Basal Types Research Articles

MP55-07 PREDICTIVE AND PROGNOSTIC ROLE OF A SIMPLE IMMUNO-PHENOTYPICAL SCORE IN CLINICAL MANAGEMENT OF PATIENTS WITH PT2 INVASIVE UROTHELIAL CARCINOMA

INTRODUCTION AND OBJECTIVE: The mapping of the genome through the TCGA collaborative project recently revealed major intrinsic molecular subtypes, Basal, Luminal, and Neural muscle-invasive urothelial carcinoma (UC), which have shown prognostic and predictive values. A standardized approach in daily practice, which is simple, fast, reproducible, and at low cost has not yet been identified. We provide to test the hypothesis that immuno-phenotypical score classification (Piescore) may discriminate Luminal from Basal from Neural UC, and to find an association of tumor Piescore with histological and clinical prognostic variables in a mono-institutional cohort of muscle-invasive (pT2) UC treated with trans-urethral resection and radical cystectomy (RC). METHODS: Primary endpoint is to stratify Luminal, Basal or Neural tumors according to a simple immuno-phenotypical score. Consecutive transurethral resection specimens harbored foci of high grade, pT2 (MIBC) from 75 pts who subsequently underwent RC, have been submitted for immunohistochemical analysis, using relevant gene-expression-based markers for Basal type (CD44, CK5/6) and Luminal type (CK20 and pPARg). Piescore divided the tumors into Basal and Luminal types when at least 3 of 4 markers were consistent with a specific phenotype. RESULTS: Luminal and Basal phenotype was present in 32 pts and 29 pts (43% and 39% respectively) while in 7 pts (9% of cases) the Piescore indentified a mixed phenotype (when 2 Luminal markers and 2 Basal markers were present simultaneously). No expression was identified in 7 pts (9%): 4 cases (5%) with morphological neuroendocrine differentiation and 3 cases (4%) with classical urothelial differentiation, all consistent with Neural phenotype. Basal Piescore with all markers consistent with basal differentiation (CD44+, CK5/6+, CK20-, pPARg-) was associated with advance stage at cystectomy (pT3-4 vs pT0-1-2, 73% vs 27% of cases respectively). Only necrosis, among histopathological parameters considered, was statistically correlated with Basal phenotype (OR: 3.3, p: 0.030), while no association with lymph-vascular invasion, papillary subtype, primary tumor, and concomitant CIS was found. CONCLUSIONS: Piescore immunophenotyping could be a simple surrogate in alternative to genome profiling, able to stratify UC-TURB patients between Luminal vs Basal type. The basal phenotype identified with immunohistochemical markers CD44, CK5/6, CK20 and pPARg seems to stratify more aggressive UC at cystectomy. Neural Piescore also identified a subset of high-grade carcinomas that harbored classical features of UC at histology. Indeed, the biological significance of the expression of multiple phenotypes in the same UC (Piescore mixed tumors) has to be further evaluate. Further studies are mandatory in order to confirm and extend our finding to clinical utility. Source of Funding: None.

Urothelial Carcinoma In Situ (CIS): New Insights.

Urothelial carcinoma in situ (CIS) is a high-grade noninvasive malignancy with a high tendency of progression. Although it is typically grouped with other nonmuscle invasive bladder cancers, its higher grade and aggressiveness make it a unique clinical entity. Urothelial CIS is histologically characterized by replacement of the urothelium by cells which fulfill the morphologic criteria of malignancy including nuclear pleomorphism, hyperchromasia, prominent nucleoli, and increased numbers of normal and abnormal mitoses. Urothelial CIS may be categorized as primary when it is not associated with any past or present urothelial carcinoma. It is termed as secondary when there is concomitant or previous urothelial carcinoma in the patient. In recent years detailed molecular studies have provided valuable data for intrinsic molecular subclassification of urothelial carcinoma into 2 broad categories namely luminal and basal types with significant implications for prognosis and therapy. Similar studies on urothelial CIS are limited but have provided crucial insight into the molecular basis of CIS. These studies have revealed that urothelial CIS may also be divided into luminal and basal subtypes, but luminal subtype is much more common. It has also been shown that in many cases, luminal type of urothelial CIS may undergo a class switch to basal type during progression to an invasive carcinoma. Additional studies may be required to confirm and further elaborate these findings.

Euconodont hard tissue: preservation patterns of the basal body

AbstractConodont elements, consisting of crown and basal tissue are the well‐known fossilized hard parts of Conodonta (extinct marine chordates), but the taphonomic processes leading to decomposition or remineralization of the basal tissue are not well understood. Here we focus on the taphonomy of basal tissue, reviewing the published record and describing new material from Asia and Europe (248 occurrences globally). These include crown and basal tissue in conjunction, and isolated basal bodies showing different stages of preservation. Some isolated specimens resemble phosphate rings similar to those assigned to Phosphannulus universalis. High‐resolution biostratigraphy indicates that the lamellar type of conodont basal tissue is found in all facies and depositional environments. Other basal tissue types, described in the literature as tubular, mesodentine, spherulitic or lamellar with canalules, are limited to the early Palaeozoic and found exclusively in siliciclastic deposits (with the exception of spherulitic tissue). Although the stratigraphic record of basal tissue spans the range of Euconodonta (Cambrian–Triassic), this study shows that most of the isolated plate and ring‐like structures are derived from early Palaeozoic coniform conodonts. Basal tissue of platform‐type elements has a much more fragile shape and is therefore rarely preserved as a recognizable isolated unit.

