.001). There were three falsepositive cases, with one each of fibroadenoma, epidermal cyst and papilloma. CONCLUSIONS: The inadequate, indeterminate, false-negative and false-positive cases showed similar histological types, notably SIDC for malignant tumors, and mastopathy, fibroadenoma and papilloma for benign cases. We need to pay particular attention to the collection and assessment of aspirates for these histological types of breast disease. In particular, several inadequate, indeterminate and false-negative cases with samples collected by aspiration were diagnosed as SIDC. These findings should encourage the use of needle biopsy rather than aspiration when this histological type is identified on imaging. Namely, good communication between clinicians and pathological staff, and triple assessment (i.e., clinical, pathological and radiological assessment), are important for accurate diagnosis of aspiration samples. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7349809170055423.     

KL-6, a Human MUC1 Mucin,as a prognostic marker for diffuse alveolar hemorrhage syndrome

Background

Diffuse alveolar hemorrhage syndrome is a life threatening condition with diverse etiologies. Sensitive prognostic markers for diffuse alveolar hemorrhage have not been well investigated. Serum KL-6 is a biomarker for various interstitial lung disease associated with disease activity and prognosis. The purpose of the present study was to evaluate the clinical utility of serum KL-6 level as a prognostic marker for diffuse alveolar hemorrhage.

Methods

We retrospectively collected 41 consecutive patients clinically diagnosed as having diffuse alveolar hemorrhage who were admitted to the Intensive Care Unit of Hiroshima University Hospital between 2004 and 2011. Correlation between prognosis and age, sex, laboratory findings including serum KL-6, radiological findings, ventilatory modes or therapeutic regimens were evaluated.

Results

Baseline and peak serum KL-6 levels were significantly higher in non-survivors compared with survivors. An increase in KL-6 levels during the initial week was associated with a subsequent deterioration of the oxygenation index. Higher baseline KL-6 levels and higher peak KL-6 levels were strongly correlated with death. With a cut-off level of 700 U/mL for peak KL-6, the sensitivity, specificity and accuracy for non-survival were 75%, 85% and 78%, respectively. In the multivariate analysis, only the peak KL-6 level ≥700 U/ml was an independent poor prognostic factor for diffuse alveolar hemorrhage.

Conclusions

Peak serum KL-6 level ≥700 U/ml may become a clinically useful marker of poor prognosis for diffuse alveolar hemorrhage.

     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

KRAS-induced actin-interacting protein regulates inositol 1,4,5-trisphosphate-receptor-mediated calcium release

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and is responsible for the proper subcellular localization of IP₃R. Since it remains unknown whether KRAP regulates the IP₃R-mediated Ca²⁺ signaling, we herein examined the effects of KRAP on the IP₃R-mediated Ca²⁺ release by Ca²⁺ imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca²⁺ release and the ATP-induced Ca²⁺ release is completely quenched by the pretreatment with the IP₃R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP₃R-mediated Ca²⁺ release. To further reveal mechanistic insights into the regulation of IP₃R-mediated Ca²⁺ release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca²⁺ content of intracellular Ca²⁺ stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca²⁺-ATPase inhibitor-induced Ca²⁺ release in the HEK293 cells, indicating that releasable Ca²⁺ content of intracellular Ca²⁺ stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP₃R-mediated Ca²⁺ release.     

Chlamydophilal antigens induce foam cell formation via c-Jun NH2-terminal kinase

Chlamydophila pneumoniae is known to be associated with atherosclerosis. Recent studies have reported that components of Chlamydophila pneumoniae (chlamydophilal antigens) induce foam cell formation in macrophages. However, the mechanism of foam cell formation induced by chlamydophilal antigens has yet to be elucidated. In this paper, we first found that mitogen-activated protein kinases including extracellular signal-regulated kinase, p38 and c-Jun NH2 terminal kinase are phosphorylated after stimulation by chlamydophilal antigens. We then showed that chlamydophilal antigens induce foam cell formation mainly via c-Jun NH2 terminal kinase. Finally, we demonstrated that foam cell formation and phosphorylation of mitogen-activated protein kinases induced by chlamydophilal antigens are mainly recognized through Toll-like receptor 2. These results collectively indicated that chlamydophilal antigens induce foam cell formation mainly via Toll-like receptor 2 and c-Jun NH2 terminal kinase.     

