Complementary role of p57kip2 immunostaining in diagnosing hydatidiform mole subtypes.

OBJECTIVES: Gestational trophoblastic disease comprises of a spectrum of pregnancy-related tumours which includes complete (CHM) and partial hydatidiform moles (PHM). Accurate diagnosis and subclassification of HM subtypes are crucial as prognosis differs. Histopathological examination using haemotoxylin and eosin (H&E) staining remains the basis for diagnosing HM, with only 80% accuracy. p57kip2 is a cyclin-dependent kinase inhibitor (CDKI) protein and is strongly paternally imprinted, being expressed from maternal allele. Therefore, complete mole (CHM) with only paternal genome has nearly absent expression of p57kip2 compared to partial mole (PHM) having both paternal and maternal genomes. This study is aimed to determine usefulness of p57kip2 immunohistochemistry (IHC) analysis in the diagnosis of HM subtypes. METHODS: A total of 82 archived paraffin embedded HM tissues with subtypes classified based on H&E staining - 39 (47.5%) CHM, 41 (50.0%) PHM and two (2.43%) unclassified molar pregnancy were retrieved. All tissue samples were subjected for p57kip2 IHC analysis and HM subtypes were then reclassified. RESULTS: A total of 66 cases (80.5%) were re-classified as CHM, 14 cases (17.1%) as PHM and two cases (2.4%) were decidual and cystic tissues. Analysis using p57kip2 immunostaining showed a diagnostic discrepancy of 33.0% from routine H&E staining and helps to improve the characterisation of the HM subtypes specifically at early gestations which have less distinctive morphologies. CONCLUSIONS: IHC using p57kip2 monoclonal antibody should be considered as a routine ancillary test to H&E in improving the diagnosis of HM subtypes particularly in developing countries with limited resources.

Helicobacter pylori cagA gene variants in Malaysians of different ethnicity.

We have defined DNA repeat variability in the 3'-terminus of the cagA gene of Helicobacter pylori strains from Malaysian patients of different ethnicities. We identified different alleles based on the EPIYA repeats. cagA types A-B-D and A-B-B-D are more similar to the sequence of Japanese strains, whereas cagA types A-B-C, A-B-C-C, A-B and A-C displayed similarity to strain 26695 sequences. A significant association was found between cagA genotypes and patients' ethnicity, with cagA type A-B-D being predominantly isolated from Chinese patients and cagA type A-B-C from Malays and Indians. Our data further corroborate the possibility that variant biological activity of CagA may affect the host specificity and/or pathogenicity of H. pylori.

Identification of Predictive DNA Methylation Biomarkers for Chemotherapy Response in Colorectal Cancer.

Resistance to 5-Fluorouracil (5-FU) is a major obstacle to the successful treatment of colorectal cancer (CRC) and posed an increased risk of recurrence. DNA methylation has been suggested as one of the underlying mechanisms for recurrent disease and its contribution to the development of drug resistance remains to be clarified. This study aimed to determine the methylation phenotype in CRC for identification of predictive markers for chemotherapy response. We performed DNA methylation profiling on 43 non-recurrent and five recurrent CRC patients using the Illumina Infinium HumanMethylation450 Beadchip assay. In addition, CRC cells with different genetic backgrounds, response to 5-FU and global methylation levels (HT29 and SW48) were treated with 5-FU and DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC). The singular and combined effects of these two drug classes on cell viability and global methylation profiles were investigated. Our genome-wide methylation study on the clinical specimens showed that recurrent CRCs exhibited higher methylation levels compared to non-recurrent CRCs. We identified 4787 significantly differentially methylated genes (P < 0.05); 3112 genes were hyper- while 1675 genes were hypomethylated in the recurrent group compared to the non-recurrent. Fifty eight and 47 of the significantly hypermethylated and hypomethylated genes have an absolute recurrent/non-recurrent methylation difference of ≥20%. Most of the hypermethylated genes were involved in the MAPK signaling pathway which is a key regulator for apoptosis while the hypomethylated genes were involved in the PI3K-AKT signaling pathway and proliferation process. We also demonstrate that 5-azadC treatment enhanced response to 5-FU which resulted in significant growth inhibition compared to 5-FU alone in hypermethylated cell lines SW48. In conclusion, we found the evidence of five potentially biologically important genes in recurrent CRCs that could possibly serve as a new potential therapeutic targets for patients with chemoresistance. We postulate that aberrant methylation of CCNEI, CCNDBP1, PON3, DDX43, and CHL1 in CRC might be associated with the recurrence of CRC and 5-azadC-mediated restoration of 5-FU sensitivity is mediated at least in part by MAPK signaling pathway.

Molecular Characterization of Somatic Alterations in Dukes' B and C Colorectal Cancers by Targeted Sequencing.

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes' B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes' B (n = 10) and Dukes' C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes' B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes' B and C). In addition, TP53 was more frequently altered in Dukes' C (n = 7) compared to Dukes' B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-ß, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape.

Surgeons' Evaluation of Colorectal Cancer Resections Against Standard HPE Protocol-Auditing the Surgeons.

The survival of Colorectal Cancer patients is very much dependent on complete tumor resection and multimodality adjuvant treatment. However, the main determinants for management plan of these patients rely heavily on accurate staging through histopathological examination (HPE). A reliable standard HPE protocol will be a significant impact in determining best surgical outcome. We evaluate surgeons' intra-operative judgment and the quality of resected specimens in the treatment of colorectal cancers. To quantify the quality of surgery by applying standard HPE protocol in colorectal cancer specimens and to assess the use of new format for pathological reporting in Colorectal Cancer using a formulated standard proforma. We perform a prospective observation of all colorectal cancer patients who underwent surgical resection over 8 month duration. Surgeons are required to make self-assessment about completion of tumor excision and possible lymph nodes or adjacent organ involvement while all pathologists followed standard reporting protocol for examination of the specimens. We evaluate the accuracy of surgeons judgment against HPE. The study involved 44 colorectal cancers comprising of 23 male and 21 female patients. The majority of these patients were Malay (50%) followed by Chinese (43%) and Indian (7%). The main presenting symptoms were bleeding (32%), intestinal obstruction (29%) and perforation (7%). Sixteen (36%) patients underwent emergency surgery.Rectal tumor was the commonest (53%) followed by sigmoid colon (22.7%). Neoadjuvant Chemoradiation were given to 8 patients and complete pathological response was observed in 1 (12.5%) of these. The final TNM classification for staging were; stage I (22.7%), stage IIa (18.2%), stage IIb (11.4%), stage IIIa (2.3%), stage IIIb (25%), stage IIIc (13.6%) and stage IV (6.8%).The commonest surgery performed was anterior resection with mesorectal excision (43.2%). Ten patients (22.7%) had laparoscopic surgery with 3 (30%) patients converted to open surgery. The surgeons claimed to have performed a curative resection with complete excision and clear margin in 40 (90%) patients. Of these, only 1 (2.5%) patient had a positive resection margin. Meanwhile, the surgeons reported involvement of resection margins in 4 cases but this was disputed by the HPE in all 4 cases. Lymph nodes involvement was detected intra-operatively in 13 (29.5%) of the cases and all were proven positive for metastases through HPE. On the other hand, of the remaining 31 patients who were reported as no obvious lymphadenopathy by the surgeons, lymph nodes positvity was found in 7 (22.5%) cases. Using standard HPE reporting protocol brings suitable evaluation of surgery in colorectal cancer treatment. Although surgeons' judgment is fairly accurate in predicting margin clearance and complete specimen excision, complete mesocolic and mesorectal excision is of utmost importance since lymph nodes metastatic involvement may not be obvious at surgery.