Morphologic analysis of false negative SurePath® slides using Focalpoint™ GS computer-assisted cervical screening technology: An Australian experience.

BACKGROUND: The trial results of BD FocalPoint™ GS computer assisted screening(FP) of BD SurePath(®) liquid based cervical cytology slides (SP) were published in Diagnostic Cytopathology in 2012.(1) METHOD: The FP-reviewed SP slides were compared to conventional cervical Pap smears (CON) in a split sample of 2198 routine specimens. In all 47 confirmed high grade (HG) cases, FP either selected fields of view (FOV) containing cells suspicious or diagnostic of HG (46/47), or prompted full screening of the slide (1/47) leading to detection of the HG lesion. In this study for the HG cases which were reported initially as negative (NEG) or low grade (LG), the original slides were reviewed to determine the reasons for the false negative reporting and to identify features characteristic of a HG pattern. RESULTS: Indicators of a high risk pattern for HG Squamous Intraepithelial lesions (HSIL) included the recognition of pale abnormal immature metaplastic cells. Indicators of a high risk pattern for HG Glandular Intraepithelial lesion (AIS) were single columnar cells with the nucleus occupying the widest area of the cell. Pavemented sheets of glandular cells and isolated mitoses were additional clues to the diagnosis. In endocervical and endometrial adenocarcinomas tumour diathesis tends to be absent in SurePath slides. Single cells with ingested neutrophils and obscured eccentric nuclei were another clue to the detection of endometrial adenocarcinoma. CONCLUSION: These morphological features are illustrated to help identify HSIL and HG glandular lesions when viewing the FOV presented by the FocalPoint(TM) GS technology.

Results of an Australian trial using SurePath liquid-based cervical cytology with FocalPoint computer-assisted screening technology.

BD FocalPoint GS™ computer-assisted screening of BD SurePath® liquid-based cervical cytology slides (SP + FP) was compared with screening an accompanying conventional cervical Papanicolaou (Pap) smear (CON) in a split sample trial of 2,198 routine specimens. The rate of unsatisfactory specimens in the SP + FP arm was 0.2% compared with 4.1% in the conventional Pap smear, a significant reduction. There was no statistically significant difference between SP + FP and CON for the detection of histologically confirmed high-grade (HG) lesions in the routine split sample specimens (n = 9). To further test the sensitivity of SP + FP for HG lesions, 38 SurePath slides from confirmed HG cases, without an accompanying CON, were interpolated among the routine smears. In every one of the 47 confirmed HG cases, either HG cells were present in the microscope fields selected by FocalPointGS™ for review by the screening cytologist (46 of 47), or full screening of the slide was indicated by the FocalPointGS™ (1 of 47), confirming the effectiveness of SP + FP technology for primary screening. In a small number of cases, the screening cytologist did not recognize the abnormality even though on review HG cells were present in fields selected by FocalPointGS™. The overall detection rate was 93% for HG squamous lesions; 89% for known HG endocervical glandular lesions; and 91% for known endometrial carcinoma. In conclusion, the SP + FP detected 100% of HG abnormalities in the trial set; significantly reduced the rate of unsatisfactory specimens; and improved the overall screening rate of detection of HG abnormalities particularly of glandular lesions when compared with other screening technologies.

Assuring the quality of quality assurance: seeding abnormal slides into the negative Papanicolaou smears that will be rapid rescreened.