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E₂ production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE₂ and PGF_2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF_1α, thromboxane B₂, PGF_2α, PGE₂, and PGD₂ by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE₂ by 7.4±0.7 ng/mg-protein/hour PGF_2α were produced. While hydrocortisone (10⁻⁴-10⁻² M) did not show a significant effect on PGE₂ production, indomethacin (10⁻⁸-10⁻⁶ M), and 5-aminosalicylic acid (2×10⁻⁴-5×10^{−3 M}) inhibited PGE₂ production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE₂ synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.     

The basis for haplotype complexity in VCBPs, an immune-type receptor in amphioxus

Innate immune gene repertoires are restricted primarily to germline variation. Adaptive immunity, by comparison, relies on somatic variation of germline-encoded genes to generate extraordinarily large numbers of non-heritable antigen recognition motifs. Invertebrates lack the key features of vertebrate adaptive immunity, but have evolved a variety of alternative mechanisms to successfully protect the integrity of “self”; in many cases, these appear to be taxon-specific innovations. In the protochordate Branchiostoma floridae (amphioxus), the variable region-containing chitin-binding proteins (VCBPs) constitute a multigene family (comprised of VCBPs 1–5), which possesses features that are consistent with innate immune-type function. A large number of VCBP alleles and haplotypes are shown to exhibit levels of polymorphism exceeding the elevated overall levels determined for the whole amphioxus genome (JGI). VCBP genes of the 2 and 5 types are distinguished further by a highly polymorphic segment (exon 2) in the N-terminal immunoglobulin domain, defined previously as a “hypervariable region” or a “hotspot.” Genomic deoxyribonucleic acid (DNA) and complementary DNA (cDNA) sequences from large numbers of animals representing different populations reveal further significant differences in sequence complexity within and across VCBP2/5 haplotypes that arise through overlapping mechanisms of genetic exchange, gene copy number variation as well as mutation and give rise to distinct allelic lineages. The collective observations suggest that mechanisms were in place at the time of divergence of the cephalochordates that could selectively hyperdiversify immune-type receptors within a multigene family.     

FAK nuclear export signal sequences

Ubiquitously expressed focal adhesion kinase (FAK), a critical component in transducing signals from sites of cell contacts with extracellular matrix, was named after its typical localization in focal adhesions. A nuclear localization of FAK has been also reported and its scaffolding role in nucleus and requirement for p53 ubiquitination were only recently described. Whereas FAK nuclear localization signal (NLS) was found in F2 lobe of FERM domain, nuclear export signal (NES) sequences have not been yet determined. Here we demonstrate that FAK has two NES sequences, NES1 in F1 lobe of FERM domain and NES2 in kinase domain. Although, both NES1 and NES2 are evolutionary conserved, and present as well in FAK-related protein kinase Pyk2, only NES2 demonstrates full biological nuclear export activity.     

TRPV1 and TRPV4 Play Pivotal Roles in Delayed Onset Muscle Soreness

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes tenderness and movement related pain after some delay (delayed-onset muscle soreness, DOMS). We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are up-regulated in exercised muscle through up-regulation of cyclooxygenase (COX)-2, and they sensitized nociceptors resulting in mechanical hyperalgesia. There is also a study showing that transient receptor potential (TRP) ion channels are involved in DOMS. Here we examined whether and how TRPV1 and/or TRPV4 are involved in DOMS. We firstly evaluated a method to measure the mechanical withdrawal threshold of the deep tissues in wild-type (WT) mice with a modified Randall-Selitto apparatus. WT, TRPV1−/− and TRPV4−/− mice were then subjected to LC. Another group of mice received injection of murine NGF-2.5S or GDNF to the lateral gastrocnemius (LGC) muscle. Before and after these treatments the mechanical withdrawal threshold of LGC was evaluated. The change in expression of NGF, GDNF and COX-2 mRNA in the muscle was examined using real-time RT-PCR. In WT mice, mechanical hyperalgesia was observed 6–24 h after LC and 1–24 h after NGF and GDNF injection. LC induced mechanical hyperalgesia neither in TRPV1−/− nor in TRPV4−/− mice. NGF injection induced mechanical hyperalgesia in WT and TRPV4−/− mice but not in TRPV1−/− mice. GDNF injection induced mechanical hyperalgesia in WT but neither in TRPV1−/− nor in TRPV4−/− mice. Expression of NGF and COX-2 mRNA was significantly increased 3 h after LC in all genotypes. However, GDNF mRNA did not increase in TRPV4−/− mice. These results suggest that TRPV1 contributes to DOMS downstream (possibly at nociceptors) of NGF and GDNF, while TRPV4 is located downstream of GDNF and possibly also in the process of GDNF up-regulation.