Sadhana Kannan

Three-dimensional conformal radiotherapy (3D-CRT) versus intensity modulated radiation therapy (IMRT) in squamous cell carcinoma of the head and neck: A randomized controlled trial

PURPOSE To compare three-dimensional conformal radiotherapy (3D-CRT) with intensity modulated radiation therapy (IMRT) in curative-intent irradiation of head-neck squamous cell carcinoma (HNSCC). METHODS Previously untreated patients with biopsy-proven squamous carcinoma of oropharynx, larynx, or hypopharynx (T1-3, N0-2b) were randomly assigned using computer-generated permuted-block design to either 3D-CRT or IMRT, with incidence of physician-rated Radiation Therapy Oncology Group (RTOG) grade 2 or worse acute salivary gland toxicity as primary end-point. RESULTS Between 2005 and 2008, 60 patients randomly allocated to either 3D-CRT (n=28 patients) or IMRT (n=32) were included and analyzed on an intention-to-treat basis. The proportion [95% confidence intervals (CI)] of patients with RTOG grade 2 or worse acute salivary gland toxicity was significantly lesser in the IMRT arm [19 of 32 patients (59%, 95%CI: 42-75%)] as compared to 3D-CRT [25 of 28 patients (89%, 95%CI: 72-97%; p=0.009)]. Late xerostomia and subcutaneous fibrosis were also significantly lesser with IMRT. There was significant recovery of salivary function over time in patients treated with IMRT (p-value for trend=0.0036). At 3-years, there were no significant differences in loco-regional control or survival between the two arms. CONCLUSION IMRT significantly reduces the incidence and severity of xerostomia compared to 3D-CRT in curative-intent irradiation of HNSCC.

Distinctive microRNA signature of medulloblastomas associated with the WNT signaling pathway

AIM Medulloblastoma is a malignant brain tumor that occurs predominantly in children. Current risk stratification based on clinical parameters is inadequate for accurate prognostication. MicroRNA expression is known to be deregulated in various cancers and has been found to be useful in predicting tumor behavior. In order to get a better understanding of medulloblastoma biology, miRNA profiling of medulloblastomas was carried out in parallel with expression profiling of protein-coding genes. MATERIALS AND METHODS miRNA profiling of medulloblastomas was carried out using Taqman Low Density Array v 1.0 having 365 human microRNAs. In parallel, genome-wide expression profiling of protein-coding genes was carried out using Affymetrix gene 1.0 ST arrays. RESULTS Both the profiling studies identified four molecular subtypes of medulloblastomas. Expression levels of select protein-coding genes and miRNAs could classify an independent set of medulloblastomas. Twelve of 31 medulloblastomas were found to overexpress genes belonging to the canonical WNT signaling pathway and carry a mutation in CTNNB1 gene. A number of miRNAs like miR-193a, miR-224/miR-452 cluster, miR-182/miR-183/miR-96 cluster, and miR-148a having potential tumor/metastasis suppressive activity were found to be overexpressed in the WNT signaling associated medulloblastomas. Exogenous expression of miR-193a and miR-224, two miRNAs that have the highest WNT pathway specific upregulation, was found to inhibit proliferation, increase radiation sensitivity and reduce anchorage-independent growth of medulloblastoma cells. CONCLUSION Expression level of tumor/metastasis suppressive miRNAs in the WNT signaling associated medulloblastomas is likely to determine their response to treatment, and thus, these miRNAs would be important biomarkers for risk stratification within the WNT signaling associated medulloblastomas.

Phenotypic alterations in Rb pathway have more prognostic influence than p53 pathway proteins in oral carcinoma

The two well-defined pathways that are shown to be prominently altered in a variety of cancers are the cell cycle regulatory pathways led by either p53 or Rb genes. The present study is undertaken to find the pathway that is more altered in oral carcinoma at protein level, with special emphasis on its prognostic significance. The expression pattern of key molecules of the Rb and p53 pathways, such as Rb, cyclin D1, CDK4, p16, p53, p21 and Bcl-2 and the proliferative marker PCNA were analysed in 348 oral carcinoma specimens by immunohistochemical technique. The expression index of these molecules and various clinicopathological factors were statistically correlated with treatment end points to assess its prognostic efficacy after following up these patients up to a maximum of 48 months with a median of 23 months. Rb pathway proteins, Rb (P=0.016), cyclin D1 (P=0.0001) and p16 (P=0.012) showed significant association with disease-free survival, and p16 (P=0.041) and cyclin D1 (P=<0.0001) with the overall survival. Among p53 pathway proteins studied, only p53 expression index showed association with both disease-free survival and overall survival. Multivariate analyses confirmed that the biological variables, cyclin D1 and p16 and the clinical variable, ‘stage of disease’ were independent predictors of disease-free survival and overall survival. Subgrouping of the patients on the basis of p16 and cyclin D1 expression revealed that the subgroup having downregulation of p16 and overexpression of cyclin D1 exhibited the worst disease-free survival and overall survival compared to the other subgroups. The present data showed that disabling of the Rb and p53 pathways were frequent events in oral carcinoma. The study also demonstrated that the Rb pathway proteins are comparatively more important than p53 pathway proteins for the prognostication of oral carcinoma patients. The combined evaluation of p16 and cyclin D1 in oral carcinoma could identify a group of patients with the worst survival who might therefore need alternate or more intense treatment strategies.

p53, p16 and cyclin D1: molecular determinants of radiotherapy treatment response in oral carcinoma.