BACKGROUND: Rapid rescreening (RR) of negative Papanicolaou smears (PS) is used in many countries as a quality-assurance measure. Seeding of abnormal slides has been suggested as a way to increase the sensitivity of this procedure. Since 2004, the authors have carried out RR with seeding before issuing reports. In this article, they describe their experience. METHODS: Abnormal seeds were sourced from the previous day's high-grade cases, both squamous and glandular. Slides were evaluated for the 'degree of difficulty' (which was defined as the number of fields required to find (fields-to-find [FTF]) the abnormality), relabeled, and redotted to make them indistinguishable from the routine RR work. The number of seeds found/missed, the identity of the screener, the type of seeded abnormality, the degree of difficulty of the seed, and the mapping technique used all were recorded. The cytologists also were surveyed about their views on seeding. RESULTS: Overall, 14.8% of 3082 high-grade seeds were missed during RR. There was no relation between seeds missed and the mapping technique used. However, the difficulty of the seed was relevant to the number missed and ranged from 8.3% when the FTF was <5 to 36% when the FTF was >10 (P = .000). The difference between intraepithelial seeds and invasive seeds was significant for squamous seeds (P = .031) but not for glandular seeds. Glandular seeds also were more likely to be missed than squamous seeds (23.1% vs 14.3%; P = .002). Most cytologists believed that seeding was a good idea and that seeds increased their level of vigilance. CONCLUSIONS: The authors' experience demonstrated that routine seeding is practicable for both conventional and liquid-based slides. With the advent of the human papillomavirus vaccine, abnormalities will become rarer, and seeding will be necessary to maintain the alertness of cytologists.

A three-armed trial of the ThinPrep Imaging System.

We compared the performance of the ThinPrep (TP) Imaging System (TIS) with manual reading of TP slides (TPM) and with manual reading of the paired conventional Pap smear (PS) in terms of sensitivity for and positive predictive value (PPV) of high-grade disease and productivity. The study consisted of 11,416 routine PS and paired TP slides as well as 103 confirmed abnormal TP slides. In terms of sensitivity for the detection of biopsy-confirmed high-grade disease, overall there was no statistically significant difference between TIS-screened TP (TPI) and TPM (81.1% vs. 86.8%). For the routine cases, TPI was significantly more sensitive than PS (73.4% vs. 57.8%). In terms of PPVs for the cytologic prediction of high-grade disease, there was no statistically, significant difference among TPI, TPM, and PS (75.6%, 73.9%, and 84.6%). For cytologic reports of possible high-grade disease, the PPVs were equivalent for TPI (45.0%) and TPM (37.0%) and there was no significant difference in PPVs between TPI and PS (61.3%). For TP slides, TIS screening showed a 27% productivity gain when compared with manual screening and a 54% productivity gain when compared with manual screening of PS slides. Use of TIS showed productivity benefits when compared with TPM and both productivity and sensitivity benefits over use of PS.

Follow-up of cytologic predictions of endocervical glandular abnormalities: histologic outcomes in 123 cases.

OBJECTIVES: To determine histologic positive predictive values (PPVs) for three categories of cytologic reports of endocervical glandular abnormalities. MATERIALS AND METHODS: We obtained histologic follow-up for 100% of 67 cytologic predictions of adenocarcinoma in situ (AIS) and 82% of 39 predictions of possible AIS (?AIS) made over a 4-year period (1999-2002) and for 25% of 105 atypical endocervical cells (AEC) predictions over a 12-month period (2000). For each category of cytologic report, we determined the histologic yields of high-grade lesions overall and of high-grade glandular lesions. RESULTS: PPVs for predictions of AIS and ?AIS for high-grade lesions overall were 91% and 75% (p = .032), respectively, and those for high-grade glandular lesions were 88% and 72% (p = .046), respectively. For a cytologic report of AEC, of those with histologic follow-up, 9% had a high-grade lesion and 7% had a high-grade glandular lesion. CONCLUSION: Cytology can accurately predict AIS.

ADAM-Integrin Interactions: potential integrin regulated ectodomain shedding activity.

ADAMs (a disintegrin and metalloprotease) are a family of cell surface proteins related to the Class III snake venom metalloproteases (SVMP). ADAMs are members of the Metazincin family which includes the matrix matalloproteases and the ADAMTS proteins. Unlike their snake venom relatives, ADAMs are expressed as transmembrane cell surface proteins. The domain structure of ADAMs suggests that these proteins posses both proteolytic and adhesive functions. Several members of the ADAM protein family have been shown to be involved in ectodomain shedding of many important cell surface proteins resulting in the release of biologically active soluble factors. The carboxyl-terminal domains, especially the disintegrin-like domain of ADAMs, have been demonstrated to support cell adhesion. The disintegrin-like domains of many ADAMs are capable of acting as integrin ligands. Integrins known to interact with ADAM disintegrin-like domains include alpha4beta1, alpha4beta7, alpha5beta1, alpha6beta1, alpha9beta1, alphavbeta3, and alphavbeta5. This integrin mediated interaction of the disintegrin-like domains with the cell surface suggests that ADAMs may function as cellular counter receptors. In this review we discuss the individual functions ascribed to members of the ADAM family especially those related to integrin interactions and the potential for integrin mediated regulation of ectodomain shedding.

