Dual Immunohistochemical Staining Research Articles

OA07.03 Prognostic Significance of Micrometastases in Mediastinal Lymph Nodes of Patients With Radically Resected Non-Small Cell Lung Cancer

Recurrence occurs in 30-50 % of patients operated for early stage non-small cell lung cancer (NSCLC), what suggests the existence of occult metastases at the time of surgery. Preoperative detection of occult micrometastases in mediastinal lymph nodes could contribute to better selection of patients appropriate for surgery. This retrospective study was undertaken to determine the prognostic significance of preoperatively detected mediastinal lymph node (LN) micrometastases in patients treated with radical surgical resection for stage I and II NSCLC. From January 2007 to December 2010, 82 patients with stage I and 67 patients with stage II NSCLC underwent transcervical extended mediastinal lymphadenectomy (TEMLA) and subsequent radical pulmonary resection. A total of 4841 mediastinal lymph nodes resected during TEMLA procedure and determined as metastases-free by hematoxylin and eosin staining were labelled to detect occult micrometastases (dual immunohistochemical staining with AE1/AE3 and BerEP4 antibodies). Micrometastases were detected in mediastinal LN of 16 patients (9,7%). 11 patients had only one LN station affected (68,8%). Subcarinal LN were most frequently affected station (11 patients, 68,8%). There was significant correlation between the presence of micrometastases and tumor size. 5-year total survival was significantly better for stage I (64,1%, p=0.0001) and stage II (44,4%, p<0.05) patients without micrometastases comparing to those with micrometastases (18,8%). By multivariate analysis, only the presence of micrometastases was demonstrated to be a significant prognostic factor for 5-year total survival. Presence of micrometastases in mediastinal LN of patients with radically resected stage I and II NSCL is associated with significantly reduced 5-year total survival. Preoprative detection of micrometastases with immunohistochemical staining of mediastinal LN resected during TEMLA procedure improves staging and may contribute to better patient selection for curative surgery.

STAT3 expression is associated with poor survival in non-elderly adult patients with newly diagnosed multiple myeloma.

BackgroundSignal transducer and activator of transcription 3 (STAT3) is not only a key signaling molecule in the regulation of growth but is also involved in malignant transformation. We investigated the prognostic significance of STAT3 expression in 94 non-elderly adult patients (aged 38 to 65 yr) with newly diagnosed multiple myeloma (MM).MethodsTumor cell-specific phosphotyrosine-STAT3 (PY-STAT3) expression at the time of diagnosis was evaluated with dual immunohistochemical (IHC) staining for PY-STAT3 and CD138.ResultsPY-STAT3 positivity was detected in 10 patients (10.6%), including three who showed strong expression. PY-STAT3-positive patients had higher serum C-reactive protein and calcium levels at diagnosis than did PY-STAT3-negative patients. PY-STAT3 positivity had predictive value for poor progression-free survival (PFS; P=0.001) and overall survival (OS; P=0.003). Among the 60 patients who received frontline autologous stem cell transplantation, PY-STAT3-positive patients had poorer PFS than did PY-STAT3-negative patients (4.2 vs. 19.2 mo, respectively; P=0.013). Multivariate analysis identified PY-STAT3 expression as an independent prognostic factor for PFS (relative risk [RR]=2.706, P=0.014) and OS (RR=3.091, P=0.044).ConclusionThese data show that PY-STAT3 positivity, as determined using dual IHC, is a marker of poor prognosis in non-elderly adult patients with MM.

Chromosome 9p24.1/PD-L1/PD-L2Alterations and PD-L1 Expression and Treatment Outcomes in Patients with Classical Hodgkin Lymphoma Treated with Nivolumab (PD-1 Blockade)

Chromosome 9p24.1/PD-L1/PD-L2Alterations and PD-L1 Expression and Treatment Outcomes in Patients with Classical Hodgkin Lymphoma Treated with Nivolumab (PD-1 Blockade)

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection

BackgroundChemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19+ B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.MethodsFlow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation.ResultsThe chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19+ cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact.ConclusionsCXCR3 is the primary chemokine receptor on CD19+ B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

Dual S-100-AE1/3 Immunohistochemistry to Detect Perineural Invasion in Nonmelanoma Skin Cancers.

