Kaempferol with Verapamil impeded panoramic chemoevasion pathways in breast cancer through ROS overproduction and disruption of lysosomal biogenesis.

BACKGROUND: Reactive oxygen species (ROS) at low level promotes cell survival through lysosome induced autophagy induction. Glucose stress induced acidosis, hypoxia, ROS, upregulates markers related to cancer stemness and multidrug resistance. Also, lysosomal upregulation is proposed to be one of the important indicators of cell survival under ROS induced stress. Studies supported that, stimulation of Lysosome-TFEB-Ca2+ cascade has important role in induction of chemoresistance and survival of cancerous cells. PURPOSE: To observe the effect of synergistic drug combination, Kaempferol and Verapamil on markers regulating chemoevasion, tumor stemness & acidosis as well as lysosome upregulation pathways, under low as well as high glucose conditions. HYPOTHESIS: Based on our earlier observation as well as previous reports, we hypothesized, our drug combination Kaempferol with Verapamil could attenuate markers related to chemoevasion, tumor stemness & acidosis as well as lysosome-TFEB-Ca2+ pathway, all of which have indispensable association and role in chemoresistance. METHODS: RNA and protein expression of candidate genes, along with ROS production and Ca2+ concentrations were measured in ex vivo models in altered glucose conditions upon treatment with KV. Also, computational approaches were utilized to hypothesize the mechanism of action of the drug combination. PCR, IHC, western blotting and molecular docking approaches were used in this study. RESULTS: The overproduction of ROS by our candidate drugs KV, downregulated the chemoresistance and tumor acidosis markers along with ATP1B1 and resulted in lysosomal disruption with reduction of Ca2+ release, diminishing TFEB expression under low glucose condition. An anomalous outcome was observed in high glucose conditions. We also observed KV promoted the overproduction of ROS levels thereby inducing autophagy-mediated cell death through the upregulation of LC3-II and p62 in low glucose conditions. The ex vivo studies also corroborate with in silico study that exhibited the parallel outcome. CONCLUSION: Our ex-vivo and in-silico studies revealed that our candidate drug combination KV, could effectively target several pathways regulating chemoresistance, that were not hitherto studied in the same experimental setup and thus may be endorsed for therapeutic purposes.

Kaempferol attenuates viability of ex-vivo cultured post-NACT breast tumor explants through downregulation of p53 induced stemness, inflammation and apoptosis evasion pathways.

Early onset of chemotherapy evasion is a therapeutic challenge. Chemotherapy-induced upregulation of stem cell markers imparts invasiveness and metastatic property to the resident tumor. The efficacy of Kaempferol in attenuating epithelial to mesenchymal transition has earlier been established in the breast cancer cell. In our study population, progression-free survival was observed to be statistically more significant in post-NACT low-grade tumors than the high-grade tumors. Further, in post-NACT TNBCs, high-grade tumors showed a preponderance of strong nuclear p53 expression and very low expression of Caspase 3, indicating that, altered p53 expression predisposes these tumors to apoptosis escape and up-regulation of stemness markers. Herein, we report the robust efficacy of Kaempferol on ex-vivo grown breast tumors, derived from post-NACT TNBC patients, through downregulation of nuclear p53, CD44, ALDH1, NANOG, MDR1, Ki67, BCL2 and upregulation of Caspase 3. Such tumors also showed concurrent deregulated RNA and protein expression of CD44, NANOG, ALDH1 and MDR1 with upregulation of Caspase 3 and cleaved Caspase 3, upon Kaempferol treatment. Validation of efficacy of the treatment dosage of Kaempferol through immunophenotyping on MDA-MB-231, suggested that Kaempferol at its IC-50 dosage was effective against CD44 and CD326 positive breast cancer through deregulating their expression. Protein-protein interaction network through STRING pathway analysis and co-expression study of candidate proteins showed the highest degree of co-expression of p53 and KI-67, CD44, NF- kappaB, ALDH1, NANOG, MDR1, and BCL2. Thus, potentially targetable oncogenic protein markers, that are susceptible to downregulation by Kaempferol, provides insight into biomarker-driven therapeutic approaches with it.

Comparison Study Between Scalpel and Electrocautery, in Causation of Seroma After Modified Radical Mastectomy.