Morphogenesis of fruits and types of fruit of angiosperms

ABSTRACTMorphological and anatomical fruit characters are used for description of morphogenetic fruit types within eight traditional fruit types – follicle, nutlet, drupe, berry, capsule, pyrenarium, amphisarca and nut, which describe the major diversity of fruits of all angiosperms. The traditional fruit types are representing levels of fruits organization and are described based on morphological characters (gynoecium type, carpels phyllotaxis, fruiting carpel dehiscence or indehiscence, number of seeds per fruit, and the presence or absence of the continuous sclerenchymatous zone in the fruit wall), whereas morphogenetic fruit types are described based on fruit wall (pericarp) anatomy. Fundamental differences between basal types of pericarp structure of the outlined morphogenetic fruit types lie in the localization (in the exocarp, specific zone of the mesocarp and/or endocarp) of the continuous sclerenchymatous zone, which secures protection of seeds, fruit dehiscence in follicles and capsules, and dissemination. Twenty seven morphogenetic fruit types are recognized in the current research: four types of follicle (Hakea, Illicium, Myristica and Talauma types), two types of nutlet (Nelumbo and Rosa types), three types of drupe (Laurus, Prunus and Rhapis types), two types of berrie (Schisandra and Nuphar types), seven types of capsule (Bombax, Eriocoelum, Forsythia, Galanthus, Hamamelis, Lilium and Nepenthes types), four types of pyrenarium (Butia, Ilex, Latania and Olea types), two types of amphisarca (Adansonia and Theobroma types), and three types of nut (Corylus, Polygonum and Centaurea types) in angiosperms. The modes of evolutionary transformations of main morphogenetic types of fruits are described.

Abstract 4408: CDK4/6 inhibitor(palbocilcib) efficacy for head and neck squamous cell carcinoma(HNSCC), including both HPV+ and HPV- cell lines

Abstract Background: Betel nut chewing might contribute to (1)strong inflammation, angiogenesis, & invasion; (2)easy recurrence. Our group had found a betel-nuts exposed HNSCC cell line, TW2.6, was resistant to chemotherapies, radiation, and EGFR inhibitors. Defective p53 mutation, p16 loss, and BCL2 overexpression were seen in TW2.6. Astragalus polysaccharides, PI3Kalpha inhibitor, AKT inhibitor, FGFR inhibitor, ALK/IGF1R inhibitor, CDK4/6 inhibitor, BCl2 inhibitor, WEE1 inhibitor, ATR inhibitor, & VEGFR2/ PDGFR/FGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, through the inhibition of mesenchymal transformation, pRB, & PI3K/AKT /mTOR signaling and the modulation of stemness & PD1/PDL1 pathway. TW2.6 might be similar to HPV- EMT subtype in TCGA. CDK4/6 inhibitor was proved to be effective in HPV-/pRB+ HNSCC and had strong immuno-modulatory effects. In our prior works, palbociclib could resensitize TW2.6 to docetaxel, afatinib, & radiation and enhance further response to BYL719 & foretinib(VEGFR2/c-MET/Axl triple inhibitor). Purpose and methods: To test palbociclib efficacy in HNSCC cell lines with different molecular phenotypes. SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, and TW2.6 evaluated for (1)in vitrosensitivity to palbociclib; (2)synergistic effect of palbociclib with other therapies by MTT assay, colony formation, flow cytometry, and western blotting. Results: Cell linesSCC25KBSASCAL27FaDuSCC15SCC9SCC4TW2.6Differ- entiationWellPoorPoorPoorPoorWellWellWellWell, but rapidly replicated, with high hyper-diploidy & complex rearrangementsHPV statusHPV 16/18HPV18--HPV 16/18--HPV 6/11-EGFR statusMediumLowHighHighMediumHighLowMedium to highUnknownDocetaxel sensitivity+++++++++++++ to +++++-+Cisplatin sensitivity+++++++++++++- to +-- to +5-FU sensitivity+++++++++++-+ to ++-- to +EGFR inhibitor sensitivity+++- to +-+++++ to +++++++++-Polo-like kinase Inhibitor sensitivity+++++++++++++ to +++- to +-- to +VEGFR2 Inhibitor sensitivity----+++++--++CDK4/6 Inhibitor response+++- to ++++++ to +++++++++++++ to +++Western blotsWeak p-AKT & VEGF-A, mild PDL1 and BMI-1, Gli-1(+)Weak p-AKT, mild PDL1 and strong VEGF-A & BMI-1, p16(+)Moderate p-AKT & BMI-1, high PDL1, mild VEGF-AHigh p-AKT & VEGF-A, mild PDL1 & BMI-1High VEGF-A, moderate p-AKT & PDL1, weak BMI-1, Gli-1(+)Weak p-AKT & VEGF-A, mild PDL1 & BMI-1Weak p-AKT, VEGF-A, & BMI-1, moderate PDL1Moderate p-AKT & VEGF-A, strong BMI-1, mild PDL1, Gli-1(+)High p-AKT, PDL1, & VEGF-A and moderate BMI-1OutcomesBest; like TCGA CL (HPV+) subtypeLike TCGA basal subtype, but responded to particular treatments eachBasalBasalLike TCGA mesen-chymal subtype (HPV+)Like TCGA CL(HPV-) subtype, different characters between these 3 cell linesCL(HPV-) subtypeCL(HPV-) subtypeWorse; like TCGA EMT subtype (HPV-) Conclusion: Palbociclib had great efficacy over SCC15(classical HPV- type with EGFR overexpression) followed by SCC25, SAS, TW2.6, & CAL27; but little efficacy over KB. In HPV+ cell lines, palbociclib had promising response on SCC25(classical HPV+ type) and little response on FaDu(HPV+ mesenchymal type) & KB(basal type). SAS & CAL27, also basal types, responded well to palbocilclib. Further NGS analysis may translate these cell lines to represent TCGA subtypes for translational research. Citation Format: Jo-Pai Chen, Jui-Ying Chang, Reuy-Long Hong. CDK4/6 inhibitor(palbocilcib) efficacy for head and neck squamous cell carcinoma(HNSCC), including both HPV+ and HPV- cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4408.

