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Introduction:Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. The success of immunotherapy in solid tumors has raised the hope for their utility in TNBCs as well. But only a subset of patients have a clinical response to check-point inhibitors. The cellular and molecular mechanisms that mediate the immunological response or tolerance are just beginning to be understood. Compared to ER/PR+ breast cancer, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The density of TILs in and of themselves do not accurately predict response to neoadjuvant chemotherapy or survival. M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. In this study, we have characterized presence of M2-TAM in the TNBC immune environment by examining expression of several biomarkers. Methods:Surgically excised tumor specimens from 88 Indian women with TNBC were accessed from the longitudinal observational series of SJNAHS tissue bank under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Transcriptome analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumour microenvironments within TNBC. CIEBERSORT tool was used to analyse for distribution of cell types. Identification of macrophages was done by Immunohistochemistry (IHC) for CD68 and CD163 markers in TNBCs and a control group of ER+HER2-.Results:NMF analysis with an enriched immune gene signature of 111 genes, yielded 3 subtypes (ST) (37%, 27% and 36% with relative proportions of ST1, ST2 and ST3) within the TNBC. The three subtypes had distinct survival patterns with ST1 having the best prognosis with enriched TH1 gene signature and ST3 had poorest (log rank p=0.5). On application of this gene signature to TNBC groups in METABRIC and TCGA data, similar pattern emerged with a significant survival pattern of ST 3 having the poorest outcome (p=0.005). A closer examination of ST 3 revealed a signature enriched for M2 macrophages also known as TAM. Immunohistochemistry for Pan macrophage marker CD68 and an M2 specific marker CD163 between TNBC and ER/PR+ revealed difference in the two groups was significantly different (Mann Whitney p=0.03), with TNBC exhibiting 2.4 fold higher expression of CD163. CD 68, was enriched in ST2 and ST3. CD163 which is an M2 specific marker was highest in ST3 as compared to ST 1& 2 (p=0.04). This group also correlated with signal molecules secreted by macrophages containing growth factors, cytokines and chemokines, such as TGF-β, VEGF, IL-10 and CXCL and interleukins like IL4 and IL6 suggestive of TAM recruitment and polarization. It is likely that the TAM enriched subgroup is likely to be unresponsive to PD1/PDL1 inhibitors but could be targeted by novel therapeutic strategies to directly target M2-TAM. In vitro and In vivo analysis to target M2-TAM to evaluate the response to these therapeutic compounds in cell lines as well as a mouse syngeneic model is underway.Conclusions and future directions: As a cell type within the tumor microenvironment, that promotes invasion, M2-TAMs makes an ideal therapeutic target in TNBC. In addition, this sub-type lends itself to easy identification by a simple IHC assay. Citation Format: Aruna Korlimarla, Jyothi Prabhu, Chantirika Ragulan, Gnanapriya Shivakumar, Krisha Desai, Maggie Cheang, Srinath BS, Ravi Diwakar, Sandhya Apachu, Rekha Kumar, TS Sridhar, Savitha RajaRajan, Rohini Kaluve, Annie Alexander, Anguraj Sadanandam. A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-37.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

Background: World over, less than a quarter of breast cancer diagnoses are in premenopausal women. However, in India premenopausal women constitute half of all women with breast cancer in most hospital case series. Most of these women present at advanced stages with aggressive subtypes of disease and hence the high mortality.The role and utility of detecting androgen receptor (AR) expression in the different sub-types of breast cancer, especially the ones without hormone receptor expression is yet to be firmly established. Evidence from previous studies is suggestive of its beneficial role in hormone receptor positive (HR+) breast cancer. The biological function of AR on the mammary epithelium is determined by the Estrogen receptor (ER) context, in that, it is found to be anti-proliferative in ER positive tumors while it is thought to promote growth in the absence of ER activity. An interesting approach to representing this interplay is as a ratio between AR/ER expressions. As expected, the ratio has been shown to be positively correlated with better outcomes in hormone receptor cancers, mostly in postmenopausal women. The effect of a high ratio in ER negative tumors seems more complicated. In this study, we have evaluated the AR/ER ratio specifically in patients younger than 50 years in whom the estrogenic influence is dominant due to their premenopausal status.Materials and Methods: Tumor samples from patients 50 years or younger were chosen from a larger cohort of 275 patients with median follow up of 72 months. Expression of ER and AR proteins were detected by immunohistochemistry (IHC), and the transcript levels of ESR1 and AR were determined by quantitative PCR. Relative normalized units of their gene expression were used to calculate the AR/ER ratio. A cut-off at the 3rd quartile was used to divide tumors into categories of high and low ratios. Clinical characteristics were compared between the low and high ratio groups along with IHC subtype distribution (HR+, HER2+ and Triple negative (TNBC)). Kaplan Meier curves was used for survival analysis and Cox proportional hazard analysis model was used to calculate the hazard ratio (HR). The results were validated in METABRIC dataset.Results: Eighty-eight (32%) patients were Citation Format: Jyothi S Prabhu, Aruna Korlimarla, Savitha Rajarajan, Annie Alexander, Ce Anupama, Rakesh Ramesh, Bs Srinath, T.S Sridhar. The prognostic utility of AR/ER ratio in young women with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-55.