Background. Perineural invasion (PNI) is an adverse prognostic histologic finding and increases the risk of local recurrence and metastasis. Objective. We aimed to determine if dual immunohistochemical (IHC) staining with S-100 and AE1/3 would increase the detection of PNI on nonmelanoma skin cancers (NMSCs). Methods. We collected 45 specimens of NMSCs in which there was clinical suspicion for PNI. Two dermatopathologists independently reviewed the tumors for the unequivocal presence of PNI. Results. Unequivocal PNI was present on 10 of the 45 tumors by H&E staining and on 15 of the 45 tumors by IHC staining. Large nerves (>0.1 mm) were involved in 3 of 10 H&E-stained cases and 3 of 15 IHC-stained cases, with 2 of the 4 cases demonstrating large nerve involvement with both staining methods. Of the 8 cases of PNI detected only on IHC, 7 were small nerves (≤0.1 mm). Limitations. All cases were selected because they were clinically suspicious for PNI, and this may be considered selection bias. Conclusions. PNI detection may be increased using dual S-100 and AE1/3 staining, but the majority of additional cases detected were small nerves. The clinical significance, given the small size of the involved nerves, is unclear.

Lymphatic Invasion Predicts Aggressive Behavior in Melanocytic Tumors of Uncertain Malignant Potential (MELTUMP)

Lymphatic invasion (LI) identified by immunohistochemical (IHC) staining is common in primary cutaneous melanoma, and LI has been shown to be an independent prognostic factor in melanoma. Its prognostic significance in melanocytic tumors of uncertain malignant potential (MELTUMPs) has not been well characterized. This study included 32 patients with provisional diagnoses of MELTUMP. Lesions were evaluated for tumor thickness, the presence of ulceration, mitotic figures, mitotic figures at the base, tumor infiltrating lymphocytes, as well as peritumoral and intratumoral lymphatic density. Dual IHC staining was used to microscopically detect lymphatic endothelium (podoplanin) containing melanoma cells (S100), with the aid of multispectral imaging in select cases. Univariate analysis was performed to identify associations between clinical and pathologic variables and melanoma-related events. The 32 patients had a median follow-up of 111 months. Two patients subsequently died of melanoma-related disease, 1 died of unknown causes, 5 developed nodal metastases, and the remainder showed no evidence of progressive disease. LI was identified in 8/32 patients (25%) by dual IHC staining, which included the 2 patients who died of melanoma-related disease, 1 patient with bulky nodal metastasis, 1/4 patients with microscopic nodal metastases, and 4 patients who showed no evidence of progressive disease. The presence of LI was associated with melanoma metastases or melanoma-related death (P=0.05). The presence of LI by dual IHC in MELTUMPs is associated with a poorer prognosis, specifically with melanoma metastasis, and may therefore serve as a useful prognostic factor for risk stratification of patients with these diagnostically challenging lesions.

P16 and Ki-67 Dual Immunohistochemical Staining in Cervical Cytology Specimens Diagnosed as Squamous Lesions Indeterminate for High Grade Dysplasia

p16 and Ki-67 Dual Immunohistochemical Staining in Cervical Cytology Specimens Diagnosed as Squamous Lesions Indeterminate for High Grade Dysplasia

Microvessel proliferation by co-expression of endothelial nestin and Ki-67 is associated with a basal-like phenotype and aggressive features in breast cancer