Race and response to neoadjuvant chemotherapy according to MammaPrint risk.

578 Background: African-American (AA) women with breast cancer have a less favorable prognosis, likely due to differences in tumor biology. This is not only driven by the higher rate of triple negative/basal tumors in patients with AA ancestry, as worse outcome has also been seen in patients with luminal tumors. The Neoadjuvant BReast Cancer Symphony Trial (NBRST, NCT01479101) was a prospective trial that has shown an association of MammaPrint/BluePrint (MP/BP) with a rate of pathologic Complete Response (pCR) of 2% in Luminal A with 95% Distant Metastasis Free Interval at 3 years. Here, we determine the MP/BP risk distribution, response to therapy, and outcome in African American (AA) and Caucasian (Cau) patients. Methods: NBRST enrolled 1,072 breast cancer patients (pts) in the US (June 2011 and December 2014), median follow-up 34.9 months. The current unplanned analysis compared clinicopathological characteristics, molecular risk assignment and outcome with neoadjuvant chemotherapy (NACT) in AA and Cau pts. Molecular subtyping groups were assessed by MP/BP as follows: Luminal A (MammaPrint Low Risk), Luminal B (MammaPrint High Risk), HER2 and Basal types. Results: Out of 1,072 pts, 157 (15%) were AA, and 780 (73%) were Cau. AA patients were younger at diagnosis (52 vs 54 yrs; p = 0.016), had a higher likelihood of having higher grade (gr 3, 65% vs 53%; p = 0.005), ER-negative (45% vs 33%; p = 0.005) and lymph node positive tumors (71% vs 51; p < 0.001). MP/BP classified more AA patients as Basal type, 45% compared to 33% of Cau patients (p = 0.004). Fewer AA patients were classified as Luminal A (15%) compared to Cau pts (33%; p = 0.004). In multivariate analysis race was a significant factor for higher pCR rates to NACT in AA compared to Cau pts, together with PR, HER2, T-stage and Grade (HR = 1.679, 95% CI = (1.057, 2.67), p = 0.028). The pCR rate to NACT in patients with Basal tumors was 38% and similar in AA and Cau patients. In patients with hormone receptor positive and HER2 negative tumors, patients classified by MP/BP as Luminal A had lower pCR (2%) compared to non-luminal A (13%) (p = 0.0015). MP low risk patients had higher 3 yr DMFS (97%) than MP high risk patients (86%; p = 0.010). DMFS for AA MP Low Risk patients was 100%. Conclusions: In this study, MP was able to identify patients with hormone receptor positive tumors with low sensitivity to chemotherapy and good outcome, irrespective of race, suggesting that this test can be helpful to characterize the tumor’s biology and select patients who will not benefit from chemotherapy independently of their ancestry. Clinical trial information: NCT01479101.

Antennal sensory structures in water bugs of Nepoidea (Insecta: Hemiptera: Nepomorpha), their morphology and function

The Nepoidea superfamily belongs to the water bugs’ infraorder (Nepomorpha) and consists of two families—Belostomatidae and Nepidae. Species from those families are the largest of all nepomorphans and are considered to be top predators in aquatic ecosystems. A characteristic feature of the group is the existence of short antennae concealed in grooves behind the eyes, which is an adaptation to the water habitat. The antennae bear many types of sensillar structures, which receive signals from the environment. Among such structures, mechanosensilla were of the greatest diversity. The antennal sensilla of species from both families were examined under the scanning electron microscope. 11 essential morphological types of sensilla were distinguished, including 5 new mechanosensilla types (sensilla paddle-like, cone-like, squamiformia, brush-like and club-like). Basal types of mechanosensilla such as trichodea, chaetica, basiconica (subtype 1) and campaniformia occur in Nepoidea and other Heteroptera. In some representatives of both families, sensilla paddle-like and sensilla basiconica type 1 were observed. Moreover, sensilla chaetica and cone-like were found in some species of Belostomatidae, whereas in Nepidae sensilla squamiformia, brush-like and club-like were observed. Apart from mechanosensilla, one type of thermo-hygrosensilla (ampullacea) and two different shaped olfactory sensilla basiconica (subtypes 2, 3) and coeloconica (subtypes 1, 2, 3) were found. It could indicate sensilla use in the identification of the water reservoirs and locating prey.