Background The study assessed the epigenetic regulation and the role of microRNA (miR) expression in locally advanced triple negative breast cancers (TNBC) and comparison with the clinico-pathological variables and survival. Methods Fifty patients of locally advanced TNBC during the period 2011–2013 were included. Expression level of test microRNA (miR-182 and miR-18a) was determined using Taqman quantitative Real time polymerase chain reaction (qRT-PCR) from formalin fixed paraffin embedded biopsy blocks. Clinical and demographic information and survival data was retrieved from the Hospital medical records. Results An improved clinical complete response (cCR) was observed in patients with age ≥ 45 years (80%), premenopausal status (70%), tumor size < 6 cms (80%), nodal status N0-N1 (95%) and grade II-III tumor (80%). A statistically significant correlation was observed on comparison of cCR with menopausal status (p-value 0.020), T category (p-value 0.018) and the clinical nodal status (p-value 0.003). pCR also correlated with clinical nodal status (p-value 0.008). Epigenetically, miR-18a under expression (< 8.84) was most commonly associated with tumor size < 6 cms (76.7%), clinical nodal status N0-N1 (90%), cCR (60%) and pCR (53.3%). A similar trend was observed with miR-182. Statistical significance was observed with T category (p-values 0.003 and 0.004), clinical nodal status (p-values 0.001 and 0.001), clinical response (p-values 0.002 and 0.002) and pathological response (p-values 0.007 and 0.006) with respect to miR-18a and miR-182, respectively. Also, the menopausal status significantly correlated with the miR-182 expression (p-value 0.009). miR-182 overexpression (≥ 6.32) was not observed in any of the postmenopausal patients. A univariate cox proportional hazard regression model also showed statistical interactions (p-values Conclusion miR-182 and miR-18a overexpression correlates with worse clinical and pathological tumor characteristics in locally advanced TNBC and hence could be used to predict the outcomes and prognosis in these patients.

PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor tyrosine kinase that participates in positive feedback signalling of the human epidermal growth factor receptors and helps in growth and migration. PTPN11/SHP2 is widely believed to be an oncoprotein, although it’s possible tumor-suppressor role is also reported. Our analysis of breast cancer metadata shows, PTPN11/SHP2 copy number loss in luminal A subtype is correlated to poor disease-specific survival and late-stage cancer at diagnosis. Analysis of the level 4 Reverse Phase Protein Array (RPPA) data available on the TCGA database resulted in positive correlations between the lower expression levels of constitutively active variant, the phospho-SHP2-Y542, of PTPN11/SHP2 and larger tumor size and lymph node positivity. We experimentally examined possible negative regulation of growth by PTPN11/SHP2 using MCF10A, a normal breast epithelial cell line. Knock-down of PTPN11/SHP2 resulted in increased cell migration, cell shape changes to mesenchymal morphology, and increased survival in cells treated with epirubicin, a DNA-damaging drug. However, it did not alter the rate of cell proliferation. It is possible that PTPN11/SHP2 might function as a tumor suppressor by potentiating proliferating cells with increased cell migration and resistance to apoptosis. Statement of Significance Molecules like PTPN11/SHP2, among many others that show dual specificity in tumorigenesis in the same tissue depending on the upstream signaling cues, present challenges in the field of targeted drug therapy. This study puts forth the importance of understanding the mechanism of one of the two outcomes and thereby helps better clinical management of a subgroup of cancer.

Introduction: In spite of rapid urbanization and modernization the family remains central in the socio-cultural structure of India. The individuals are enmeshed into this unit and tend to be interlinked financially, emotionally and socially. The head of this family unit tends to be a male more often than not. As is well known, despite recent attempts by the governments at the state and centre at providing health coverage for cancer through regional cancer centres, a majority have to raise the money for cancer care by themselves. We have examined the role of the family in treatment decision making and in the strategies employed to raise the money and cope with the financial stress imposed by a diagnosis of breast cancer. Method: 378 women with breast cancer were enrolled into a longitudinal study at first diagnosis between the years 2008-2012, at two tertiary care hospitals in Bangalore, India. The median follow up as of May 31st 2017 is 78 months with only 2% loss to follow-up over the past 8 years. Follow-up was maintained by frequent meetings between a counselling psychologist (AA) and the patient and/or a family member. The frequency of meetings was monthly during the initial treatment and then quarterly over the next 5 years. Information on demographics was collected during the treatment phase and information on the psychosocial aspects was collected in non-structured interactions subsequently. This information included details of support structure, decision making, and financial arrangements. Results: This is a predominantly urban cohort with 80% being urban. The median age of patients at first diagnosis was 55 years. Almost all of our patients (99%) had the support of one or more family members. We analysed the pattern of decision making for treatment and in half of all cases either the husband or the son were the decision makers. In an additional 15% daughters and other relatives were the primary decision makers. Approximately a third of women made the decision concerning treatment themselves, and these women tended to be college educated (51% vs 16%) and employed (53% vs 12%). 30% of the patients met the costs incurred through medical insurance plans purchased by the family. Another quarter of patients were able to meet the costs from their savings. 45% had difficulty in finding the money for treatment and 15% took personal loans while 30% had to sell land/gold ornaments or take loans against assets of these sorts. Only (3%) discontinued the treatment due to financial difficulties. As in the case of decision making those who had the financial resources tended to be more educated (41% vs 11%), and were employed (31% vs 21%). Conclusion: The data from a predominantly urban cohort of breast cancer enrolled between 2008-2012, supports the general belief that in India the family remains the fulcrum of an individual during crises, and not surprisingly education and employment lead to both psychological and economic emancipation of women. Citation Format: Alexander A, Kaluve R, Prabhu JS, Korlimarla A, BS S, Manjunath S, Patil S, KS G, Sridhar TS. Treatment decision making, and strategies for coping with financial stress in Indian women diagnosed with breast cancer and their families [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-10-12.

