Beena Kumar

The prognostic significance of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 expression in early stage non-small cell lung cancer

Background Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 has been functionally associated with a stem cell phenotype in normal and malignant cells. The prevalence of such cells in solid tumours should therefore correlate with recurrence and/or metastasis following definitive surgical resection. The aim of this study was to evaluate the prognostic significance of ALDH1A1 and CD133 in surgically resected, early stage non-small cell lung cancer (NSCLC). Methods A retrospective analysis of ALDH1A1 and CD133 expression in 205 patients with pathologic stage I NSCLC was performed using immunohistochemistry. The association between the expression of both markers and survival was determined. Results We identified 62 relapses and 58 cancer-related deaths in 144 stage 1A and 61 stage 1B patients, analysed at a median of 5-years follow-up. Overexpression of ALDH1A1 and CD133, detected in 68.7% and 50.7% of primary tumours, respectively, was an independent prognostic indicator for overall survival by multivariable Cox proportional hazard model (p=0.017 and 0.039, respectively). Overexpression of ALDH1A1, but not of CD133, predicted poor recurrence-free survival (p=0.025). When categorised into three groups according to expression of ALDH1A1/CD133, patients with overexpression of both ALDH1A1 and CD133 belonged to the group with the shortest recurrence-free and overall survival (p=0.015 and 0.017, respectively). Conclusions Expression of ALDH1A1 and CD133, and coexpression of ALDH1A1 and CD133, is strongly associated with poor survival in early-stage NSCLC following surgical resection. These data are consistent with the hypothesis that expression of stem cell markers correlates with recurrence as an indirect measure of self-renewal capacity.

E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis

Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.

Lymphatics in the human endometrium disappear during decidualization

BACKGROUNDnThe mammalian placenta plays a central role in maternal tolerance of the semi-allogeneic fetus and fluid balance between the maternal and fetal compartments. The lymphatics play a role in both these function. The aim of this study was to describe the distribution of lymphatic vessels in human decidua, with particular focus on the lymphatics that surround remodelling spiral arteries during decidualization and trophoblast invasion.nnnMETHODSnPlacental bed and non-placental bed (decidua parietalis) biopsies were obtained from 41 women undergoing elective termination of pregnancy at 6-18 weeks gestational age as well as placental bed biopsies from 5 women undergoing elective Caesarean section at term. In addition to routine haematoxylin and eosin staining, double immunohistochemical labelling was performed on serial 3-µm sections to identify lymphatic vessels in conjunction with one of the following: blood vessels, smooth muscle, epithelial and trophoblast cells or proliferating cells. Representative photomicrographs of all sections were obtained from a total of 273 areas (46 samples, average 6 range 3-15 areas per sample). Descriptive findings of the organization of lymphatics in human placental bed and decidua parietalis were made from a total of 1638 images.nnnRESULTSnLymphatic vessels positive for podoplanin were abundant in non-decidualized hypersecretory endometrium at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics could be observed at the boundary between non-decidualized hypersecretory and decidualized endometrium. Lymphatic vessels were notably absent from the vicinity of spiral arteries that were surrounded by decidualized stromal cells. Lymphatic vessels in non-decidualized hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. Placental bed lymphatics exhibited limited and variable staining with LYVE-1 at all stages of pregnancy apart from term.nnnCONCLUSIONSnWe have made novel observations on lymphatics in the placental bed and their relationship with other structures throughout pregnancy. Endometrial stromal cell decidualization results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arteries.

Biodosimetric quantification of short-term synchrotron microbeam versus broad-beam radiation damage to mouse skin using a dermatopathological scoring system

OBJECTIVESnMicrobeam radiotherapy (MRT) with wafers of microscopically narrow, synchrotron generated X-rays is being used for pre-clinical cancer trials in animal models. It has been shown that high dose MRT can be effective at destroying tumours in animal models, while causing unexpectedly little damage to normal tissue. The aim of this study was to use a dermatopathological scoring system to quantify and compare the acute biological response of normal mouse skin with microplanar and broad-beam (BB) radiation as a basis for biological dosimetry.nnnMETHODnThe skin flaps of three groups of mice were irradiated with high entrance doses (200 Gy, 400 Gy and 800 Gy) of MRT and BB and low dose BB (11 Gy, 22 Gy and 44 Gy). The mice were culled at different time-points post-irradiation. Skin sections were evaluated histologically using the following parameters: epidermal cell death, nuclear enlargement, spongiosis, hair follicle damage and dermal inflammation. The fields of irradiation were identified by γH2AX-positive immunostaining.nnnRESULTSnThe acute radiation damage in skin from high dose MRT was significantly lower than from high dose BB and, importantly, similar to low dose BB.nnnCONCLUSIONnThe integrated MRT dose was more relevant than the peak or valley dose when comparing with BB fields. In MRT-treated skin, the apoptotic cells of epidermis and hair follicles were not confined to the microbeam paths.