Разнообразие клеточных типов у гаплотипа H4 Placozoa sp.

Эволюционная ветвь Пластинчатых (Placozoa) имеет большое значение для понимания происхождения и эволюции Metazoa. Представители типа Placozoa обладают простым строением тела, однако полное отсутствие органов, нервной системы и мышечных клеток резко контрастирует с достаточно сложными поведенческими реакциями, в том числе динамическими изменениями формы тела. Известно 19 гаплотипов Placozoa, включающих два вида — Trichoplax adhaerens (H1) и Hoilungia hongkongensis (H13) — и пока ещё малоисследованные штаммы, обозначаемые как H2–H19 Placozoa sp. Хотя все Placozoa имеют похожий фенотип (пластинчатую дископодобную форму тела), анализ клеточной организации был сделан только для H1 (Trichoplax adhaerens) с идентификацией шести типов клеток. В этой работе мы, используя конфокальную и электронную микроскопию, исследовали организацию штамма H4, который близок к новому роду — Hoilungia. У H4 обнаружены все шесть основных типов клеток, идентифицированных ранее у H1. Между тем мы выявили у штамма H4 большее разнообразие клеток по форме, плотности и, впервые, по мембранному потенциалу митохондрий, чем ранее было описано для H1. Это позволяет нам подчеркнуть важность объединения методов молекулярно-генетических исследований и микроскопии для уточнения клеточной систематики, включая параметры митохондриальной активности для классификации клеток и их состояний. Сравнительно-эволюционное изучение клеточных типов у разных экологических групп Placozoa и их сопоставление с функциями расширяют понимание того, как относительно простой организм формирует сложное поведение и как типы клеток эволюционируют.

Peculiarities of the genital organs in girls with aplasia of the vagina and uterus (Mayer-Rokitansky-Kyustner-Hauser syndrome)

Objective - to evaluate the structural features of the external and internal genital organs in girls with aplasia of the vagina and uterus. Materials and methods. The study included 64 adolescent girls aged 15-18. The examination included a general and gynecological with an assessment of the structural features of the perineum, external genital organs and urethra, including using a specially designed measurement scheme for anatomical landmarks. An ultrasound study was performed with the aiming assessment of uterine horns and, when identifying signs of functioning, laparoscopic removal of them with histological study. In order to optimally assess the type of tissue lining the vaginal fossa zone, an analysis was carried out of the material obtained by scraping from the studied zone using liquid cytology. Results. Every third patient (34.3%) was diagnosed with urinary system pathology. In most patients, the external opening of the urethra was typical. Almost everyone had hymen (95%). Gynecological examination showed that in the vast majority of patients in the area of intended vaginal entry, was a blindly ending vaginal fossa (95.3%). The length of the fossa was on the average 1.7±0.1 cm. According to the smear scrapings from the vaginal fossa, flat cells of the superficial, intermediate, and parabasal and basal types were present. Representatives of the lactobacillus and coccal microflora were revealed. A topographic-anatomical analysis of the orientations of the perineum in girls with aplasia of the vagina showed that the depth of the vaginal fossa weakly depended from the age of the patients and did not depend on any of the suggested guide points. According to the results of instrumental methods of investigation of the pelvic organs, 68.75% of the girls had a muscular cord at the uterus place. Moreover, 22% of girls had signs of the functioning of rudimentary horns. Ovaries, as a rule, were located high, at the entrance to the small pelvis. Laparoscopy was performed in all patients with cyclic pain syndrome and closed functioning uterine rudiments, removal of the uterine horns with fallopian tubes were prefered. Histological examination of the obtained preparations was characterized by the detection of endometrial stroma and active endometrial glands. Summary. The authors emphasize that a multilateral examination of this cohort of patients is extremely important for the diagnosis and optimized treatment.

Dermal papillae flattening of thigh skin in Conus Cauda Syndrome.