Placental structure and function determine birth outcomes. Placental mass does not always correlate with fetal birth weight (BW) in uncomplicated pregnancies which raises the possibility of other variables such as placental shape and cord insertion being the determinants of placental efficiency. In total, 160 women with singleton pregnancy, recruited into a pregnancy cohort were studied. Placental weight (PW) was measured and other data were obtained from clinical records. Birth outcomes were classified as small for gestational age (SGA) and appropriate for gestational age (AGA) based on fetal gender, gestational age (GA) and BW. High-resolution images of the chorionic plate were recorded. The shape of the placenta and the insertion of the cord were measured using eccentricity index (EI) and cord centrality index (CCI). Only placentae with eccentrically inserted cords (n=136) were included. The mean BW and PW were 2942 (±435) g and 414 (±82) g with average GA of 38.6 weeks. The mean CCI and EI was 0.483 (±0.17) and 0.482 (±0.16). Neither of these correlated with placental efficiency. However, EI showed negative correlation with placental surface area and breadth. Upon sub-grouping the cohort into SGA (n=32) and AGA (n=104), the SGA babies with the highest EI (third tertile) had significantly lower BW than those with the least eccentric placentae (first tertile). Although eccentric-shaped placentae were present in both SGA and AGA groups, the effect on BW was observed only in the SGA group.

Structural or functional defects in the placenta, are the primary cause of growth restriction of the fetus. Morphological examination of such placentas from intrauterine growth restricted (IUGR) fetuses often appears deceptively normal. Evaluation of angiogenesis and fetoplacental vasculature is critical to understand the underlying pathogenesis of fetal growth restriction in both idiopathic as well as cases where it is thought to be secondary to complications like preeclampsia (PE). We analyzed the immaturity of fetoplacental vasculature using CD15, which is a stage specific embryonic antigen known to be expressed in immature endothelium.One hundred and twelve placentas (81 from IUGR and 31 from gestationally appropriate samples (appropriate for gestational age (AGA)) were collected based on stringent inclusion criteria, and subjected to detailed examination of morphology and microscopy along with immunostaining for CD15. IUGR placentas known to have villous immaturity such as those associated with gestational diabetes, Rh negative pregnancies and anemia were excluded. The time of clinical onset of IUGR, associated complications like PE and oligohydramnios along with clinical variables were recorded. CD15 expression was scored in both distal and proximal vasculature and the values in IUGR and AGA pregnancies were compared and correlated with clinical variables.The mean CD 15 scores in both proximal vasculature (PV) as well as distal (DV) vasculature were significantly higher in the IUGR group compared to AGA (17.7 versus 5.16 in PV and 50.8 versus 23.7 in distal vasculature (DV)). Gestational age had no influence on CD15 staining in PV or DV in IUGR group, whereas preterm AGAs expressed higher CD15 only in the distal vessels. PE, oligohydramnios and the time of onset of IUGR did not influence the fetal vascular immaturity, as measured by CD15 scores. Although none of the clinical or obstetric factors influenced CD15 staining in AGA, fetal vessel immaturity in the IUGR group remained high even after adjusting for confounding variables like maternal age, gestational age and birth weight. Histological features suggestive of chronic hypoxia were significantly higher in IUGR placentas, compared to AGA and correlated positively with CD15 expression.Fetoplacental endothelium in both PV and DV is immature in IUGR irrespective of the gestational age or any other associated factors and CD15 immunodetection is a valuable marker for assessment of immaturity.

Background Glucocorticoid receptor (GR) is shown to have variable frequency of expression in invasive tumors of the breast. Investigation of additional nuclear receptors like GR in receptor negative tumors like triple negative breast cancer (TNBC) may have prognostic and therapeutic significance. Methods Expression of GR was evaluated by immunohistochemistry in 175 tumors of invasive breast cancer with long term follow up. GR Expression was separately evaluated in invasive tumor cells, stromal cells and tumor infiltrating lymphocytes (TIL's). Staining pattern was categorised as positive when more than 1% of the cells stained in each subpopulation of cells. Disease free survival was analysed between GR positive and negative status by Kaplan Meier analysis. Results Of the 175 tumors, 121 (70%) were ER positive, 53 (30%) were ER negative and 29% (51) were triple negative. 74% (130/175) tumors showed expression of GR in invasive tumor cells while (84%) 147/175 had expression in TIL's. No significant difference in distribution of GR was noted between ER positive and ER negative tumors (78% vs 66%, p-0.1). Of the TNBC's 54% (28/51) and 70% (36/51) showed expression of GR in invasive tumor and TIL's respectively. Overall, GR positive tumors had significant better survival than GR negative tumors (mean survival time of 85 vs 59 months respectively, p-0.04) Contrary to the reports that GR expression in TIL's are associated with immunosuppressive activity in model systems, TNBC's with increased expression of GR in immune cells were associated with better survival (Mean survival time 74 vs 41 months, log rank test- p-0.03). TNBC tumors which were GR negative had higher lymph node metastases (p-0.04) and none of the other clinical features like age, menopausal state, tumor size and grade were different between GR positive and negative tumors within TNBC. Conclusions Glucocorticoids (GC) are often used to alleviate the adverse symptoms during chemotherapy. Determining the GR status is of importance due to the pro cell survival effect of the glucocorticoids mediated through GR during chemotherapy. Though GC mediated effects on chemotherapy are controversial, our results indicate favourable effects in TNBC.

MicroRNA mediated aberrant gene regulation has been implicated in several diseases including cancer. Recent research has highlighted the role of epigenetic modulation of the complex process of breast cancer metastasis by miRNAs. miRNAs play a crucial role in the process of metastatic evolution by facilitating alterations in the phenotype of tumor cells and the tumor microenvironment that promote this process. They act as critical determinants of the multi-step progression starting from carcinogenesis all the way to organotropism. In this review, we focus on the current understanding of the compelling role of miRNAs in breast cancer metastasis.