ADAM disintegrin-like domain recognition by the lymphocyte integrins alpha4beta1 and alpha4beta7.

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin alpha4beta1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins alpha4beta1, alpha4beta7 and alpha9beta1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported alpha4beta1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin alpha4beta7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with alpha4beta7. All three disintegrin domains supported alpha9beta1-dependent cell adhesion. Recognition by both alpha4beta1 and alpha4beta7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins alpha4beta1, alpha4beta7 and alpha9beta1. ADAM33 exclusively supported only alpha9beta1-dependent adhesion.

Matrix-dependent plasticity of the malignant phenotype of bladder cancer cells.

The purpose of this study was to investigate the effect of cancer- and normal basement membrane-derived extracellular matrix to modulate the phenotype of bladder cancer cell lines. Five lines, varying in malignancy from papilloma to highly undifferentiated and invasive and immortalized human urothelial cells, were grown on two extracellular matrix preparations, Matrigel and SISgel. Matrigel represents matrix remodeled by malignancy while SISgel, obtained from small intestine submucosa (SIS), represents the normal matrix supporting differentiated cell growth. On Matrigel, regardless of the content of growth factors, the invasive lines displayed an invasive phenotype, while the low grade lines grew as papillary structures. In contrast, when the same cells were grown on SISgel, they grew as a layer of cells one to 5 cells thick, failed to invade, and expressed cell-surface E-cadherin. Unlike breast cancer cells, neutralization of beta 1, beta 4 and alpha 6 integrins altered cell-cell and cell-matrix adhesiveness but did not alter the phenotype. When invasive cells were grown on mixtures of SISgel and Matrigel, the phenotype changed gradually, not abruptly, indicating that factors within the gel reversibly alter the phenotypic expression of invasion. In summary, the phenotype of bladder cancer cells growing in tissue-like 3-dimensional culture is highly plastic, and malignant properties such as invasion and papillary growth can be suppressed by the matrix.

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain.

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.

In vitro selection of fibronectin gain-of-function mutations.

Directed protein evolution, which employs a combination of random mutagenesis, phage display, and in vitro selection, was used to identify second-site suppressors of the fibronectin (Fn) cell binding domain mutation Asp1495Ala (RGA). The mutations in the Fn 9th (3fn9) and 10th (3fn10) type III repeats obtained after selection on purified integrins alphaIIbbeta3(D119Y) and alpha5beta1 are reported. The 3fn9-10(D1495A) phage with substitution mutations at Asp1418, which is located within the linker region between 3fn9 and 3fn10, enhanced binding to the integrins alphaIIbbeta3 and alpha5beta1, but not alphavbeta3. The substitution mutations identified at residue Asp1418 were introduced into the native recombinant 3fn9-10 sequence and found to augment binding to alphaIIbbeta3, demonstrating that the observed gain-of-function phenotype was independent of the multivalent character of the phage. These results support the following conclusions. First, regions of Fn in addition to the RGD loop are in close proximity to alphaIIbbeta3 and alpha5beta1 and are capable of participating in the binding to these integrins. Secondly, the conformational relationship between the 3fn9 and 3fn10 modules may be an important factor in the binding of Fn to these two integrins. Thirdly, other altered properties of Fn-integrin interactions, such as integrin specificity, may also be selected. This is the first description of Fn mutations that augment binding to integrins. The ability to select for particular phenotypes in vitro and the subsequent characterization of these mutations should further our understanding of the molecular details involved in the association of integrins and their ligands. Additionally, these higher-affinity 3fn9-10 ligands provide a starting point for further in vitro evolution and engineering of integrin-specific modules.

The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1.

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.