Worldwide, breast cancer is the commonest malignancy in female population, and an increasing number of women are undergoing modified radical mastectomy (MRM) as a treatment for breast cancer. Though for most patients mastectomy goes uneventful, but for some patients complications of mastectomy are seen that cause morbidity, prolong hospital stay, and delay the adjuvant treatment. Seroma is encountered as a commonest complication after mastectomy. Though various factors are suspected in causation of seroma, in this prospective study, we tried to evaluate outcome of two different surgical technique of MRM in causation of seroma formation. We randomized all patients of breast cancer undergoing MRM in to two groups; in one group, we used electrocautery for raising the skin flap and axillary dissection while in another group we used scalpel to raise the skin flap along with aid of scissors and suture ligation for axillary dissection. Incidence of seroma formation was compared in both the groups. Incidence of seroma was significantly more with use of electrocautery. Results in both the groups were compared by chi-square method, and statistically significant difference in incidence of seroma formation was found between two groups. So in breast surgery, there should not be an injudicious use of electrocautery.

Clinicopathological characteristics of patients of certain molecular subtypes and elevated postoperative cancer antigen 15.3 levels and its correlation with menopausal status.

CONTEXT: It is well established that breast cancer subtypes differ in their outcome and treatment response. AIM: To observe tumor characteristics of different molecular subgroup and patients with postoperative (PO) raised cancer antigen 15.3 (CA 15.3) group and variation of tumor nature between pre- and post-menopausal breast cancer patients. MATERIALS AND METHODS: Blood samples and tumor blocks were collected from 95 nonmetastatic female breast cancer patients. Immunohistochemical stains for estrogen receptors (ER), progesterone receptor (PR), and HER2/Neu were used to classify molecular subtypes. CA 15.3 level was detected by ELISA. Significance levels were ascertained by Pearson Chi-square test. RESULTS: Prevalence of luminal A tumor with grade 3 was high. Triple negative and ER positive (ER+) types showed tumors with high grade and high lymph node (LN) metastasis. More nodal involvement was noticed in patients with PO raised CA 15.3. In addition, premenopausal patients with triple-negative and ER+ subtypes exhibited more aggressive tumors which were characterized by high grade and large numbers of LN metastasis. CONCLUSION: Clinicopathological characteristics of certain molecular subtypes and influence of menopausal status on it can predict disease recurrence or overall survival of breast cancer patients.

Anorectal Malformation: Paediatric Problem Presenting in Adult.

This is a case report of 22-year-old girl admitted with abdominal distension, vomiting, and chronic constipation since birth. Abdomen was distended, and perineal examination revealed imperforate anus with vestibular fistula (ARM). So far worldwide very few cases have been reported about anorectal malformation presenting in adulthood, and thus extremely little data is available in the literature about an ideal management of anorectal malformation in adults. In our case in the treatment instead of conventional procedure of posterior sagittal anorectoplasty (PSARP) anal transposition was done and till two years after the definitive treatment during follow-up patient has been doing well with Kelly's score of six. Our experience suggests that anal transposition provides satisfactory outcome in adults presenting late with anorectal malformation.

Primary Squamous Cell Carcinoma of Thyroid: A Rare Entity.

Primary squamous cell carcinoma (PSCC) of thyroid is an extremely rare malignancy of thyroid. Herewith, we describe a case report of female patient who presented with neck swelling; FNAC misdiagnosed it as papillary carcinoma of thyroid but, after resection, biopsy revealed it to be a case of squamous cell carcinoma of thyroid. After extensive investigations no possible primary focus of squamous cell carcinoma was found elsewhere, so diagnosis of primary squamous cell carcinoma of thyroid was made. Patient underwent chemoradiation but still patient succumbed to death within a year.

Decreased Expression of BRCA2 Accelerates Sporadic Breast Cancer Progression.