Both intrauterine growth restriction (IUGR) and pre-eclampsia (PE) are accompanied by alterations in the vascular structures of the umbilical cord (UC). However, it is unclear if the vasculature is significantly different when both conditions co-exist. Digitized sections of 77 UC from four groups of women were analyzed morphometrically. The groups included women with PE (group I), IUGR and PE (group II), IUGR (group III) and women with uncomplicated pregnancy as controls (group IV). The effect of PE, IUGR and their combination on UC parameters were examined using two-way ANOVA and the correlation of birth weight and placental weight in these parameters were measured. There were 12 cases in group I, 22 in group II, 26 in group III and 17 in group IV. The umbilical vein (UV) parameters like wall thickness (0.33 vs 0.42 mm, p = 0.04), cross sectional area, (2.9 vs 4.1 mm2, p = 0.01), diameter (2.2 vs 2.6 mm, p = 0.04) and muscle cross sectional area (1.53 vs 2.4 mm2, p = 0.01) were lower in the IUGR group as compared to other groups. In the group with PE, UV wall:lumen ratio (0.28 vs 0.2, p = 0.05), UA D (0.77 vs 0.63 mm, p = 0.04) and UA CSA (0.99 vs 0.8 mm2, p = 0.04) were significantly higher compared to other groups. The interaction effect of PE and IUGR was not significant for any of the umbilical vessel parameters. The UV dimensions are significantly smaller in IUGR and PE pregnancies. These differences were mainly seen in the IUGR group and the presence of PE did not amplify the differences.

Background: MicroRNA mediated molecular alterations are involved in the initiation and progression of cancer. Altered expression of multiple microRNAs is associated with endocrine resistance in hormone receptor positive HER2 negative (HR+/HER2-ve) cancer. The role of miR-221 in inducing epithelial to mesenchymal transition (EMT) is well documented especially in cell line model systems. However, the detailed mechanism of specific microRNAs in intrinsic and acquired resistance to endocrine therapy needs to be worked out. In addition, more needs to be done in the documentation of these mechanisms in human breast cancer specimens with complete clinical documentation and long-term follow-up. In this study, we have evaluated the clinical significance of miR-221 and its mechanistic role in EMT using human specimens and cell line models. Materials and Methods: Formalin fixed paraffin embedded tumor from 129 HR+/HER2-ve breast cancer patients with a median follow up of 63 months were used for estimation of miR-221 by quantitative real time PCR. Expression levels of genes which are direct targets of miR-221 and related genes in EMT were analysed from these tumors. Survival between miR-221 high and low groups was compared by Kaplan Meier survival curves and prognostic relevance was estimated by Cox proportional hazard model. Cell line experiments to investigate the role of miR-221 in inducing EMT through integrin β6 are underway in both wild type and tamoxifen resistant MCF-7 cell lines (A gift from Prof Ben Ho Park, Johns Hopkins University School of Medicine). Results: A significant elevated level of miR-221 was observed in small proportion (14%) of HR+/HER2-ve tumors. miR-221 expression had an inverse correlation with both ER protein and ESR1 mRNA levels within HR+/HER2-ve tumors. Tumors with high levels of miR-221 showed significantly higher expression of integrin β6 which is a robust marker of EMT. Patients with high expression of miR-221 had a poorer survival in Kaplan Meier analysis. Results of interrogation of EMT mediated through integrin related pathways involving miR-221 in cell line models will be presented. Discussion: The association between miR-221 and integrin β6 in HR+/HER2-ve breast cancer with endocrine resistance suggests a potential link between an epigenetic regulator and a mediator of tumor-stromal interaction. The other mediators involved in this pathway are being investigated. miR-221 could be potentially used as a marker for identification of a poor prognostic subtype within HR+/HER2-ve breast cancers. Citation Format: Prabhu JS, Kaul R, Korlimarla A, Desai K, Gangadharan C, Rajarajan S, Nair MG, Alexander A, Kaluve R, Manjunath S, Correa M, Prasad MSN, Patil S, Srinath BS, Sridhar TS. Epithelial mesenchymal transition associated with high miR-221 and integrin β6 leads to poor prognosis in hormone receptor positive HER2 negative breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-10.

Aims Placental physiology and morphology is critically regulated by DNA methylation. As such, placental global DNA methylation and transcript abundance of placental DNA methyltransferases ( DNMT1 and DNMT3A ) may relate to placental and fetal growth in human pregnancies. We aimed to test correlations of human fetoplacental parameters and birth weight with the placental expression of DNA methyltransferases ( DNMT1 and DNMT3A ) and placental global methylation. Subjects and methods Placentae (n = 109) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 56 SGA and 53 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of DNMT1 , DNMT3A and DNTMT3B normalized to a panel of reference genes. LINE-1 methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 68). Associations of placental transcript abundances of DNMT1, DNMT3A and DNMT3B and of LINE-1 methylation levels with maternal, placental and neonatal parameters were tested. Results Placental DNMT1 transcript abundance associated positively with placental weight (β = 10.21, P = 0.013). This association was specific to the AGA births (β = 12.77, P = 0.022) and was absent in the SGA births. Association of DNMT1 expression with placental weight and birth weight within the AGA births was specific to the female gender (Birth weight: β = 83.61, P = 0.043; Placental weight: β = 23.92, P = 0.025). Placental DNMT1 transcript levels were not different according to SGA status or gender. Placental DNMT3A transcript levels and LINE-1 methylation levels did not show any associations with maternal, placental and neonatal parameters. Conclusions Placental DNMT1 expression was found to be associated positively with placental weight and birth weight, specifically in the female AGA births. Thus, we hypothesize that placental DNMT1 participates in fetoplacental growth in a fetal gender-specific manner.