BackgroundTo quantify tumour angiogenesis, microvessel density (MVD) has been widely used. We here present a novel angiogenesis marker, microvessel proliferation (MVP), based on dual immunohistochemical staining of nestin and Ki-67. Immature endothelial cells express nestin, and when co-expressed with the proliferation marker Ki-67, the number of proliferating immature blood vessels can be measured. Materials and methodsMicrovessel proliferation was evaluated in 178 breast cancer samples and estimated by vascular proliferation index (VPI), the ratio between the number of vessels containing proliferating endothelial cells and the total number of immature vessels. ResultsHigh VPI was strongly associated with several markers of aggressive breast cancer, such as negative oestrogen receptor (ER) status (p = 0.003), high tumour cell proliferation by Ki-67 (p = 0.004), high p53 expression (p = 0.001), and five profiles for the basal-like phenotype (odds ratios (OR); range 3.4–6.3). Also, high VPI was significantly associated with interval detected breast cancer compared with screening detected lesions (p < 0.0005), and adverse outcome in univariate and multivariate survival analysis (p = 0.034 and p = 0.022, respectively). ConclusionMicrovessel proliferation is a novel marker of ongoing angiogenesis and was associated with aggressive tumour features, basal-like phenotypes, interval presentation, and prognosis in this series of breast cancer.

Post-weaning social isolation attenuates c-Fos expression in GABAergic interneurons in the basolateral amygdala of adult female rats

Previous studies have found that adolescent social isolation of rats can lead to an increased anxiety state during adulthood, while chronic anxiety states are associated with dysregulated local GABAergic inhibition within the basolateral amygdala (BL). Therefore, we investigated the effects of post-weaning social isolation of female rats, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142), on a subset of GABAergic interneurons in the BL in adulthood using dual immunohistochemical staining for c-Fos and parvalbumin. Juvenile female rats were reared in isolation or in groups of three for a 3-week period from weaning to mid-adolescence, after which all rats were group-housed for an additional 2weeks. Group-reared rats and isolation-reared rats injected with FG-7142 had increased c-Fos expression in GABAergic interneurons in the anterior part of the BL compared to group-reared rats and isolation-reared rats, respectively, injected with vehicle. Isolation rearing had a main effect to decrease c-Fos expression in GABAergic interneurons in the anterior part of the BL compared to group-reared rats. These data suggest that post-weaning social isolation of female rats leads to dysregulation of a parvalbumin-containing subset of local GABAergic interneurons in the anterior part of the BL, which have previously been implicated in the pathophysiology of chronic anxiety states. These cellular changes may lead to an increased vulnerability to stress- and anxiety-related responses in adulthood.

Post-weaning social isolation of female rats, anxiety-related behavior, and serotonergic systems

Our previous studies have shown that post-weaning social isolation of male rats leads to sensitization of serotonergic systems and increases in anxiety-like behavior in adulthood. Although studies in humans suggest that females have an increased sensitivity to stress and risk for the development of neuropsychiatric illnesses, most studies involving laboratory rats have focused on males while females have been insufficiently studied. The objective of this study was to investigate the effects of post-weaning social isolation on subsequent responses of an anxiety-related dorsal raphe nucleus (DR)-basolateral amygdala system to pharmacological challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the γ-aminobutyric acid (GABA)A receptor). Juvenile female rats were reared in isolation or in groups of three for a 3-week period from weaning to mid-adolescence, after which all rats were group-reared for an additional 2weeks. We then used dual immunohistochemical staining for c-Fos and tryptophan hydroxylase in the DR or single immunohistochemical staining for c-Fos in the basolateral amygdala. Isolation-reared rats, but not group-reared rats, injected with FG-7142 had increased c-Fos expression within the basolateral amygdala and in serotonergic neurons in the dorsal, ventrolateral, caudal and interfascicular parts of the DR relative to appropriate vehicle-injected control groups. These data suggest that post-weaning social isolation of female rats sensitizes a DR-basolateral amygdala system to stress-related stimuli, which may lead to an increased sensitivity to stress- and anxiety-related responses in adulthood.