Changes in aldehyde dehydrogenase-1 expression during neoadjuvant chemotherapy predict outcome in locally advanced breast cancer

IntroductionAlthough neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can improve operability and local disease control, there is a lack of reliable biomarkers that predict response to chemotherapy or long-term survival. Since expression of aldehyde dehydrogenase-1 (ALDH1) is associated with the stem-like properties of self-renewal and innate chemoresistance in breast cancer, we asked whether expression in serial tumor samples treated with NAC could identify women more likely to benefit from this therapy.MethodsWomen with locally advanced breast cancer were randomly assigned to receive four cycles of anthracycline-based chemotherapy, followed by four cycles of taxane therapy (Arm A), or the same regimen in reverse order (Arm B). Tumor specimens were collected at baseline, after four cycles, and then at surgical resection. ALDH1 expression was determined by immunohistochemistry and correlated with tumor response using Fisher’s exact test while Kaplan-Meier method was used to calculate survival.ResultsA hundred and nineteen women were enrolled into the study. Fifty seven (48%) were randomized to Arm A and 62 (52%) to Arm B. Most of the women (90%) had ductal carcinoma and 10% had lobular carcinoma. Of these, 26 (22%) achieved a pathological complete response (pCR) after NAC. There was no correlation between baseline ALDH1 expression and tumor grade, stage, hormone receptor, human epidermal growth factor receptor 2 (HER2) status and Ki67 index. ALDH1 negativity at baseline was significantly associated with pCR (Pu2009=u20090.004). The presence of ALDH1(+) cells in the residual tumor cells in non-responding women was strongly predictive of worse overall survival (Pu2009=u20090.024). Moreover, serial analysis of specimens from non-responders showed a marked increase in tumor-specific ALDH1 expression (Pu2009=u20090.028). Overall, there was no survival difference according to the chemotherapy sequence. However, poorly responding tumours from women receiving docetaxel chemotherapy showed an unexpected significant increase in ALDH1 expression.ConclusionsALDH1 expression is a useful predictor of chemoresistance. The up-regulation of ALDH1 after NAC predicts poor survival in locally advanced breast cancer. Although the chemotherapy sequence had no effect on overall prognosis, our results suggest that anthracycline-based chemotherapy may be more effective at targeting ALDH1(+) breast cancer cells.Trial registrationACTRN12605000588695

Mitochondrial DNA plasticity is an essential inducer of tumorigenesis.

Although mitochondrial DNA has been implicated in diseases such as cancer, its role remains to be defined. Using three models of tumorigenesis, namely glioblastoma multiforme, multiple myeloma and osteosarcoma, we show that mitochondrial DNA plays defining roles at early and late tumour progression. Specifically, tumour cells partially or completely depleted of mitochondrial DNA either restored their mitochondrial DNA content or actively recruited mitochondrial DNA, which affected the rate of tumorigenesis. Nevertheless, non-depleted tumour cells modulated mitochondrial DNA copy number at early and late progression in a mitochondrial DNA genotype-specific manner. In glioblastoma multiforme and osteosarcoma, this was coupled with loss and gain of mitochondrial DNA variants. Changes in mitochondrial DNA genotype affected tumour morphology and gene expression patterns at early and late progression. Importantly, this identified a subset of genes that are essential to early progression. Consequently, mitochondrial DNA and commonly expressed early tumour-specific genes provide novel targets against tumorigenesis.

Infusional dose-adjusted epoch plus bortezomib for the treatment of plasmablastic lymphoma