Aberrations in placental vascular development compromising fetal supply of oxygen and essential nutrients can be a significant contributor to intrauterine growth restriction (IUGR). The development of placental vascular tree is under the influence of two families of growth factors, namely the vascular endothelial growth factor (VEGF) family and angiopoietin/TEK family. In this study, we have examined the expression of angiogenesis-related growth factors, mainly VEGF family and angiopoietin-TEK (endothelial-specific receptor tyrosine kinase) family genes in placentae from IUGR pregnancies uncomplicated by preeclampsia (PE) compared to normal pregnancies.Placentae from normotensive IUGR (n = 42) and appropriate for gestational age (AGA) pregnancies (n = 47) were collected and examined histologically. Clinical parameters were obtained from the medical records. Real-time quantitative PCR was performed to assess placental transcript abundance of VEGF, PGF, FLT1, ANGPT1, ANGPT2, and TEK normalized to a panel of reference genes. Associations of placental transcript abundance of the genes with maternal, placental, and neonatal parameters were tested.Placental transcript abundance for VEGF (relative expression 10.81 versus 12.98, p.001), PGF (12.14 versus 13.8, p.001) and ANGPT2 (3.67 versus 9.55, p = .002) were significantly lower in IUGR placentae compared to AGA. The transcript level of VEGF showed significant negative correlation with birth weight (r = -0.419, p = .006), placental weight (r = -0.318, p = .040), placental length (r = -0.389, p = .011) and breadth (r = -0.308, p = .047) only in the IUGR group. Presence of histopathological features of hypoxia correlated with significantly higher transcript levels of PGF in IUGR placentae (12.6 versus 10.9, p = .046).The low levels of VEGF transcripts may be responsible for the impaired angiogenesis in IUGR placentae. The significance of higher relative expression of PGF in the presence of chronic hypoxia needs to be explored.

Background: Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. The hallmark of TNBC is genomic instability with very high rates of TP53 mutation. BRCAness has been defined by Ashworth et al (2004 &2014), as the phenocopying of BRCA1&2 mutation by alternate genetic or Epigenetic mechanisms. The consequence of this is DNA damage repair (DDR) deficiency. Though PARP inhibitors have been of utility in treating this subset of TNBC, trials have in general not found an unequivocal support for the use of platinum. We have developed an assay for BRCAness, the BRCA1 deficiency score (BDS) (Korlimarla et al 2016). In this study, we have applied BDS assay in tandem with whole exome sequencing to a small retrospective series of TNBC patients more than half of whom were treated with Platinum. Methods: 40 TNBC primary specimens along with complete clinical data from a retrospective series at the SSCHRC were obtained under all IERB approvals and informed patient consent for BDS assay and mutational analysis. BDS assay is a multianalyte assay involving measurement of BRCA1 (Transcript and protein) as well as additional epigenetic regulators of BRCA1, mir182 and ID4. Whole Exome Sequenced (WES) using the Agilent Sureselect V6 kit on Illumina HiSeq platform. Variant calling analysis was performed using GATK Mutect2 with Hg38 reference genome following the best practice workflow. Variants annotated as protein affecting based on their functional impact prediction and mapped to known cancer related genes (from COSMIC), were selected. Results: Clinical details: Mean age of patient in the series was 49. 37/40 qualified for BDS assay. 21/37 (57%) patients were treated with Carboplatin in combination with Docetaxel in an adjuvant setting. Remaining patients were treated with Cyclophosphamide Adriamycin with or without Docetaxel. 15/37 (45%) were BRCA1 deficient and10/15 were treated with Platinum and 8 (80%) were responders. 39 passed QC and considered for analysis. Spectrum of variants were missense mutations (86.5%), followed by stop gained (5.91%) and frame shift (3.76%). Results were compared to TCGA TNBC set (n=123). The most frequently mutated gene was TP53 (62%) as reported in TCGA. We also report higher frequency of deleterious mutations on DNA damage repair (DDR) genes like ATM and BRCA2 (15%). Response to Platinum therapy in this subset, correlated with Mutations in DNA damage repair genes (p ≤ 0.003). 4/6 samples mutated were also In the BDS deficient group. BRCAness score for predicting Platinum response also correlated significantly (p ≤ 0.05). Other genes which showed significant alterations were KMT2A and KMT2D (15% and 13%) which encode the histone methyl transferase and are responsible in altering the chromatin structure and RECQL4 (10%) which is a helicase involved in DDR Conclusion: Both BRCAness and mutation profile identified a DDR deficient group within TNBC patients. In addition to TP53 which is the most frequently mutated gene, our small sample set has shown higher frequencies of mutations in DDR genes like ATM and BRCA2. This group of patients showed favourable response to platinum therapy. Since very little is known about the molecular heterogeneity of TNBCs in Indian patients, our analysis aids in identification of actionable mutations in TNBCs and may be of use in selection of patients for platinum therapy. CHARACTERSn%Platinum Treatment - ( n )Treatment response%BDS3721Deficient154513BRCA1 deficient response861Proficient19518BRCA1 Proficient response450Undet34 Citation Format: Aruna Korlimarla, Sabarinathan Radhakrishnan, Snijesh VP, Jyothi S Prabhu, Naveen Luke Demonte, Savitha Rajarajan, Yatish Patil, Ravi Diwakar, Sandhya Apachu, BS Srinath, Sridhar TS, Anguraj Sadanandam. Whole exome sequencing and BRCAness estimation in TNBCs and their correlation with response to platinum [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-19.

