Bright-field <i>in situ</i> hybridization detects gene alterations and viral infections useful for personalized management of cancer patients

<p><b>Introduction</b>: Bright-field <i>in situ</i> hybridization (ISH) methods detect gene alterations that may improve diagnostic precision and personalized management of cancer patients.</p> <p><b>Areas covered</b>: This review focuses on some bright-field ISH techniques for detection of gene amplification or viral infection that have already been introduced in tumor pathology, research and diagnostic practice. Other emerging ISH methods, for the detection of translocation, mRNA and microRNA have recently been developed and need both an optimization and analytical validation. The review also deals with their clinical applications and implications on the management of cancer patients.</p> <p><b>Expert commentary</b>: The technology of bright-field ISH applications has advanced significantly in the last decade. For example, an automated dual-color assay was developed as a clinical test for selecting cancer patients that are candidates for personalized therapy. Recently an emerging bright-field gene-protein assay has been developed. This method simultaneously detects the protein, gene and centromeric targets in the context of tissue morphology, and might be useful in assessing the <i>HER2</i> status particularly in equivocal cases or samples with heterogeneous tumors. The application of bright-field ISH methods has become the gold standard for the detection of tumor-associated viral infection as diagnostic or prognostic factors.</p

Simian virus 40 (SV40) genome and the two selected peptides from the early coding region employed in indirect ELISA.

<p>The upper panel represents a circular map of the SV40 genome with map units from 0 to 100 running in a clockwise direction (inner circle, black). The nucleotide (nt) sequence and numbers refer to the 5,243 nucleotide genome of SV40 strain 776 (<a href="http://www.ncbi.nlm.nih.gov/genome" target="_blank">http://www.ncbi.nlm.nih.gov/genome</a>). Ori marks the origin of viral DNA replication (0/5243 nt). SV40 early and late genes are transcribed in anti-clockwise and clockwise directions, respectively (gray arrows); numbers indicate nt. The large T antigen (T) and small t antigen (t) are encoded by the early region. T antigen exon 1 is encoded from 5,163–4,918 nt and exon 2 from 4,571–2,691 nt, with intron 1 from 4,917 to 4,572 nt. Small t antigen is encoded from 5,163 to 4,639 nt. Viral capsid proteins 1–3 are codified by the late region (VP1, 1,499–2,593 nt; VP2, 562–1,620 nt; and VP3, 916–1,620 nt). A portion of the early coding region is expanded at the bottom of the figure. The selected Tag peptides, Tag A and Tag D, are from exon 2; Tag A encompasses amino acids (a.a.) 669–689 (21 a.a.), and Tag D a.a. 659–682 (24 a.a.). These two Tag epitopes overlap from a.a. 669 to 682.</p

Structural characteristics of large T-antigen peptides.

<p>(A) Tag A and (B) Tag D linear peptides were characterized by secondary structure folding domains identified by PSIPRED analysis. (C and D) Tertiary structures of SV40 large T antigen computationally determined with I-TASSER modeling algorithm, with a C-score = –2. Tag A and D both show a partial overlapping localization near the N-terminal native protein domain (magenta sticks in panel C and D, respectively). (E and F) Mesh representation of the large T antigen surface (N-terminal portion) on which Tag A and Tag D are mapped.</p

Polyomavirus phylogenetic tree based on the large Tag a.a. sequences.

<p>The similarity of large Tag sequences among different polyomaviruses is shown. Note that SV40 (simian virus 40) Tag is more closely related to those of JCV (JCPyV, JC polyomavirus), SA12 (simian agent virus 12), and BKV (BKPyV, BK polyomavirus) than to the Tags of other polyomaviruses: KIV (KIPyV, KI polyomavirus), WUV (WUPyV, WU polyomavirus), HPyV11/STLPyV (human polyomavirus 11), HPyV10 (human polyomavirus 10), HPyV7 (human polyomavirus 7), HPyV6 (human polyomavirus 6), HPyV9 (human polyomavirus 9), LPV/AGMPyV (B-lymphotropic polyomavirus), TSV (TSPyV, Trichodysplasia spinulosa-associated polyomavirus), MCPyV (Merkel cell polyomavirus), HPyV12 (human polyomavirus 12), and NJPyV (New Jersey polyomavirus, not shown) (<a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id510624" target="_blank">http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id510624</a>).</p

Similarity among SV40-specific Tag mimotopes (Pep Tag A and Pep Tag D) and other polyomavirus Tag sequences, Panel A and B, respectively.