Human tumor suppressor BRCA2 has been known to function in the repair of DNA double strand break. Germ line mutation in BRCA genes predisposes individuals to familial breast and ovarian cancer. The aim of present study was to characterize the implication of BRCA2 expression in cases of non - hereditary (sporadic) breast cancer. Female breast cancer patients aged between 20 and 75 years who underwent surgery were randomly selected. Patients with Stage IV disease at time of primary diagnosis or previous history of any other malignancy other than breast carcinoma or undergoing neoadjuvant chemotherapy were excluded from the study. Total 48 patients full filled these criteria. Patients were treated with either modified radical mastectomy or breast conservation therapy. Concentration of BRCA2 was measured by Sandwiched method of ELISA. Immunohistochemistry was used to determine the hormonal receptor status. Stage of the disease was not found to have any significant correlation on the BRCA2 expression. In patients with higher grade of tumors the level of BRCA2 expression was found to be low. Decreased expression of BRCA2 was found to be triple negative, had aggressive features and associated with higher chances of axillary metastasis. Genetic instability caused by decreased expression of BRCA2 could trigger mutations in sporadic breast cancer cases and mutations in turn leads to uncontrolled proliferation and invasive growth.

Cytological and morphometric study of urinary epithelial cells with histopathological correlation.

This study has been done to asses the utility and accuracy of urinary cytology and morphometric study of exfoliated cells in early detection and follow-up of urothelial neoplasms and thereby help to reduce the disease-related mortality and morbidity. A total 100 patients with urinary symptoms were studied by cytological examination of urine along with morphometric analysis of suspicious epithelial cells. Immunostaining to detect CK-20 expression and p53 over expression was done in smears showing atypical cells. Histopathological confirmation was done in cases which were suspicious on cystoscopy. P value was determined by using unpaired t-test. Statistically significant difference was found between neoplastic and non-neoplastic lesions of the urinary bladder as far as morphometry is concerned. Urinary cytology along with morphometry is an important tool in early detection of urothelial neoplasms. It is also helpful to find out the recurrences during post-operative follow-up period. Overexpression of CK-20 and p53 immunostain in cytology can act as an adjunct to the cytological diagnosis.

Role of Cyclooxygenase 2 (COX-2) in Prognosis of Breast Cancer.

COX-2 regulates tumour growth, invasion and metastasis in breast cancer. This study investigated the association between COX-2 expression in human breast cancer versus the expression of ER, PR, HER-2/neu, as well as its association with other established prognostic indicators like age, menopausal status, tumour size, lymph nodal status, stage, grade, NPI and histological subtype, and aims to validate the role of overexpression of COX-2 as a prognostic marker in patients with breast cancer in Indian subcontinent. In this hospital based study of 123 breast cancer patients (Group-A) and 76 female patients with benign breast disease (Group-B) attending a Comprehensive Breast Clinic at a reputed institute in Eastern India, COX-2 protein expression was measured from breast tissue using the Western Blot Technique. COX-2 mRNA expression was measured by RT-PCR Technique. ER, PR and HER-2/neu status was measured by immunohistochemistry methods. COX-2 was not expressed in the control group. The proportion of COX-2 positive tumours was significantly higher in patients of age >50 years [52(91.2 %), p < 0.01], postmenopausal status [64(90.1 %), p < 0.01], advanced stage of disease (p < 0.01), higher grade (p < 0.01), larger tumors (p < 0.01), metastatic lymph nodes (p < 0.01) and NPI ≥ 5.4 (p < 0.01). COX-2 expression was seen in ER-negative [66(95.7 %), p < 0.01], PR-negative [76(92.7 %), p < 0.01], and HER-2/neu positive tumours [29(100.0 %), p < 0.01]. Risk of COX-2 positivity was found to be 2.74 times more for postmenopausal status, 6.90 times more for large size tumours (≥ 2.5), 34.37 times more for node positive tumours, 9.26 times more with ER negative patients and 5.88 times more for PR negative patients. COX-2 expression is associated with established indicators of poor prognosis such as postmenopausal status, age >50 year, advanced stage of disease, large tumour size, higher grade, lymph node metastasis, NPI ≥ 5.4, ER negativity, PR negativity and HER-2/neu positivity. Thus, COX-2 expression implies aggressive tumour biology, and may play an important role as a prognostic marker.

Can molecular subtyping replace axillary nodal status as prognostic marker in breast cancer?