Integrin αvβ6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin β6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvβ6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin β6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvβ6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho–Rac pathway including downstream genes (ACTR2,ACTR3) and effector proteinases (MMP9,MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2‐ and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho–Rac pathway (P‐values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease‐free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9–8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node‐positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho–Rac pathway through increased expression of MMP9 and MMP15.

Background: Generation of BT-474 and MDA-MB-231 xenografts in immunocompromised mice provides one means of dissecting the molecular events involved in local invasion versus distant metastasis. Recently, Glunde et al have shown an interdependence of genes involved in cell-cell and cell-matrix adhesion (E-cadherin and integrin β1) and extra-cellular matrix degradation (MMP-2 and 9) in allowing cells to metastasize. Further, Fite el al have identified a set of microRNAs (miRs) up-regulated by E-cadherin (a pre-EMT marker) and down-regulated by Vimentin (post-EMT marker) in acquiring an invasive phenotype. We have performed a detailed analysis of integrins, matrix metallo-proteases and key miRs to better understand the molecular events underlying these disparate behaviours. Methods: We injected BT-474 (N=5) and MDA-MB-231 (N=5) cells orthotopically into SCID mice. Xenografts were assessed for local growth rate and monitored for distant metastasis. The implanted tumors as well as the distant metastatic foci were harvested. Markers involved in local invasion, distant metastasis and tumor-stroma interactions including miRs were compared between BT-474 and MDA-MB-231 cell lines and their xenografts by q-RT-PCR, immunofluorescence and immunohistochemistry. Results: As expected BT-474 xenografts showed a higher rate of tumor growth when compared to MDA-MB-231. Histological examination of BT-474 tumors confirmed only locally invasive tumor growth with infiltrated blood capillaries and vessels; no macro and microscopic metastases were observed in the organs collected. On the contrary, MDA-MB-231 xenografts showed highly undifferentiated tumor growth and frank lung metastasis and extra-pulmonary tumor growth in one of the five mice injected despite slow rate of local growth. Expression of matrix metalloproteases – MMP-2 & 9 was more than 30 fold upregulated in MDA-MB-231 xenografts as compared to BT-474. Elevated level of E-cadherin was observed in BT-474 but was absent in MDA-MB-231. The most interesting differences were seen in the levels of miRs and cell-surface integrins. High levels of miR-18a, miR-93 and miR-182 were observed in BT-474 implants when compared to MDA-MB-231 which had a much lower level of these miRs. On the contrary, higher levels of integrin β3, and β1 were observed in MDA-MB-231 tumors when compared to BT-474. Integrin β6 was absent in both. The reciprocal relationship between these markers is being examined and compared between locally invasive tumors and metastatic triple negative breast cancers from our case series of human specimens (N=250). Conclusion: miRs and integrins known to be involved in invasion are differentially regulated in tumors that are locally invasive compared to ones with distant metastasis. The level of the key targets of these miRs as well as additional integrins is being examined. Understanding the epigenetic regulations leading to metastasis via tumor-stroma interaction might help in discerning differential tumor behaviour. Citation Format: Lawrence PV, Desai K, Prabhu JS, Korlimarla A, Nair MG, Sridhar TS. Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-11.

The presence of a nuclear localization signal (NLS) in proteins can be inferred by the presence of a stretch of basic amino acids (KRKK). These NLSs are termed classical NLS (cNLS). However, only a fraction of proteins containing the cNLS pattern are transported into the nucleus by binding to importin α. Hence, there must exist, additional structural determinants that guide the appropriate interaction between putative NLSs containing cargo and importin α. Using 52 protein structures containing cNLS obtained from RCSB PDB, we assembled a training set and a validation set such that both sets were comprised of a combination of proteins with proven nuclear localization and ones that were non-nuclear. We modeled the interface between cargoes containing cNLS and importin α. We conducted rigid body docking and produced induced-fit modes by allowing both side chain and the backbone to be flexible. The output of these studies and additional determinants such as energy of interaction, atomic contacts, hydrophilic interaction, cationic interaction, and penetration of the cargo protein were used to derive a 26 parameter quantitative structure activity relationship based regression equation. This was further optimized by a step-wise backward elimination approach to derive a 15 parameter score. This NLScore was not only able to correctly classify confirmed nuclear and non-nuclear localized proteins but it was able to perform better than currently implemented algorithms like NucPred, Euk-mPLoc 2.0, cNls Mapper, and NLStradamus. Leave-one-out cross validation (LOOCV) showed that NLScore correctly predicted 78.6% and 81.6% of non-nuclear and nuclear proteins respectively. Graphical abstract NLScore: a novel quantitative algorithm based on 3 dimensional structural determinants to predict the probability of nuclear localization in proteins.

