SPAK Deficiency Attenuates Chemotherapy-Induced Intestinal Mucositis

IntroductionSte20-related protein proline/alanine-rich kinase (SPAK) affects cell proliferation, differentiation, and transformation, and sodium and chloride transport in the gut. However, its role in gut injury pathogenesis is unclear.ObjectiveWe determined the role of SPAK in chemotherapy-induced intestinal mucositis using in vivo and in vitro models.MethodsUsing SPAK-knockout (KO) mice, we evaluated the severity of intestinal mucositis induced by 5-fluorouracil (5-FU) by assessing body weight loss, histological changes in the intestinal mucosa, length of villi in the small intestine, pro-inflammatory cytokine levels, proliferative indices, and apoptotic indices. We also evaluated changes in gut permeability and tight junction-associated protein expression. Changes in cell permeability, proliferation, and apoptosis were assessed in SPAK siRNA-transfected 5FU-treated IEC-6 cells.Results5-FU-treated SPAK-KO mice exhibited milder intestinal mucositis, reduced pro-inflammatory cytokine expression, increased villus length, good maintenance of proliferative indices of villus cells, decreased apoptotic index of enterocytes, reduced gut permeability, and restoration of tight junction protein expression (vs. 5-FU-treated wild-type mice). Under in vitro conditions, siRNA-mediated SPAK-knockdown in IEC-6 cells decreased cell permeability and maintained homeostasis following 5-FU treatment.ConclusionSPAK deficiency attenuated chemotherapy-induced intestinal mucositis by modulating gut permeability and tight junction-associated protein expression and maintaining gut homeostasis in murine small intestinal tissues following gut injury. The expression of SPAK may influence the pathogenesis of chemotherapy-induced intestinal mucositis.

Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications

Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML. Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis. Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients. Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications. Disclosures Ramaekers: Nordic-MUbio: Current Employment.

Molecular Signatures and Their Clinical Utility in Pancreatic Neuroendocrine Tumors

Pancreatic neuroendocrine tumors (PNETs) are classified based on their histologic differentiation and proliferative indices, which have been used extensively to determine prognosis. Advances in next-generation sequencing and other high-throughput techniques have allowed researchers to objectively explore tumor specimens and learn about the genetic alterations associated with malignant transformation in PNETs. As a result, targeted, pathway-specific therapies have been emerging for the treatment of unresectable and metastatic disease. As we continue to trial various pharmaceutical products, evidence from studies using multi-omics approaches indicates that clinical aggressiveness stratifies along other genotypic and phenotypic demarcations, as well. In this review, we explore the clinically relevant and potentially targetable molecular signatures of PNETs, their associated trials, and the overall differences in reported prognoses and responses to existing therapies.

Mitotic and Proliferative Indices in WHO Grade III Meningioma

Meningiomas with inherently high mitotic indices and poor prognosis, such as WHO grade III meningiomas, have not been investigated separately to establish interchangeability between conventional mitotic index counted on H&E stained slides (MI) and mitotic index counted on phosphohistone-H3 stained slides (PHH3 MI). This study investigates the agreement of MI and PHH3 MI and to analyze the association of progression-free survival (PFS) and MI, PHH3 MI, and the proliferative index (PI, Ki-67) in WHO grade III meningioma. Tumor specimens from 24 consecutive patients were analyzed for expression of Ki-67, PHH3 MI, and MI. Quantification was performed independently by two observers who made replicate counts in hot spots and overall tumor staining. Repeatability in replicate counts from MI and PHH3 MI was low in both observers. Consequently, we could not report the agreement. MI, PHH3 MI and hot spot counts of Ki-67 were associated with PFS (MI hot spot HR = 1.61, 95% CI 1.12–2.31, p = 0.010; PHH3 MI hot spot HR = 1.59, 95% CI 1.15–2.21, p = 0.006; Ki-67 hot spot HR = 1.06, 95% CI 1.02–1.11. p = 0.004). We found markedly low repeatability of manually counted MI and PHH3 MI in WHO grade III meningioma, and we could not conclude that the two methods agreed. Subsequently, quantification with better repeatability should be sought. All three biomarkers were associated with PFS.

BIOM-51. FACTORS ASSOCIATED WITH DECLINING SEX DISCREPANCY IN MENINGIOMA WITH INCREASING TUMOR AGGRESSIVENESS: RETROSPECTIVE REVIEW OF A RURAL PENNSYLVANIA COHORT

INTRODUCTION Meningioma is the most common benign primary brain tumor in adults comprising 37% of all primary CNS tumors. Approximately 81.1% of meningiomas are grade I, 16.9% - grade II, and 1.7% - grade III. While females are 2.5 times more likely to have meningiomas, males more commonly have aggressive grades. Loss of progesterone receptor (PR) and presence of androgen receptor (AR) are associated with tumor aggressiveness. We observed a declining sex-discrepancy with increasing tumor grade. We present an ongoing assessment of a rural population to define potential factors contributing to this observation. METHODS We completed a single institutional retrospective review of 406 pathology confirmed meningiomas in rural Northeast Pennsylvania. We compared WHO grade, hormone receptors, and proliferative indices between males and females. We further evaluated tumor location and comorbid endocrine disease for possible association. RESULTS Our patient cohort had a high frequency of aggressive tumors 73.5% (n= 297) grade I, 24% (n= 99) grade II, and 2.5% (n= 10) grade III. The ratio of females versus males was 3.1:1, 1.3:1, and 1:1 in grades I, II, and III respectively. Factors contributing to closing the sex-gap were: (1) grade II: males expressed less PR than females (76% vs 94%); (2) grade III: females expressed less AR than males (0% vs 100%), while PR expression remained equivalent; and (3) males had higher tumor proliferative indices compared to females (7% and 22% vs 2% and 14%), grades I and II respectively. There was no relationship determined for tumor location or comorbid endocrine disease between the sexes. CONCLUSION The association between declining sex discrepancy in meningioma with increasing tumor aggressiveness is potentially related to the expression PR, AR, and increased tumor proliferation indices with increasing meningioma grade.