Lymphatic Invasion Is Independently Prognostic of Metastasis in Primary Cutaneous Melanoma

Lymphatic invasion (LI) in primary cutaneous melanomas was recently found to be common. In this study, we evaluated LI as an independent prognostic factor. This study included 251 patients with vertical growth phase (VGP) primary cutaneous melanomas who had paraffin-fixed lesional tissue and were in a prospective cohort seen between 1972 and 1991, had no clinical evidence of regional nodal disease at diagnosis, and had at least ten years of follow-up. Dual immunohistochemical staining was used to detect lymphatic endothelium (podoplanin) and melanoma cells (S-100). Multivariate logistic regression for ten-year metastasis was used to define independent prognostic factors, and a prognostic tree was developed to characterize and discriminate risk groups. Kaplan-Meier disease-free survival curves for those with and without LI within current American Joint Committee on Cancer stages were compared using the log-rank statistic. LI was observed in 43% (108 of 251) of the study melanomas. The multivariate model for ten-year metastasis identified four independent prognostic factors: tumor thickness, mitotic rate, LI, and anatomic site. The prognostic tree identified a group of patients with thin (≤1 mm thick) melanomas and poor prognosis: stage IB melanomas with LI. Survival curves for time to first metastasis showed significantly poorer prognosis for patients with LI compared with those without it for both stages IB and IIA. LI is common across the range of tumor thicknesses in primary VGP melanomas. It is an independent prognostic factor and significantly increases the risk of metastasis in patients in clinical stages IB and IIA.

Cofilin and Slingshot Localization in the Epithelium of Uterine Endometrium Changes During the Menstrual Cycle and in Endometriosis

Regulation of the actin cytoskeleton is essential for epithelial cell polarity and protein trafficking within human uterine epithelium. The actin-binding protein cofilin is involved in regulation of actin dynamics by promoting actin branching and cytoskeleton reorganization. Dual immunohistochemical staining of cofilin and G-actin (represented by DNAse I staining) revealed cofilin-G-actin colocalization in the apical side of luminal epithelial cells of human uterine endometrium during the proliferative phase of the menstrual cycle. Interestingly, during the secretory phase of the menstrual cycle, cofilin was only present on the basolateral side. To determine whether the disease endometriosis causes a different pattern of actin remodeling, we investigated an established baboon model of induced endometriosis. The cofilin pattern in the secretory phase of baboons with endometriosis was similar to the proliferative phase in normal animals; cofilin was observed in the apical parts of luminal and glandular epithelium. A phosphatase regulating the activity of cofilin, slingshot (SSH1), revealed a similar staining pattern within these tissues. These patterns were confirmed through quantitative image analysis. Quantification of messenger RNA (mRNA) detected upregulated SSH1 and suggested a progesterone resistance-related pattern of nuclear steroid hormone receptors, but no change in membrane progesterone receptors (mPR alpha or mPR beta) was observed in endometriosis. Our data indicate that the severe dyssynchrony during menstrual cycle phases in endometriosis is connected with improper cytoskeleton rearrangements. We suggest that cofilin-mediated actin reorganization in uterine epithelial cells might be important in preparation for blastocyst implantation; dysregulation of this reorganization may lead to decreased fertility in endometriosis.

Vascular proliferation is a prognostic factor in breast cancer

Angiogenesis is important for the growth and spread of malignant tumors, and anti-angiogenesis treatment is currently being evaluated for breast cancer and other tumors. Although microvessel density is the most commonly used tissue-based marker of tumor associated angiogenesis, it has significant limitations and has not proven effective as a predictive factor in selecting patients for treatment. We here wanted to explore the significance of vascular endothelial cell proliferation in breast carcinoma. We examined microvessel proliferation in breast cancer by dual immunohistochemical staining, using the pan-endothelial marker Factor-VIII combined with proliferation of endothelial cells by Ki-67 expression, in three independent series of breast cancer, including a total of 499 patients and 141 events during follow-up. Common statistical tests of associations as well as univariate and multivariate regression analysis of patient survival were used. By counting vessels with actively proliferating endothelium, we show that microvascular proliferation is a significant predictor of disease progression in breast cancer, especially among high-grade and ER-negative tumors. Our findings indicate that this novel marker of active tumor angiogenesis might be of value in patient management and should be further studied in the context of patient selection for anti-angiogenesis treatment.