Our previous studies have shown that severely atrophic Quadriceps muscles of spinal cord injury (SCI) persons suffering with complete conus and cauda equina syndrome, and thus with permanent denervation-induced atrophy and degeneration of muscle, were almost completely rescued to normal size after two years of home based Functional Electrical Stimulation (hbFES). Since large surface electrodes were used to stimulate the denervated thigh muscles, we wanted to know if the skin was affected by this peculiar long-term treatment. Indeed, we demonstrated by two approaches that the epidermis decreases in thickness in the long term denervated persons, while it increased to almost pre-SCI values in hbFES compliant SCI persons. Here we report data of morphometry of skin biopsies from both legs of 18 SCI persons, harvested at enrolment in the Project RISE, to test if the Interdigitation Index, a simple measurement of the epidermal‐dermal junction, may provide a further precise quantitative evidence of the flattening of the skin in those SCI persons. The Interdigitation Index of the 36 skin biopsies shows a higly significant linear correlation with the years of SCI (p < 0.001). Furthermore, when the 18 SCI persons are divided in two groups (1 to 3.9 versus 4.1 to 8.0 years from SCI, respectively) and the data are compared, the later Group presents a statistically significant -22% decrease (p, 0.029) of the Interdigitation Index. On the other hand counting the papille do not provide the same strong evidence. In conclusion, the Interdigitation Index is an additional sound quantitative structural biomarker of skin atrophy and flattening occurring in SCI. The result correlates with the much severe extent of atrophy of the permanently denervated thigh muscles, as determined at both macro and microscopic levels.We are confident that the Interdigitation Index will provide sound evidence that the effects of hbFES, we previously reported on skeletal muscle and epidermis thickness, will be extended to the dermal layer of the skin, suggesting a coordinated negative effects of SCI on skeletal muscle and skin, and an improvement of both tissues after hbFES. Incoming analyses will be extended to basal lamina, collagene types, elastic fibers and skin annexes in the subcutaneous layer.

Fractal-Based Computation of Heat Source Depths and Temperatures for the Soutpansberg Basin, South Africa

The fractal-based approach was used for computing magnetic depths and temperatures for the Soutpansberg Basin in South Africa. The average depth to the top Zt and basement depth Zo for the Soutpansberg Basin were 4.36 &amp;plusmn; 0.28 km, 10.43 &amp;plusmn; 0.65 km, respectively. The average temperature at depth Zt was 184.69 &amp;plusmn; 7.66 &amp;ordm;C. Magnetic source depths and basal temperatures that were in the Curie point range were determined, to be within 20.35 km to 21.68 km and 549.34 &amp;ordm;C to 585.24 &amp;ordm;C, respectively. Increasing the value of the fractal parameter &amp;beta; from 0 to 4, had an effect of retaining deeper depths and higher temperatures. The fractal parameter values of &amp;beta; &amp;gt; 3 retained Curie point depths and temperatures that indicated basal rock types with an igneous predisposition. The fractal-based approach proved to be an improved technique as compared to the conventional centroid method.

ANTHECOLOGY OF SOME SPECIES OF THE GENUS ALLIUM L. UNDER MOSCOW PROVINCE CONDITIONS

Different perennial species of Allium L. have been studied under Moscow Province conditions as new vegetables and melliferous plants. The results of long-term studies of anthecology of seven species of Allium L.: A. nutans L., A. flavescens Bess., A. odorum L., A. montanum F.W. Schmidt, A. fistulosum L., A. angulosum L., A. schoenoprasum L. are presented in the paper. It is shown that the objects of study differ in terms of the beginning of flowering and length of the growing season. The early-flowering (A. fistulosum, A. montanum, A. schoenoprasum), and late-flowering species (A. angulosum, A. nutans, A. odorum, A. flavescens) were identified. The species features in the daily dynamics of flowering, the timing of development of pistils and the duration of susceptibility of their stigmas are revealed. The significant influence of weather conditions on these processes is established. By nature of blooming flowers within a single inflorescence type are revealed apical (A. schoenoprasum, A. fistulosum) and basal (A. odorum, A. nutans, A. montanum, A. flavescens) types of inflorescence. Besides, groups of species differing with the dynamics of disclosure of flowers in inflorescences are noted. It was found that the daily course of opening flowers within the species, as a whole, is constant, but depends on weather conditions. It is shown that the maximum number of flowers opening per day in all species falls on the middle of the flowering period. The species composition of pollinators and entomophages for the studied species of Allium L. was determined. The taxonomic diversity of the composition of insects visiting the inflorescences of onions is shown. The role of separate insects in the pollination process is established. The main pollinators of early-flowering and late-flowering species of onions are determined. It is stated that the most important as pollinators are species related to the orders of Hymenoptera (bumblebees, bees) and Diptera (flower flies, tachina flies), the activity of which largely depends on weather conditions and time of day.

Abstract 989: Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages and can form chimeric mammary glands in vivo

Abstract Use of cell culture models to investigate the development of cancer associated with familial breast cancer syndromes is challenging because many culture methodologies do not maintain all of the lineages that are present in vivo, particularly the progenitor cells that may be the specific cancer cell-of-origin. We evaluated the ability of organoid culture technology to preserve stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Tissue samples were obtained from elective reduction mammoplasties or from prophylactic mastectomies for cancer prevention, and were histologically normal. These were grown using conditions similar to organoid cultures for other epithelial cell types, as has been recently described (Sachs et al., Cell 2018). We found that basal stem and luminal progenitor cells could be cultured long-term, and differentiated in culture to generate mature basal and luminal cell types. Analysis of matched organoid cultures and native tissues by mass cytometry (CyTOF) for 38 markers at single-cell resolution confirmed the presence of multiple mammary epithelial cell types in the organoids, and demonstrated that protein expression patterns of the tissue of origin were largely preserved in culture. In addition, we generated a panel of over 40 mammary organoids derived from patients harboring inherited mutations in the cancer predisposition gene BRCA1 and from unaffected controls. Despite interindividual variability, an expansion in luminal progenitor populations associated with BRCA1 mutation could be detected in cultured organoids. Human mammary organoids heterozygous for BRCA1 could be engrafted into the murine mammary gland, resulting in mixed ductal and acinar structures. These studies indicate that this new model system is well suited for studies of aberrant phenotypes associated with BRCA1 mutation and approaches to prevent cancer development in these genetically predisposed tissues. Citation Format: Jennifer M. Rosenbluth, Ronald Schackmann, Carman Li, Norman Sachs, Deborah Dillon, Andrea Richardson, Jane Brock, Judy Garber, Gary Kenneth Gray, Jason Zoeller, Mackenzie Boedicker, Hendrick Johannes Kuiken, Hans Clevers, Joan Brugge. Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages and can form chimeric mammary glands in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 989.