Subtypes are an established prognostic factor of BC in western population but its significance in Indian BC patients has not been evaluated. Thus this study provides an insight into the prognostic significance of molecular classification and its effect on the survival of BC patients in Eastern India. In this hospital based study 242 BC patients attending a Comprehensive Breast Service Clinic of a reputed institute in Eastern India and having IDC were studied over a period of 7 years (January 2007 to October 2013). Nonluminal HER-2-positive and Triple negative tumors were associated with advanced stage of disease, metastatic lymph nodes and NPI ≥5.4, whereas Luminal 1 and Luminal 2 tumors were associated with early stage, uninvolved lymph nodes and NPI <5.4. Better survival was observed for the patients with Luminal 1 [OS = 57.1 % (n = 36)] and Luminal 2 [OS = 60.0 % (n = 6)], compared to Triple negative [OS = 33.6 % (n = 38)] and nonluminal HER-2-positive tumors [OS = 32.1 % (n = 18)]. This study provided some idea about the pattern of BC on the basis of classification by molecular profiling. Our study indicated that Triple negative and nonluminal HER-2-positive tumors have reduced DFS and OS compared with luminal 1 and 2 subtypes. In our patients, Triple negative and nonluminal HER-2-positive tumors were associated with established unfavorable prognostic indicators and this reflects the data in the western literature. The results suggest that the molecular subtypes are an independent prognostic and predictive marker in Indian BC patients. Whether or not molecular subtyping of breast cancer can replace axillary lymph nodes as the standard in prognosis remains to be seen, but if molecular subtyping can provide more information than the axilla about the prognosis and treatment option, it may well be the future of prognostication.

Morphometric and histological study of breast lesions with special reference to proliferative activity and invasiveness.

Breast carcinoma is the most common cause of carcinoma death in women. Sometimes, difficulty arises to differentiate between premalignant lesions and carcinoma by routine histopathology. Our study was done to establish the role of morphometry and immunohistochemistry to solve this problem. In this study, total 60 cases of different breast lesions were included and 10 controls were also included to compare the results with the normal findings. They were studied by hematoxylin and eosin-stained sections for morphometry and routine histological study; as well as by proliferative markers such as proliferating cell nuclear antigen and p53. Invasiveness was studied using immunohistochemical staining with 34 ßE12 monoclonal antibody. Statistically significant differences were found in morphometric parameters and in expression of proliferative markers between most of them. Morphometry and immunohistochemistry help in the proper diagnosis of different breast lesions that lie in the gray zone on routine histopathology.

Prognostic significance of HER-2/neu and survival of breast cancer patients attending a specialized breast clinic in Kolkata, Eastern India.

INTRODUCTION: The worldwide incidence of breast cancer has increased rapidly in recent years. The scenario of Eastern India is also showing the same trend. It is necessary to study the utility of HER-2/neu as a prognostic factor in breast cancer survival. However, there have not been detailed studies in this respect with the breast cancer patients of Eastern India. Thus this study was conducted. MATERIALS AND METHODS: In this hospital-based study 86 breast cancer patients attending a breast clinic of a reputed institute of Eastern India and having invasive ductal carcinomas were observed for a period of 5 years after surgery. Associations between 5 years observed survival and status of ER, PR and HER-2/neu of the patients were critically evaluated. RESULTS: There was statistically significant association between survival pattern for 5 years and the HER-2/neu status (p=0.00001). Better survival was observed for the patients with HER-2/neu negative tumors 67(100%) compared to HER-2/neu positive tumors 7(36.8%). CONCLUSION: There is strong interaction between survival and HER-2/neu expression of breast cancer patients. Thus the patients with HER-2/neu positive tumors need to be treated aggressively.

Is immediate postoperative CA15.3 assay a predictive marker of early postoperative recurrence of carcinoma breast?

Breast cancer is still an enigma. Systemic metastasis is an important prognostic factor. Tumour marker can predict occult systemic metastasis. To evaluate the immediate postoperative CA15.3 as predictor of early recurrence, a study was carried out in 48 patients of carcinoma breast in whom immediate postoperative marker level was done. In follow-up, recurrence was noted and relation with tumour size and stage done. Null hypothesis and 't' test were used for analysis. Relation of tumour size with marker is weak but strong relation exists between tumour stage with marker and recurrence with marker. CA15.3 predicts tumour load, can also predict occult residual/occult metastatic disease better than other prognostic markers which only predict tumour behaviour.