<p>SV40 (simian virus 40) Tag sequences were compared to the Tags of the following polyomaviruses: BKV (BKPyV, BK polyomavirus), HPyV6 (human polyomavirus 6), HPyV7 (human polyomavirus 7), HPyV9 (human polyomavirus 9), HPyV10 (human polyomavirus 10), HPyV11/STLPyV (human polyomavirus 11), HPyV12 (human polyomavirus 12), JCV (JCPyV, JC polyomavirus), KIV (KIPyV, KI polyomavirus), LPV/AGMPyV (B-lymphotropic polyomavirus), MCV (MCPyV, Merkel cell polyomavirus), SA12 (simian agent virus 12), TSV (TSPyV, Trichodysplasia spinulosa-associated polyomavirus), WUV (WUPyV, WU polyomavirus), and NJPyV (New Jersey polyomavirus, not shown). (<a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10624" target="_blank">http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10624</a>).</p

Intra-run and inter-run variability of OD values of human serum antibody reactivity to Tag A and D peptides.

<p>Data are presented as scatter dot plot of OD readings at λ 405 nm, mean and standard error of the mean (SEM) marked by short horizontal lines for each peptide. (A) OD value variability, intra-run. Tag A: mean = 0.19, SEM = 0.006; Tag D: mean = 0.18; SEM = 0.005. (B) OD value variability, inter-run. Tag A: mean = 0.18, SEM = 0.005; Tag D: mean = 0.19, SEM = 0.005.</p

KIR gene profiles in our study populations.

<p>A total of 42 KIR gene profiles were identified (panel A). Genotype ID number reported are those from the reference database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.ref028" target="_blank">28</a>]. The presence of KIR genes is indicated by grey shanding. Genotypes AA and BX according to criteria reported in material and method section are indicated in the first column.°Only ID Genotypes present in at least two patients were reported in tables. Distribution of genotype ID1 and ID4 for HCV-negative patients and for patients with HCV infection and different outcomes (panel B). Distribution of KIR3DL1, KIR2DL3, KIR2DS4, KIR2DS3 genes ID4 for HCV-negative patients and for patients with HCV infection and different outcomes (panel C). * Fisher’s Exact test, p<0.05.</p

Distribution of KIR3DL1 gene and combination with HLA-B for patients with different HCV related disease outcomes.

<p>It is showed a negative trend association between the KIR3DL1/HLA-Bw4 with the presence of HCV-related lymphoma.</p

Centromeric and telomeric halves of KIR genotypes (panel A).

<p>A stretch of 14 kb DNA that interconnects KIR3DP1 and KIR2DL4 divides the KIR genotype into two halves. The centromeric half is delimited by 3DL3 and 3DP1, while the telomeric half is delimited by 2DL4 and 3DL2. There is different KIR gene content, due to a recombination of these genes, in KIR genotypes across individuals and populations. The framework genes, present in all genotypes are shown in grey boxes; genes encoding activating KIR are in red color; and those for inhibitory receptors are in blue color. KIR2DL4 encodes a receptor that has both inhibitory and activating functions The KIR2DP1 and 3DP1 (green) are pseudogenes that do not express a receptor. <b>Pairwise D’ LD based on Cramer’s V correlation coefficient between the presence and absence of different KIR genes in four groups of patients (panel B-E)</b> B: HCV negative; C: Chronic HCV; D: Hepatocellular carcinoma; E: Lymphoproliferative disease. The KIR cluster genetic polymorphism is considered as the presence or absence of KIR genes.</p

Cent/Tel KIR gene profiles in HCV-negative patients and patients with HCV infection and different outcomes.

<p>Distribution of Cent2 and Cent6 locus (panel A, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t002" target="_blank">Table 2</a>). Distribution of Tel3 and Tel6 loci (panel B, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t002" target="_blank">Table 2</a>). Distribution of Cent/Tel1 loci (panel C, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t002" target="_blank">Table 2</a>). Distribution of Cent/Tel1 with A/B KIR2DL5 variant (panel D, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t003" target="_blank">Table 3</a>). Distribution of Cent-2DS3/5 (1) loci (panel E, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t003" target="_blank">Table 3</a>). Distribution of Tel-2DS4 (3) AND Tel-2DS4 (8) locus (panel F, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117420#pone.0117420.t003" target="_blank">Table 3</a>). * Fisherx’s Exact test, p<0.05.</p