EGCG promotes cell growth inhibition and reprograms mesenchymal‐epithelial transition by restoring CCN5/WISP2 in triple negative breast cancer cells in vitro and in vivo

Background and ObjectiveCCN5/WISP‐2, a matricellular secretory protein that is localized in both cytoplasm and nucleus, is mainly expressed in luminal cells rather than basal cell types in breast cancer tumors. Endogenous CCN5 plays critical role in promoting growth arrest and reprograming Mesenchymal to Epithelial Transition(MET), inhibition of migration and invasion, overall inducing the invasive to non‐invasive phenotypes. Thus, we hypothesize that targeting endogenous CCN5 restoration in Triple‐negative‐breast cancer (TNBC) cells could be an ideal strategy to prevent the progression of TNBC. To test the hypothesis, we set out to identify a molecule that has the capability to restore CCN5 in TNBC cells. Here we report that CCN5, which is normally absent in TNBC cells can be restored by a common dietary supplement epigallocatechin‐3‐gallate (EGCG), an ester of epigallocatechin and gallic acid, and is a type of catechin, to promote growth arrest and reverse EMT.MethodsEffect of EGCG on breast cancer cells were characterized by in vitro protein and gene expression profilings, cytotoxic assays, colony formation assays, migration assays, anchorage independent growth assays. In vivo effect of EGCG on tumorigenesis was investigated in xenograft model of immunosuppressive mice.Result and ConclusionEGCG treatment in breast cancer cells significantly elevated and restored CCN5 expression in TNBC cells along with inhibition of cell proliferation, migration and colony formation and induction of apoptosis. Reprogramming of mesenchymal to epithelial transition(MET) was also observed in EGCG‐treated TNBC cells. Oral administration of EGCG resulted in drastic inhibition of tumor growth and propagation in immunosuppressive mouse xenografts of MDA‐MB‐231 cells, along with restoration of CCN5 expression. Altogether, these studies clearly indicate that EGCG is an ideal molecule to restore CCN5 in TNBC cells in preventing the TNBC growth, progression and possibly make these cells susceptible to anti‐estrogen treatment, as restoration of CCN5 causes estrogen receptor(ER)‐α in TNBC cells. Moreover, it is reasonable to speculate that the CCN5 pathways probably play an important part in the anti‐tumorigenic role of EGCG in TNBC cells.Support or Funding InformationSupported by Midwest Biomedical Research Foundation and VA Merit Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clinical Outcomes of Muscle-Invasive Bladder Cancer According to the BASQ Classification

Background: We evaluated the clinical efficacy and prognosis of muscle-invasive bladder cancer according to the basal/squamous-like (BASQ) classification system based on immunohistochemical staining [CK5/6( ), CK14( ), GATA3(-), and FOXA1(-)]. Methods: One hundred patients diagnosed with muscle-invasive urothelial carcinoma of the urinary bladder were included in the study. All patients underwent radical cystectomy after transurethral removal of bladder tumor. Immunostaining was performed for CK5/6, CK514, FOXA1, and GATA3 antibodies on tissue microarray slides, and expression patterns were quantitatively analyzed using a scanning program. Findings: The median follow-up time was 47.4 (interquartile range 6-89.9) months. The mean age of the patients was 65.1 ± 11.2 years. FOXA1 or CK14 expression greater than 1% was respectively positively and negatively correlated with overall survival (OS; P = 0.011 and P = 0.042, respectively), cancer-specific survival (CSS; P = 0.050 for both), and recurrence-free survival (RFS; P = 0.018 and P = 0.040, respectively). For CK5/6 and GATA3- or FOXA1- expression, 10% CK5/6 cells was negatively correlated with OS (P = 0.032 and P = 0.039, respectively) and with RFS in combination with FOXA1- only (P = 0.050). Interpretation: FOXA1 and GATA3 were associated with a better prognosis, and CK14 and CK5/6 were associated with a poor prognosis in patients with muscle-invasive bladder carcinoma. Accordingly, basal types such as CK5/6 and GATA3-, or CK5/6 and FOXA1- were associated with a poor prognosis. A new classification system using molecular characteristics of bladder cancer can improve personalized treatment strategies and prediction of the therapeutic response. Funding Statement: This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (Grant number: 2016R1A2B4011623). This study was supported by the Korean Urologic Oncology Society Grant (16-01). Declaration of Interest: No conflict of interest. Ethics Approval Statement: This study was approved by the Institutional Review Board (IRB No. H-1806-081-951).