Role of nuclear factor-κB in female breast cancer: a study in Indian patients.

INTRODUCTION: The nuclear factor κB (NF-κB) is a super family of transcription factors which plays important roles in development and progression of cancer. The present investigation concerns NF-κB /p65 activity in human breast cancers with overexpression of ER, PR, HER-2/neu, as well as the significance of p65 expression with regard to menopausal status, stage, grade, tumor size, nodal status, and NPI of invasive ductal carcinomas in Eastern India. MATERIALS AND METHODS: In this hospital based study 57 breast cancer patients attending a Breast Clinic of a reputed institute of Eastern India were assessed for p65 protein expression in breast tumor tissue samples by Western blotting. ER, PR and HER-2/neu expression was determined by immunohistochemistry. RESULTS: NF-κB/p65 was significantly associated with advanced stage, large tumor size (≥5 cm), high grade, negative ER, negative PR, and positive HER-2/neu. High NF-κB/p65 expression was more frequent in patients with a high NPI (NPI≥5.4, 84.6%) compared with low NPI (<5.4, 44.4%) and this association was statistically significant (p=0.002). CONCLUSION: NF-κB/p65 overexpression was associated with advanced stage, large tumor size, high grade, and high NPI which are poor prognostic factors linked to enhanced aggressiveness of the disease. NF-κB/p65 expression implies aggressive biological behavior of breast cancer and this study validates significant association of NF-κB /p65 overexpression with negative estrogen and progesterone receptor status and overexpression of HER-2/neu oncoprotein. In our good clinical practice, patients with NF-κB positive tumors need to be treated aggressively.

Can cyclo-oxygenase-2 be a useful prognostic and risk stratification marker in breast cancer?

Cyclo-oxygenase-2 (COX-2) is a prostaglandin synthease that catalyses the synthesis of prostaglandin G2 (PGG2) and PGH2 from arachidonic acid. COX-2 plays an important role in tumourigenesis of different carcinoma types and it is thought to take part in breast carcinoma. In this study, the aim was to investigate the relationship of COX-2 with clinical parameters such as menopausal status, tumour size, grade, nodal status, Nottingham prognostic index (NPI), oestrogen receptor(ER), progesterone receptor (PR), human epidermal growth factor receptor type 2 (HER-2/ neu). The patients were divided into two groups, first group (group A) comprised 57 primary breast cancer patients and the second group (group B) comprised control group 27 patients consisting of fibro-adenoma and benign breast disease. In control groups COX-2 (0%) is not over expressed and we observed that high frequency of COX-2 (73.68%) over expressed in breast carcinoma. In high grade, large tumour size and positive lymph node metastasis, COX-2 expression rate was 78.6%, 59.5% and 90.5% respectively. COX-2 expression is directly correlated with ER negative (88.1%, p = 0.001) and also associated with higher NPI value (78.6%, p = 0.006). In invasive ductal carcinoma (IDC) COX-2 over expression had a significant relationship with HER-2/neu over expression (p < 0.001). The results indicated that COX-2 over expression correlates with aggressive phenotypic features, such as high histological grade, large tumour size, higher NPI value, ER negativity and HER-2/neu positivity.

Study of immunohistochemistry in prostatic lesions with special reference to proliferation and invasiveness.

Prostatic lesions on routine staining sometimes cause diagnostic dilemma especially in premalignant lesions like atypical adenomatous hyperplasia and prostatic intraepithelial neoplasia. Benign small acinar lesions also may be difficult to differentiate from small acinar adenocarcinoma. An important differentiating point is the loss of basal cell layer in adenocarcinoma and its presence in benign lesions. Basal cell markers (e.g. 34ßE12 cytokeratin) & proliferative markers (e.g. AgNOR and PCNA) can help in this regard. Total 60 cases of different prostatic lesions studied. After history taking, clinical examination, radiological & other investigations were done. Routine H&E staining, immunohistochemical staining against 34ßE12 cytokeratin & proliferative markers (AgNOR & PCNA) was performed. Statistically significant differences found in expression of 34ßE12 cytokeratin and proliferative markers between benign, premalignant and malignant prostatic lesions. Basal cell markers and proliferative markers are important parameters to distinguish between different benign, premalignant and malignant prostatic lesions.