Management of oral cancer by radiotherapy has witnessed promising advances in the past few years, with patient‐tailored radio fractionation regimens. Different fractionation schedules, conventional and altered regimes, have been used in curative radiotherapy. Although contribution of biological markers on radio response has been evaluated, its unique influence on various radio fractionation schemes has not been accounted so far. Our study analyses a set of proteins that previously demonstrated radio response influence for their possible prognostic value in decision‐making process between the respective fractionation schemes. Expression patterns of regulatory proteins such as p53, cyclin D1, p16, Cdk4, p21, Rb, bcl‐2 and PCNA were determined by immunohistochemistry utilizing monoclonal antibodies in 125 patients who received curative radiotherapy dose. Among these 125 patients, 90 (72%) received altered fractionation, whereas 35 (28%) received conventional fractionation. p53 over‐expression correlated with local treatment failure among the patients treated with conventional fractionation whereas cyclin D1 over‐expression and p16 underexpression were associated with local treatment failure as well as overall survival in altered fractionation treated cases. Our findings suggest that wild‐type p53 status may be an important parameter for achieving high local control in those patients undergoing conventional fractionation, where as intact p16 and cyclin D1 status may be beneficial for effective local control in patients who are treated with altered fractionation. Furthermore, it can be assumed that conventional fractionation employs p53‐mediated apoptosis, whereas altered fractionation activates the functional G1 cell‐cycle checkpoint for tumor growth suppression.

Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma.

Purpose: Clinico-epidemiological studies show that the behaviour of the tongue cancer is different from the cancer originating at other sites of the oral cavity. However, studies identifying the reason for such difference are lacking in the literature. Methods: In the present study, we have attempted to see whether any difference existed in the cell cycle regulatory mechanism of these tumours by comparing immunohistochemically the expression of major cell cycle regulatory proteins in 147 buccal and 94 tongue carcinoma (anterior two-third of tongue) prospectively. Results: On comparison of buccal and tongue carcinoma, expression of p16 and p21 showed significant difference. In combined analysis, simultaneous down regulation of p16 and p21 was seen in 47% of tongue cancer cases as against 28% in buccal carcinoma (P=0.004). In univariate analysis, none of the clinico-biological variables studied showed significant association with survival in tongue carcinoma, whereas, some of the clinico-biological variables associated with survival in buccal carcinoma. Among the biological markers, the overexpression of cyclin D1 (P=0.007) and p53, detected using both the clones of antibodies—DO7 (P=0.008) and PAb240 (P=0.014) and the down regulation of p16 (0.033), showed significant association with shorter disease free survival (DFS) in these cases. Whereas in the case of overall survival (OS), overexpression of p53 [DO7 (P=0.031) and PAb240 (P=0.017)] and cyclin D1 (P=0.001) associated with poor survival. In multivariate analysis, the expression pattern of p53 and p16 protein influences the DFS whereas cyclin D1 expression showed independent association with the OS in buccal carcinoma. Conclusions: Thus, tongue and buccal cancers represent different biological subentities, and such differences should be considered in oral cancer management.

H-Ras mutation modulates the expression of major cell cycle regulatory proteins and disease prognosis in oral carcinoma

Activating mutations of the Ras is a moderately frequent event in oral carcinogenesis in Indian patients. Ras pathway has essential roles in regulation of various phases of the cell cycle, especially at G1 phase. Despite a large body of in vitro evidence, the multidimensional interaction between mutated Ras pathway and G1 cell cycle regulatory proteins in tumours in vivo is poorly determined. In the present study, DNA samples were screened for mutations in hot spot exons of B-Raf and hot spot codons 12, 13 and 61 of H-, K- and N-Ras by PCR-SSCP. Mutations were confirmed by direct sequencing. Expression of G1 cell cycle regulatory proteins—cyclin D1, CDK4, Rb, p53, p16 and p21 and proliferation marker PCNA was analysed immunohistochemically. The results revealed the absence of B-Raf mutations in oral carcinoma in spite of 12.5% of the samples showing H-Ras mutation. The H-Ras mutant cases showed significantly low cyclin D1 (P=0.027) and CDK4 (P=0.046) expression and overexpression of Rb (P=0.011) and p16 (P=0.026). H-Ras mutant carriers also had significantly high recurrence-free survival (P=0.033). In summary the present study demonstrated an epistatic interaction between H-Ras mutation and G1 cell cycle regulatory proteins in vivo. H-Ras mutation, thus, defines a molecular subtype of oral carcinoma with favourable outcome and unique biology.

Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular classification of formalin-fixed paraffin embedded medulloblastomas

BACKGROUND Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers. METHODS Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay. RESULTS The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P < .0001) overexpression of miR-193a-3p, miR-224, miR-148a, miR-23b, and miR-365. Reliable classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification. CONCLUSIONS The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.