Introduction: The initial identification of HER2 as a driver in a subset of breast cancers was at the level of DNA (amplification), and subsequently noted at the level of transcripts and protein as well. However, the clinical selection of patients for treatment with Trastuzumab, has been through either IHC (protein) or FISH (DNA amplification) and not through transcript abundance. Interestingly, in most studies that have estimated transcript abundance in primary tumors, the proportion of patients that demonstrate increased transcript levels (termed HER2 Enriched) have tended to be slightly larger than the clinical HER2+ category. A more clinically useful measure might be proof of HER2 downstream activity that might help separate tumors being driven significantly by HER2 from ones where its role is supportive. One of the many consequences of HER2 over-expression is activation of the oncomiR, miR-21 via the MAPK pathway. miR-21 in turn is known to epigenetically regulate multiple targets including the tumor suppressors PTEN and PDCD4. While these molecular mechanisms have been demonstrated convincingly in breast cancer cell lines, clinical studies of these alterations in large numbers are yet to be reported. In this study we have examined the relationship between clinical HER2 positivity and miR-21 levels in 124 surgically excised breast cancer specimens. Methods: We selected 124 surgically excised specimens of primary breast cancers from our cohort that by HER2 immunohistochemistry (IHC) comprised 42 positive, 62 negative and 20 equivocal. Relative abundance of miR-21 was assessed using a TaqMan qRT-PCR, with normalization by RNU48. Relative transcript abundance of a set of 6 genes (HER2, GRB7, MLN64 and 3 reference genes) were evaluated by SYBR Green real time qPCR. Results: The majority of tumors that were clinically HER2+ over expressed miR-21. A concordance with an AUC of 96% at 100% sensitivity and 85% specificity was noted. There is a highly significant differential expression of miR-21 between HER2 positive, negative and equivocal samples (P < 0.0001). HER2 enriched score determined by using the expression levels of 3 genes (HER2, GRB7, MLN64) identified 35% (44/124) of the samples to be HER2 enriched. 72% of these (32/44) were also clinical HER2 positive by IHC. As expected, miR-21 was significantly over expressed in these tumors as well (P To identify all samples which might show HER2 downstream activity, a logistic regression model was built using expression of miR-21, HER2, MLN64 and GRB7 as the determinants of HER2 status. The best fitting model classified 91% (38/42) of HER2 +, 95% (59/62) of the HER2 negative accurately with 94% specificity and an AUC of 0.96. The model helped identify 10% of clinical HER2 negative samples (6/20 equivocal & 3/62 HER-2 negative) to have a high probability of being HER2+. Conclusion: Identification of HER2+ tumors with evidence of downstream activity may help identify patients with tumors being driven significantly by HER2 from ones where its role is supportive. The possibility of targeting miR-21 raises the tantalizing prospect of effecting change by altering the epigenetic regulation of multiple targets including tumor-suppressors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-09.

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

Background: Clinical epidemiology studies of breast cancer in India have reported younger age at detection, presentation at a later stage with a greater proportion of Triple Negative Breast Cancer (TNBC). The aim of this study was to examine the standard clinic-pathological variables in the hormone-receptor based sub-types for patterns indicative of intrinsic differences from that reported in Western, Caucasian women. Methods: Clinico-pathological variables from 645 patients who were diagnosed with breast cancer during 2012 at the regional cancer were retrospectively analyzed for clinical and immunohistochemistry details. Results: The median age at first diagnosis is 48 years which is decade earlier than that reported in Western case-series, 65% were lymph-node positive, and 33% of all cases were Triple negative Breast Cancers. Sub-type specific examination of tumor size and lymph-node (LN) status showed the HER2 positive tumors to have the highest proportion of tumors that were pT4 and 75% were LN positive. Conversely, despite 92% of TNBCs being grade 3, 40% of them were LN negative. Conclusion: We confirm the three cardinal clinical epidemiological features reported by other Indian centres. The clinical behavior of the HER2 positive and TNBC sub-types are no different from that reported in Western caseseries suggesting that these aspects are innate and conserved.

There is an ever increasing demand and innovation worldwide in the area of materials research in aerospace industry. Research on materials is the focused area in aerospace sector because reducing aircraft/spacecraft weight and/or increasing the temperature capabilities is the key factor in increasing the performance. Hence there is always an urge on the development of advanced materials especially in the area of light weight, High temperature resistant materials. There are different ways of keeping abreast on current state of art on the development of advanced materials namely reading Journal publications, attending conferences/seminars or performing intellectual property (IP) analysis specifically looking at patents. Patent portfolio indicates the technology and research direction of a company. Intellectual property plays a key role in the present global competitive world. It is not only an asset for the company but also helps in increasing the bottom line of an organization. Hence, an attempt was made to look at the global patent scenario of materials used for aerospace industry. A high level scan of aerospace materials patent portfolio in the past 20 years was done to understand the current technology focus and trend of materials research. The study was restricted to Aerospace/Aviation Players. Four different types of Materials are considered for the study namely Light weight metals (includes Al and Ti alloys), Superalloys, High temperature coatings and Composites. A further deep dive analysis on the above materials has been done to understand the Industry focus on various technology areas such as Manufacturing, Inspection, Application etc. This analysis helps technologists in understanding the current state of art and research focus in Materials area specific to Aerospace/Aviation Industry