Oncoplastic Breast Surgery - Our Experiences in the Breast Clinic, IPGME&R, Kolkata.

Oncoplastic breast surgery is the fusion of oncological and surgical principles to gain successful breast tumour excision with good cosmesis. It is an widely accepted and popular method in the western world. However, the picture is different in India. The major issues here, like late detection and advanced tumours, poor socio-economic status overriding quality of life issue and shortage of dedicated oncoplastic surgeons result in a poor acceptance and practice of oncoplastic breast surgery. This article explores the use of various oncoplastic techniques in clinical practice and discusses future directions in this emerging field in an Indian perspective. In our institute (breast care unit, I.P.G.M.E&R,Kolkata), we performed a retrospective study over a period of five years (2005-2009). It included a sample size of 30 patients with diagnosed breast cancer or Phylloides Tumor (PT). The study focused on the indications, type of oncoplastic procedure used, cosmetic outcome (shape / volume replacement, ptosis correction & chest wall coverage), complications faced and response to subsequent therapy. The indications, for which oncoplastic techniques were applied, were DCIS (2), LCIS (1), IDC (19) and phylloides tumor (8). Of the 30 patients, RAT was used in 8, LDMF (of various types and volume) was used in 16, pedicled TRAM flap was used in 3 and reduction mastopexy in 3.The procedural indications of reconstruction were total glandular replacement by TRAM flap, mini-LDMF to fill volume loss after BCS or wide local excision, rotation advancement technique for reshaping / symmetry maintenance after BCS or wide local excision, LDMF for chest wall coverage after MRM and reduction mastopexy after wide local excision. From patient's point of view the outcome of surgery was highly satisfactory (score 3 or more) in 19 out of 30 patients (63.33%). LDMF was the most commonly used (16 out of 30) oncoplastic procedure with least complication rates (0 out of 16). 4 out of 30 patients had complications related to the procedure. Chest wall coverage after MRM still forms the main indication of oncoplastic surgery in this country.

Role of proliferative markers in breast lesions.

BACKGROUND: Carcinoma of breast is a leading cause of death in women throughout the world. Difficulty in diagnosis occurs in routine Hematoxylin and Eosin (H&E) stained sections to differentiate premalignant lesions like epitheliosis and malignant lesions like carcinoma in situ which may affect prognosis. AIMS: Our study was done to assess the role of proliferative markers in this field. MATERIALS AND METHODS: In this study, total 66 cases of different breast lesions were studied by H&E stained sections as well as proliferative markers like silver staining nucleolar organizer region (AgNOR) and proliferating cell nuclear antigen (PCNA). RESULTS: Statistically significant differences found in expression of proliferative markers between most of them. CONCLUSION: Study of proliferative markers help in the proper diagnosis of different breast lesions which lies in the grey zone on routine histopathology.

Preoperative ultrasound guided needle localisation for non-palpable breast lesions.

BACKGROUND: Breast cancer is a major cause of cancer-related morbidity and mortality in Indian women. Most cases present late and thus survival in Indian patients is poor compared to the western world. In the absence of a screening, early detection of breast cancer is a challenge in Indian subcontinent. METHOD: Though much is known about management of any palpable lump in breast, clear guidelines in dealing with non-palpable lesions of breast is still obscure. Careful imaging of breast followed by assignment of standard Breast Imaging Reporting and Data Systems (BIRADS) category to the finding can go in a long way to predict chances of malignancy in a non-palpable breast lesion (NPBL). Total 22 patients with impalpable lesions in the breast were localised with US guided needle and lesion excised. Applicability of Ultrasonography (USG) to detect early breast lesions and comparison with mammography in predicting malignancy was assessed by tests of proportions (z test). RESULTS: Total of three early breast cancers and four borderline lesions were diagnosed by this method among 22 properly selected cases. Needle localisation of the lesion on sonographic guidance followed by lumpectomy can be both an accurate diagnostic and therapeutic method to deal with occult lesion in our scenario. CONCLUSION: US guided lumpectomy is a feasible alternative to other methods and is effective in Indian scenario to diagnose early subclinical breast cancers.