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

Clinico-pathological features of cervical lesions.

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

Immunohistochemical staining patterns of ROBO1, ROBO2 and SLIT2.

<p>(<b>A</b>) In normal cervical epithelium the basal and parabasal layer stained intensely for all the three proteins, whereas the intensity and frequency of stained cells reduced with further differentiation in the spinous layer. (<b>B</b>) In primary CACX expression pattern of these proteins were concordant with respective molecular alterations. (<b>C</b>) In SiHa cells ROBO1 and ROBO2 were membrane localized, whereas SLIT2 was present mostly in the cytoplasm. T: primary CACX sample; scale bars for both 20X and 40X is 50 µm; original magnifications are indicated in parenthesis. Magnification of panel <b>C</b> is 40X.</p

Kaplan-Meier analysis of survival (up to 5 years) of CACX patients.

<p>Alterations of (<b>A</b>) <i>SLIT2</i> and/or <i>ROBO1</i> and (<b>B</b>) <i>SLIT2</i> and/or <i>ROBO2</i> ligand-receptor pairs were significantly associated with poor patient outcome [OS]. (<b>C</b>) Representation of Cox Multivariate analyses of genetic, clinical and etiological parameters in predicting outcome of CACX patients. N: total number of samples and P<0.05 denotes statistical significance.</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