Introduction: Breast cancer is the leading cancer diagnosed in urban Indian women. Historically the proportion of women presenting with advanced disease has been in excess of 50% at most regional cancer centres. The past 20 years has witnessed rapid economic growth and urbanization with significant gains in women's education and access to health. However, it is not clear if this has translated to earlier seeking of care in the educated. The aim of this study was to examine if higher education (College) was a determinant in the time of seeking first medical consultation, and whether there were significant differences between women under the age of 40 versus women over 60 years old at the time of diagnosis. Methods: The data for analysis were obtained from a prospective longitudinal observational study conducted between 2008-2013 at a medical teaching hospital and a tertiary specialized cancer care centre. A total of 460 patients have been enrolled so far. All patients provided informed consent and the study has been reviewed and approved by the institutional ethics committees at these institutions. We have collected from the patients and their medical records information about their age at diagnosis, educational level, stage of disease, histopathology reports and clinical details. Results: Data from 194 patients have been used for the analysis. Patients were divided into one group of < = 40Y of age (Group I, N = 58) and the second of > = 60 years of age (Group II, N = 136). Mean age at presentation for the groups was 35 and 68 years respectively. As expected almost 1.5 times as many women under the age of 40 (36%) were college educated compared to the women over 60 (22%) (p = 0.05). The proportion of LABC in the two groups was not different with group I having 27% and group II 25%. The proportion of older women with LABC and a college degree was 20%. However, rather than the expected decrease in proportion of women with high education and LABC, 31% of young women with LABC had a college degree. LABC in the college educated was not different in the group I when compared to group II. (p = 0.39). Conclusion: The determinants of seeking health care are complex and influenced by a variety of factors including socio-economic status, access to health care, education, cultural beliefs, and personal preferences. While urban India's steady economic growth has been highlighted by the scholarly as well as the lay press, the data presented here suggest that there is not an automatic and linear transfer between education, economics and healthcare seeking behaviours. We suggest that while a lot of attention has been paid to awareness and screening, we may need to focus on local cultural factors, and perhaps provide support from female counsellors and care providers as critical components of attempting to bring our women to the hospital at the earliest. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-08-12.

Introduction Imprinted genes play an important role in mammalian fetoplacental growth and development. We have evaluated whether the placental expression of two imprinted genes, growth factor receptor-binding protein 10 ( GRB10 ) and pleckstrin homology-like domain, family A, member 2 ( PHLDA2 ) correlate with human fetoplacental growth parameters. Methods Placentae (n = 77) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 36 SGA and 41 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of GRB10 and PHLDA2 normalized to a panel of reference genes. Results Placental GRB10 transcript abundance associated positively with placental weight (r = 0.307, P = 0.007), birth weight (r = 0.267, P = 0.019) and neonatal head circumference (r = 0.280, P = 0.014). Placental GRB10 transcript levels were significantly lower in male SGA placentae compared to the male AGA placentae. Placental PHLDA2 transcript abundance did not show any associations with maternal, placental or neonatal parameters. Discussion Placental GRB10 expression was found to be associated positively with placental weight, birth weight, and neonatal head circumference, especially in males. Hence, we speculate that placental GRB10 plays a role in regulating fetoplacental growth and thereby in the pathophysiology of fetal growth restriction in the context of fetal gender.

The purpose of the present study was to evaluate the frequency of microsatellite instability (MSI) in colorectal cancers in an Indian cohort. Paraffin embedded tissue samples of colorectal cancers from 46 patients were assessed for mismatch repair protein expression (hMLH1 and hMSH2) by immunohistochemistry. Subsequently, MSI analysis was done after PCR amplification of five Bethesda markers. Amongst 46 cases studied, only 5 patients (10.8%) showed MSI. Out of these, two (4.3%) had high microsatellite instability (MSI-H) and three (6.5%) showed low microsatellite instability (MSI-L). Out of 46 cases, 41 were microsatellite stable (MSS). In the 46 cases tested by immunohistochemistry, 7 (15.7%) showed the absence of hMLH1 and 1 case showed the absence of hMSH2. Our study indicates a similar rate of incidence of MSI in colorectal cancers in the Indian cohort compared to the West (10–15%) despite lower incidence of colorectal cancers and predominance of rectosigmoid tumors in the Indian population.

Kinetic data are reported for the reaction of cyclopropane with hydrogen over a series of nickel catalysts supported on alumina and silica-alumina. Metal areas of the catalysts were measured by hydrogen chemisorption. Both hydrogenation to propane and hydrocracking to equimolar amounts of ethane + methane occur. Over all the catalysts, both reactions are zero order in hydrogen and approximately 0.6 order in cyclopropane. Activation energies are given and it appears the activation energy for the hydrocracking reaction is approximately 2–4 kcal greater than that for the hydrogenation reaction. The selectivity of the catalyst (rate of hydrogenation/rate of hydrocracking) appears to be quite sensitive to the nature of the support and the metal dispersion. The total specific activity based on nickel area is however approximately the same for all the catalysts studied. It is tentatively suggested that both reactions proceed through a series path involving a common overall rate-determining step.

Kinetic data are presented for the reaction of cyclopropane with hydrogen over four supported nickel catalysts. Both hydrogenation and hydrocracking reactions occur to yield propane, ethane, and methane; but, on the basis of the kinetic evidence, it is suggested that the reactions proceed through a series mechanism involving a common rate controlling step. Analysis of the kinetic data by the method of Hougen and Watson gave, as the best fit rate equation for all four catalysts, r = kKcpc(1 + Kcpc)−1. The values of the cyclopropane adsorption equilibrium constant Kc obtained from the kinetic data by regression analysis are quite similar for all four catalysts and agree well with independently measured values derived from experimental equilibrium isotherms for cyclopropane adsorption. It is suggested that the rate determining step is the cleavage of the cyclopropane ring to form the 1–3 diadsorbed species from a physically adsorbed or weakly chemisorbed precursor. This mechanism appears to be consistent with evidence from deuterium exchange studies. Results of catalyst pretreatment studies are briefly summarized.

We provide evidence for the downregulation of phospho-tyrosine phosphatases (PTPases) in malignancy. AK-5, a rat macrophage tumor, shows the downregulation of the transcripts of two non-receptor-type PTPases, PTP-1 and PTP-S. Though downregulated fourfold, the genomic organization of PTP-S is unaltered. There is no gross alteration of the PTPase activity in AK-5 as compared to macrophages. Immunoblot analysis reveals no significant change in the total phospho-tyrosine levels in AK-5, but there is a qualitative difference in the pattern between AK-5 and macrophages. Our results lend credence to the conjecture that PTPases also might be involved in malignancy.