Inactivation of SLIT2-ROBO1/2 Pathway in Premalignant Lesions of Uterine Cervix: Clinical and Prognostic Significances

The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion: ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation: SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease

Domestication technique of commercially important freshwater mud eel, Monopterus cuchia (Hamilton, 1822)

The research was conducted to develop the domestication technique of Monopterus cuchia collected from Sherpur district, Bangladesh. A total of 300 young M. cuchia (average 95±6.4 g) was reared under nine treatments as earthen pond with dead fish (EpDf), earthen pond with live fish (EpLf), earthen pond with formulated feeds (EpFf), mud treated plastic tank with dead fish (PmDf), mud treated plastic tank with live fish (PmLf), mud treated plastic tank with formulated feeds (PmFf), plastic tank with dead fish (PtDf), plastic tank with live fish (PtLf), and plastic tank with formulated feeds (PtFf) with three replicates each. Size of each earthen pond was 1m3 and plastic circular tank was 0.322m3. Stocking density was maintained at 10 fish in each replicate by random selection. Survival, weight gain (WG), specific growth rate (SGR%/day), feed conversion ratio (FCR), feed conversion efficiency (FCE), and protein efficiency ratio (PER) were measured. The best survival (100±0), WG (61.17±1.83), SGR (0.118±0.026), FCR (2.44±0.17), FCE (35.75%±2.90), and PER (1.75±0.14) were obtained in EpLf, which was significantly higher (P<0.05) than others. This is the first report of domestication of M. cuchia and indicates that earthen pond with live fish might be suitable for domestication of M. cuchia

RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic significance

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4–9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) &#x003E; MLH1 (46%) &#x003E; RBSP3 (45%) &#x003E; ITGA9 (41%), methylation: RBSP3 (25%) &#x003E; ITGA9 (24%) &#x003E; STAC (19%) &#x003E; MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN → Stage I/II and for ITGA9 from CIN → Stage I/II and also from Stage I/II → Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (&#x2265;5)/early sexual debut (#x2264;19 years) coupled with RBSP3 alterations/RB1 deletion predicted worst prognosis. Thus, inactivation of RBSP3 might be one of the early events in cervical carcinogenesis

Representative autoradiographs showing deletion and microsatellite size alteration (MA) of cervical lesions, at different marker loci.

<p>(i) LOH: loss of heterozygosity, (ii) MA-1: microsatellite size alteration of one allele. (iii) LOH + MA: loss of one allele and microsatellite size alteration of the other. (iv) Hemizygous (HE) deletion of <i>ROBO2</i> locus as shown by D3S2515. (v) & (vi) HE deletion as shown by exonic markers (EM) from <i>ROBO1</i> and <i>ROBO2</i> respectively, <i>SST</i> used as control. The sample numbers and marker loci are indicated above and below the figure respectively. →: allelic loss, “*”: allelic size alteration. Deletion of <i>ROBO1/2</i> and <i>SLIT2</i> analyzed by microsatellite and exonic markers in (<b>B</b>) CIN and (<b>C</b>) CACX. T: Tumor DNA, N: DNA from normal cervix/PBL. (<b>D</b>) Pattern of deletion and methylation of <i>ROBO1/2</i> and <i>SLIT2</i> during disease progression. Asterisk denotes statistical significance (P<0.05). (<b>E</b>) Overall alteration patterns of the individual genes, <i>SLIT2-ROBO1</i> and <i>SLIT2-ROBO2</i> ligand-receptor pairs, during disease progression. Asterisk denotes statistical significance (P<0.05).</p

Correlation between deletion/methylation and reduced expression (RNA/protein) of <i>ROBO1/2</i> and <i>SLIT2</i> in CIN/CACX.

<p>Samples C1, C2 and C3 are CIN lesions.</p><p>D+/−, Deletion (HE, HM, LOH) positive/negative; M+/−, methylation positive/negative; Del/Meth: Deletion or methylation. ↓: Reduced mRNA expression (≥2 folds); ↑: Increased mRNA expression (≥2 folds); -: Reduced/Increased mRNA expression (<2 folds). nd, not done due to insufficient/scanty paraffin embedded tumor tissue; ND, Fresh tissues unavailable for RNA isolation. *, statistically significant (<i>P</i><0.05).</p

Clinico-pathological features of cervical lesions.

§<p>According to The international federation of gynecology and obstetrics (FIGO) classification.</p