Expression of n-MYC, NAMPT and SIRT1 in Basal Cell Carcinomas and their Cells of Origin.

Deregulated Hedgehog signalling is a driver of basal cell carcinomas. One effector of the Hedgehog pathway is n-MYC. c/n-MYC proteins, NAMPT and DBC1 are linked to SIRT1 in a positive feedback loop that may contribute to tumorigenesis of basal cell carcinoma. In 5 basal cell carcinoma types immunohistochemistry revealed n-MYC, NAMPT and SIRT1 expression. DBC1 was homogenously expressed in all epithelial cells. NAMPT, SIRT1 and c-MYC were expressed in the stratum basale of human and murine skin. In hair follicles NAMPT and SIRT1 were expressed together with c-MYC and n-MYC, except for the matrix, where n-MYC was strongly positive, but c-MYC expression was absent. Therefore, a common pathway connecting n-MYC, NAMPT and SIRT1 may be active in basal cell carcinomas and in their cells of origin. This pathway may contribute to the development of basal cell carcinomas. Targeting factors in the feedback loop may offer novel therapeutic options.

Clinicopathologic and Immunohistochemical Study of Combined Small Cell Carcinoma and Urothelial Carcinoma Molecular Subtype.

Muscle invasive bladder cancer, an aggressive disease with heterogeneous molecular profiles, has recently been subclassified into three major molecular subtypes -basal, luminal and "p53-like" urothelial carcinomas (UCas), which bear prognostic and therapeutic implication. Similar to breast cancer, basal and luminal subtype UCas are designated by basal (CK5/14) and luminal (CK20) markers. The "p53-like" subtype presents with wild-type p53 gene with upregulated p53 pathways and is implicated in chemoresistance. Urinary bladder is one of the most common primary sites of extrapulmonary small cell carcinoma (SmCC). Bladder SmCC frequently coexists with UCa; however, the relation of SmCC with specific UCa molecular subtypes has not been studied. The aim of this study is to investigate the clinicopathology and immunophenotypes of the combined SmCC and UCa molecular subtypes. A total of 22 combined SmCC and UCa cases were studied for the clinicopathology and immunohistochemical (IHC) profiles by luminal and basal cell markers as well as Her2/Neu and p53. Our results demonstrated that all the urinary bladder SmCCs were associated with high grade UCas. They were more commonly seen in older male patients with a smoking history and had a poor prognosis. Based on the reported molecular subtyping, the UCas could be immunohistochemically subclassified into luminal, basal, dual and null types, which showed different clinicopathologic and IHC features. Compared to non-SmCC associated UCa, the subtypes of UCa in the combined SmCCs and UCas were characterized by: 1) Although overall luminal type was still relatively more common in men, basal marker-expressing subtypes were significantly increased in incidence and were more common in women. 2) Her2/Neu overexpression was more commonly observed in luminal than basal cell marker-expressing UCas. 3) IHC overexpression of p53 was common in all the subtypes, with UCas and SmCCs sharing the same p53 expression pattern. Although limited by relatively a small number of cases, the results of this study will enhance our understanding of the combined SmCC and UCa entity and potentially lead to a future therapeutic management.

Adjustment of medium composition and iso-osmotic potential in direct-shoot organogenesis produces true-to-type oil palm (Elaeis guineensis Jacq.) plantlets

Clonally propagating elite lines of oil palm via multiple shoot organogenesis is promising due to genetic stability of regenerants. In this study, we tested the effects of medium composition using three basal media types (Murashige and Skoog (MS), woody plant medium (WPM), and Y3) with concentrations of sucrose and different ionic strengths derived from sucrose, sorbitol, or both sucrose and sorbitol on the growth and development of oil palm plantlets derived from direct-shoot organogenesis and zygotic embryo-derived seedlings (control). We used histological assays to better understand the medium formulation’s efficiency for shoot organogenesis. The shoot induction percentage was the highest when plantlets were cultured on ½ MS medium supplemented with 5% sucrose. A minimum osmotic potential of -2.0 MPa is essential to efficiently drive shoot maturation of oil palm. Sorbitol appeared to be less effective than sucrose for shoot formation of oil palms. To determine the rate of somoclonal changes between these two micropropagation methods, we investigated the rate of DNA methylation in oil palm plantlets using the methylation-sensitive amplification polymorphism (MSAP) technique. Our MSAP results showed that polymorphisms between in vitro seedling-derived plantlets and direct-shoot organogenesis-derived plantlets were considerably low. We conclude that regenerated plantlets derived from this established protocol are reliable for trueto- type propagation of elite oil palm clones.