Influence of genetic polymorphisms on frequency of micronucleated buccal epithelial cells in leukoplakia patients

Micronuclei (MN) are extensively used as an indicator of chromosomal damage and early biomarker of cancer risk. The genetic host factors are known to influence the level of chromosomal alterations consequently affecting MN frequencies. Hence, in the present study, we investigated the extent of chromosomal damage by analyzing micronucleated cell (MNC) frequency in exfoliated buccal epithelial cells (BEC) and its possible relationship with genetic polymorphisms in patients with oral leukoplakia (OL). The study group comprised of habit-free (NHC, n=39), habit controls (HC, n=62) and OL patients (n=66). Micronucleus assay was carried out to determine the MNC frequency and the genotyping was performed by PCR-RFLP for metabolizing (CYP1A1, GSTM1, GSTT1, GSTP1) and DNA repair (hOGG1, XRCC1, XPD) genes. The correlation between MNC frequency and genetic polymorphisms was analyzed. We found significant increase in overall MNC frequency in OL patients as compared to habit-matched controls (p=0.01). The higher proportion of multiple micronucleated cells (>5 MN per cell) indicate increased DNA damage in the buccal mucosa of OL patients than the controls (p=0.004). The polymorphic alleles of XPD751 and hOGG1 showed significant association with total MNC frequency in OLs (p=0.034 and p=0.03 respectively). In conclusion, the extent of chromosomal damage in target tissues is higher in patients with OL. MNC frequency in combination with genetic polymorphisms in DNA repair genes may serve as better predicator of risk.

Cyclophosphamide plus total body irradiation compared with busulfan plus cyclophosphamide as a conditioning regimen prior to hematopoietic stem cell transplantation in patients with leukemia: a systematic review and meta-analysis

BACKGROUND AND OBJECTIVES Cyclophosphamide plus total body irradiation (CYTBI) and oral busulfan plus cyclophosphamide (BUCY) are commonly used conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (HSCT) in patients with leukemia. However, there is conflicting data on the superiority of one regimen over the other. Our aim was to critically appraise and synthesize available evidence regarding the efficacy and safety of CYTBI compared to BUCY as a conditioning regimen. DESIGN AND SETTING Systematic review and meta-analysis of randomized, controlled trials (RCTs) comparing BUCY with CYTBI. METHODS We did a systematic search of the indexed medical literature using appropriate keywords to identify potentially relevant articles. The primary outcome of interest was efficacy measured by overall survival (OS) and disease-free survival (DFS). Acute and late toxicity were secondary endpoints. Meta-analysis was attempted only on RCTs. A relative risk or risk ratio (RR) and 95% confidence interval (CI) was calculated for each outcome in the meta-analysis. RESULTS Fifteen non-randomized comparative studies involving 6280 patients were included in a narrative review without attempting a pooled analysis, in view of the potential for significant bias. Outcome data from seven RCTs involving 730 patients randomly assigned to either CYTBI or BUCY was pooled using meta-analytic methods. CYTBI was associated with a modest but non-significant reduction in all cause mortality (RR=0.82, 95%CI: 0.64-1.05; P=.12) and relapse of leukemia (RR=0.89, 95%CI: 0.72-1.10; P=.28). Transplant-related mortality (TRM) was significantly lesser with CYTBI compared to oral BUCY (RR-0.53, 95%CI: 0.31-0.90; P=.02). The cumulative incidence of major complications was not significantly different between the two regimens, but specific complications varied according to the conditioning regimen. TBI-based regimens were associated with more severe late effects on growth and development in children. CONCLUSION This analysis represents the largest comparative analyses of CYTBI with BUCY as a conditioning regimen prior to HSCT in the indexed medical literature. Conditioning regimen and disease (type and setting) can significantly affect outcomes. TRM is significantly lesser with CYTBI, but this does not translate into a significant survival benefit. There remain valid concerns regarding the late effects of TBI, particularly in children. Although not overly superior, the weight of evidence favors CYTBI over BUCY as a first choice-conditioning regimen in patients with leukemia.

Significance of AgNOR Count in Differentiating Malignant Cells from Reactive Mesothelial Cells in Serous Effusions

OBJECTIVE To distinguish reactive mesothelial cells from malignant cells in serous effusions using silver staining of nucleolar organizer regions (AgNOR) applied to ethanol-fixed cytologic preparations. STUDY DESIGN One hundred aspirated samples of benign and malignant effusions were studied using the one-step silver staining method. Eight cytologically atypical samples were also included in the study. RESULTS In malignant cells the mean AgNOR count was 4.72 +/- 0.76 (+/- SD), and the AgNORs were irregular in shape, while in benign mesothelial cells AgNORs were comparatively larger, single dots with a mean count of 1.92 +/- 0.23. Of the cytologically atypical samples, five were in the malignant range. The others were within benign limits. Repeat cytology of the second aspirate confirmed that finding. CONCLUSION AgNOR study appears to be clinically useful as an additional diagnostic tool for use in ascitic and pleural fluid samples when the cytologic diagnosis is difficult.