Dear Editor, Plasmablastic lymphoma (PBL) is a rare disease with a poor prognosis with standard chemotherapy. We report an HIV-negative patient with a diagnosis of stage IV PBL successfully treated with bortezomib in combination with infusional dose-adjusted EPOCH (V-EPOCH). A 72-year-old man presented with weight loss of 10 kg over 3 months and progressive lethargy. Performance status was ECOG 0. His medical history includes rheumatoid arthritis, stable on a combination of prednisolone, leflunomide, sulfasalazine, hydroxychloroquine and etanercept. Laboratory testing revealed a mild normocytic anaemia, raised lactate dehydrogenase (LDH) of 302 U/L (100–200), and raised serum kappa free light chains at 349.1 mg/L (3.3– 19.4) with normal lambda light chains and normal serum protein electrophoresis. Serology was positive for EBV IgG and negative for CMV, HIV, hepatitis B and C. PET/CT identified extensive thoracic and abdominal lymphadenopathy; a large left sided pleural effusion and extra-nodal involvement of the spleen, stomach, bone and pleura (Fig 1). CT-guided biopsy was diagnostic for plasmablastic lymphoma with strong positivity for CD138, kappa-ISH and EBV-ISH. The Ki67 proliferative index was 75 %. Fluorescence in situ hybridisation revealed the presence of t(8;14). Bone marrow biopsy was uninvolved. Final disease staging was IVB. Treatment was commenced with dose-adjusted EPOCH [1] in combination with bortezomib 1.3 mg/m2 subcutaneously day 1 and 8 of each cycle. Supportive care included filgrastim 300 mcg, prophylactic valacyclovir and trimethoprim/sulfamethoxazole. The patient received a total of 6 cycles of V-EPOCH and six doses of intrathecal methotrexate (12.5 mg) as CNS prophylaxis. Treatment was well tolerated and he achieved a complete metabolic remission after cycle two (Fig 1). He remains in clinical remission more than 2 years later. The lack of efficacy of CHOP or CHOP-like regimens in PBL has led to the use of more intensive therapies, including infusional DA-EPOCH, following evidence of its efficacy in DLBCL [2]. Bortezomib has well-established efficacy in the treatment of multiple myeloma with emerging evidence of potential efficacy in relapsed/refractory non-germinal centre DLBCL [3]. PBL is typified by the expression of the plasma cell transcription factors Irf4, Blimp1 and Xbp1 [4–6]. Expression of Xbp1 and Blimp1 has been linked with efficacy of bortezomib in myeloma [7, 8], likely due to driving antibody production and hence reliance on the unfolded protein response [9]. In addition, Xbp1 mutations have been shown to lead to disease resistance [8]. The successful use of V-EPOCH has recently been reported in a small case series of two HIV-associated and one non-HIV case of PBL [10]. Our case adds further support to the efficacy of this approach, and demonstrates its tolerability in an older patient. Although the retrospective report of four patients is inadequate for definitive conclusions, the consistency of our experience with that of Castillo and colleagues is encouraging. The reduced frequency of bortezomib dosing (days 1 and 8 * Pasquale L. Fedele pasquale.fedele@monashhealth.org

Endoscopic ultrasound-guided fine-needle aspirate-derived preclinical pancreatic cancer models reveal panitumumab sensitivity in KRAS wild-type tumors

Pancreatic cancer (PC) is largely refractory to existing therapies used in unselected patient trials, thus emphasizing the pressing need for new approaches for patient selection in personalized medicine. KRAS mutations occur in 90% of PC patients and confer resistance to epidermal growth factor receptor (EGFR) inhibitors (e.g., panitumumab), suggesting that KRAS wild‐type PC patients may benefit from targeted panitumumab therapy. Here, we use tumor tissue procured by endoscopic ultrasound‐guided fine‐needle aspirate (EUS‐FNA) to compare the in vivo sensitivity in patient‐derived xenografts (PDXs) of KRAS wild‐type and mutant PC tumors to panitumumab, and to profile the molecular signature of these tumors in patients with metastatic or localized disease. Specifically, RNASeq of EUS‐FNA‐derived tumor RNA from localized (nu2009=u200920) and metastatic (nu2009=u200920) PC cases revealed a comparable transcriptome profile. Screening the KRAS mutation status of tumor genomic DNA obtained from EUS‐FNAs stratified PC patients into either KRAS wild‐type or mutant cohorts, and the engraftment of representative KRAS wild‐type and mutant EUS‐FNA tumor samples into NOD/SCID mice revealed that the growth of KRAS wild‐type, but not mutant, PDXs was selectively suppressed with panitumumab. Furthermore, in silico transcriptome interrogation of The Cancer Genome Atlas (TCGA)‐derived KRAS wild‐type (nu2009=u200938) and mutant (nu2009=u2009132) PC tumors revealed 391 differentially expressed genes. Taken together, our study validates EUS‐FNA for the application of a novel translational pipeline comprising KRAS mutation screening and PDXs, applicable to all PC patients, to evaluate personalized anti‐EGFR therapy in patients with KRAS wild‐type tumors.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2−ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success. Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples. We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors. Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype. Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer

SummaryPreclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (nu2009=u20095) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IRu2009+u2009C) or standard (IRu2009+u2009C) treatment, to a maximum of 6xa0cycles. The second line patients were added to the study and treated with open label HA-IRu2009+u2009C. Tumour response was measured after every 2xa0cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39–83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28xa0weeks, respectively (pu2009=u20090.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44su2009+u2009cancer stem-like cells provide an early hint towards a potential novel target.