Clinicopathological study of premalignant and malignant lesions of cervix along with apoptotic index and Ki-67 expression

Background: Cervical cancer is known to have a good response to radiotherapy. The response and prognosis are dependent on the level of apoptosis. Pap smear and histopathology are cost-effective methods in diagnosing premalignant and malignant lesions of cervix but not accurate in classifying and estimating the progression of the disease, especially in premalignant lesions. Therefore this study was undertaken to know the role of Ki-67 expression and apoptotic index in classifying accurately the premalignant lesions for better management.Methods: The study included 540 cases diagnosed histologically as cervical intraepithelial neoplasia or carcinoma. The apoptotic index is calculated for all the 540 cases using light microscopy on Haematoxylin and Eosin stained sections. Ki-67 immunohistochemical staining was done for 100 cervical biopsies. Ki-67 expression was graded and the Ki-67 labelling index was calculated. Statistical evaluation was done using the unpaired t-test.Results: The Apoptotic index increased with increasing grade of dysplasia. There is a significant difference in the mean apoptotic index between premalignant and malignant lesions of the cervix. The ki-67 index increased with increasing grade of dysplasia. There is a significant difference in the mean Ki-67 index between premalignant and malignant lesions of the cervix.Conclusions: Apoptotic index and proliferative indices have been found useful in distinguishing between premalignant and malignant lesions of the cervix and gives an idea about the proliferative activity of the tumour for better management of the patient and to determine prognosis.

Correlation Between Digital and Manual Determinations of Ki-67/MIB-1 Proliferative Indices in Human Meningiomas

Objective. Proliferative activity in tumor tissues is assessed as the percentage of Ki-67/MIB-1-positive cells, or the proliferative index (PI). The PI is routinely assessed manually. However, the subjectivity of manual assessments might result in poor reproducibility. We hypothesized that digital assessments might reduce the error. Method. In our study, we assessed Ki-67/MIB-1 PIs, both manually and digitally, with tissue microarrays constructed from 141 human meningioma samples. Spearman-rank correlation and κ statistics were applied for correlation and agreement analyses, respectively. Mann-Whitney U tests were used to compare MIB-1 PIs between groups. Prognostic ability was assessed with Kaplan-Meier and Cox regression analyses. Results. We found a significant, high correlation (Spearman ρ = 0.832, P < .01) and moderate agreement (κ coefficient = 0.617, observed agreement = 80.9%) between the 2 methods. Both methods found significantly different Ki-67/MIB-1 PIs for different World Health Organization grades ( P < .05). Neither method showed significant prognostic value. Conclusion. Digital determinations of Ki-67/MIB-1 PIs in human meningiomas are feasible for the daily routine.

Genotoxicity of cisplatin and carboplatin in cultured human lymphocytes: a comparative study

Cisplatin and carboplatin are integral parts of many antineoplastic management regimens. Both platinum analogues are potent DNA alkylating agents that robustly induce genomic instability and promote apoptosis in tumor cells. Although the mechanism of action of both drugs is similar, cisplatin appears to be more cytotoxic. In this study, the genotoxic potential of cisplatin and carboplatin was compared using chromosomal aberrations (CAs) and sister-chromatid exchange (SCE) assays in cultured human lymphocytes. Results showed that cisplatin and carboplatin induced a significant increase in CAs and SCEs compared to the control group (p<0.01). Levels of induced CAs were similar in both drugs; however, the magnitude of SCEs induced by cisplatin was significantly higher than that induced by carboplatin (p<0.01). With respect to the mitotic and proliferative indices, both cisplatin and carboplatin significantly decreased mitotic index (p<0.01) without affecting the proliferative index (p>0.05). In conclusion, cisplatin was found to be more genotoxic than carboplatin in the SCE assay in cultured human lymphocytes, and that might explain the higher cytotoxicity of cisplatin.

The impact of proliferation and cancer stem cell upon the resistence to chemotherapy in salivary mucopepidermoid carcinoma

Background and aim: Mucoepidermoid carcinoma is a common salivary tumor that affects both adults and children. Proliferation is one of the most fundamental biological processes of growth and maintenance of tissue homeostasis. CD-44 may be used as an indicator of aggressive behavior of some human malignancy. Multidrug resistance is one of the major obstacles for successful cancer chemotherapy. The present study was carried out for evaluation of the biological rules and the clinicopathological significance of Ki-67, CD-44 and MDR-1 expression in the different histopathological grades of MECs.Patients and methods: Eighty paraffin embedded MEC tissues were collected and classified to three groups according to their histological grades. Tissue sections were stained with Ki-67, CD-44 and MDR-1 then examined microscopically and analyzedstatistically.Result: High grade MEC cases showed the highest expression for Ki-67, CD-44 and MDR-1. Additionally, significant differences were found between the histopathological grades as well as between lymph node stages of the studied cases and the expression of the three utilized markers.Conclusion: Ki-67, CD-44 and MDR-1can be used to evaluate the degree of differentiation and to predict the prognosis of MECs, furthermore, high grade MEC cases with high proliferative indices might be resistant to chemotherapy.