Response to Pre-Operative Radiotherapy in Relation to Gene Expression Patterns in Breast Cancer Patients

Current risk assessment for local recurrence after breast conserving therapy for breast cancer is based on clinicopathological factors. In addition, variability in tumor radiosensitvity is likely to influence local control. Genomic markers may help to predict the response to radiotherapy (RT) and may facilitate individualized treatment by identifying which patients will benefit from RT. The aim of our study is to identify a radiosensitivity signature and changes in gene expression after RT. Fresh frozen tumour material was collected from low-risk breast cancer patients (age ≥ 60 years; cT1-2pN0; unifocal lesion) treated within the multi-institution Pre-operative Accelerated Partial Breast Irradiation (PAPBI) trial (NCT01024582). Patients were treated with pre-operative RT (10x4 Gy in 10 days/5x6 Gy in 5 days) and a lumpectomy 6 weeks thereafter. The tumour response to RT was evaluated by pathological assessment by comparing pre-RT biopsies to surgical resection specimens. RNA-seq using next-generation sequencing was performed on all available samples. Unsupervised and supervised clustering was performed to analyze the gene expression data. Differential mRNA expression in pretreatment biopsies between responders and non-responders and between paired pre-and post RT samples was evaluated (DESEQ2). Genes with a false-discovery rate <0.01 were considered as differentially expressed. 72 samples of 58 patients were analyzed: 48 pre-RT biopsies and 24 post-RT surgery specimens. Based on the biopsies, 14 tumours were grade 1 and 29 grade 2. 41 tumours were luminal A, 4 luminal B and 3 basal type. 6 patients had a (near) complete pathological response; 22 patients any response; 16 patients no or very limited response. 2 of 3 basal types showed a complete pathological response. For 16 patients (7 no response, 9 partial response) both pre-treatment biopsies and surgical resection specimens were available. Unsupervised hierarchical clustering showed co-clustering of pre- and post RT samples of 11 patients and very close clustering of 2 patients. The biopsies of responders and non-responders did not cluster in separate groups.No genes were differentially expressed between responders and non-responders. Comparison of pre-and post RT samples identified significant changes in gene expression of 485 genes of which 146 genes were involved in connected pathways. Most genes were involved in p53 signaling, cell cycle, DNA repair and inflammatory response. To date, this is the largest study of gene expression profiles associated with response to pre-operative RT. Using supervised classification no gene expression profile associated with response to RT could be identified. Larger studies will be required to identify such a predictive gene expression profile, which can be of considerable clinical utility in personalized treatment. RT results in altered gene expression of genes involved in p53 signaling, cell cycle, DNA repair and inflammation.

Abstract 1748: Organ-specific epigenetic reprogramming of leukemia cells: Clues to chemoresistance

Abstract We previously reported our observation that tumors composed of leukemic cells found in breast mimic solid breast cancers in gross and histologic appearance (single-filing of cells and distinctive keloid-like fibrosis), invasiveness, metastatic pathway, and high mortality. They are resistant to anti-leukemia drugs; only 4% treated without excision lived 4 years but survivals over 20 yrs resulted when tumor and its microenvironment were excised. The incidence of occult leukemic tumors, still unknown as scans are not yet routine, was estimated near 20% in 1970s autopsy studies. We hypothesized there may be similarly altered genes in leukemic and epithelial breast tumors directing clinical behavior and resistance that could provide targets for curative treatment. We performed RNA sequencing on 11 FFPE breast tumors from annotated AML cases, all IHC-negative for hormones and E-cadherin, compared to normal breast samples. Differential expression analysis identified 3647 genes that were significantly downregulated (adjusted p &amp;lt; 0.05 and log2 fold change &amp;lt; -2). Among the 100 genes downregulated 8-10 fold in leukemic breast, FOXA1, CA12, CXCL14, SFRP1, SFRP4, DCN, LUM, PHLDA1, NTN4, GABRP, TAGLN, AZGP1, and ARRDC3 have been associated with poor prognosis in breast cancer, mainly triple-negative (TNBC)/basal types. Compared to TNBC cases in the TCGA database, 851 genes were similarly deregulated; significantly enriched KEGG pathways include neuroactive ligand-receptor interaction, focal adhesion, ECM-receptor interaction, and regulation of lipolysis in adipocytes. A role for altered stroma is suggested as many of the similarly downregulated genes were reported in dissected breast cancer stroma and silenced SFRP1 and CXCL14 were found in keloid fibroblasts. Silencing of SFRP1 and E-cadherin in AML marrows was reported in 30-50% and each was correlated with poor survival. Reversal of SFRP1 silencing has been achieved by epigenetic-modifying drugs in cancers and keloid fibroblasts. We validated loss of SFRP1 and RGS6 in leukemic breasts by RT-PCR. Loss of RGS6 in breast and other cancers promotes doxorubicin resistance and DNMT1 upregulation leading to silencing of tumor suppressor genes. These data open a new view of resistance of AML cells to marrow-directed drugs through interaction with aberrant microenvironment. Solid tumors of epithelial and leukemic cells may share unrecognized treatable targets. Adding drugs directed at tumor, including available epigenetic-modifying agents, to marrow-effective protocols, may finally decrease the persistently high rate of AML treatment failure. Citation Format: Isabel Cunningham, Diane Hamele-Bena, Audrey Papp, Yan Guo, Antony B. Holmes, Yu Shyr, Rory A. Fisher. Organ-specific epigenetic reprogramming of leukemia cells: Clues to chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2017-1748