Swim stress activates serotonergic and nonserotonergic neurons in specific subdivisions of the rat dorsal raphe nucleus in a temperature-dependent manner

Physical (exteroceptive) stimuli and emotional (interoceptive) stimuli are thought to influence stress-related physiologic and behavioral responses through different neural mechanisms. Previous studies have demonstrated that stress-induced activation of brainstem serotonergic systems is influenced by environmental factors such as temperature. In order to further investigate the effects of environmental influences on stress-induced activation of serotonergic systems, we exposed adult male Wistar rats to either home cage control conditions or a 15-min swim in water maintained at 19 °C, 25 °C, or 35 °C and conducted dual immunohistochemical staining for c-Fos, a marker of immediate-early nuclear activation, and tryptophan hydroxylase (TPH), a marker of serotonergic neurons. Changes in core body temperature were documented using biotelemetry. As expected, exposure to cold (19 °C) swim, relative to warm (35 °C) swim, increased c-Fos expression in the external lateral part of the parabrachial nucleus (LPBel), an important part of the spinoparabrachial pathway involved in sensation of cold, cutaneous stimuli, and in serotonergic neurons in the raphe pallidus nucleus (RPa), an important part of the efferent mechanisms controlling thermoregulatory warming responses. In addition, exposure to cold (19 °C) swim, relative to 35 °C swim, increased c-Fos expression in the dorsal raphe nucleus, ventrolateral part/periaqueductal gray (DRVL/VLPAG) and dorsal raphe nucleus, interfascicular part (DRI). Both of these subregions of the dorsal raphe nucleus (DR) have previously been implicated in thermoregulatory responses. Altogether, the data are consistent with the hypothesis that midbrain serotonergic neurons, possibly via activation of afferents to the DR by thermosensitive spinoparabrachial pathways, play a role in integration of physiologic and behavioral responses to interoceptive stress-related cues involved in forced swimming and exteroceptive cues related to cold ambient temperature.

Vascular proliferation is increased in basal-like breast cancer

Based on molecular sub-classification, basal-like breast cancer is associated with aggressive behavior. These tumors are frequently triple negative and lack traditional treatment targets. Angiogenesis, one of the hallmarks of cancer, is important for the local growth and spread of malignant tumors and is now a treatment target. The aim of this study was to explore whether angiogenesis is increased in relation to certain molecular subtypes of breast cancer with special focus on the basal-like category. Altogether, we analyzed a total of 431 breast cancers from two independent series after dual immunohistochemical staining of Factor VIII for endothelial cells and Ki-67 for proliferating cells. We then determined vascular proliferation in the most vascularized areas of the tumor. In both Series I and II, high vascular proliferation index (VPI) was significantly associated with expression of cytokeratin 5/6 (P = 0.001, 0.010), P-cadherin (P < 0.0005, <0.0005), epidermal growth factor receptor (P = 0.003, 0.001), the basal-like subtype (P = 0.001, 0.011), and the core basal phenotype (P = 0.002, 0.002), respectively. In Series I, high VPI was associated with the triple negative phenotype (P = 0.004) and p63 expression (P = 0.008). Tumor angiogenesis, as measured by vascular proliferation, was increased in the basal-like subtype in two independent breast cancer series and may thus be a possible treatment target in this category. Studies are required to evaluate whether this novel angiogenesis marker can be used to stratify patients for anti-angiogenesis treatment.

Transcription Factor ets-2 Plays an Important Role in the Pathogenesis of Pulmonary Fibrosis

Ets-2 is a ubiquitous transcription factor activated after phosphorylation at threonine-72. Previous studies highlighted the importance of phosphorylated ets-2 in lung inflammation and extracellular matrix remodeling, two pathways involved in pulmonary fibrosis. We hypothesized that phosphorylated ets-2 played an important role in pulmonary fibrosis, and we sought to determine the role of ets-2 in its pathogenesis. We challenged ets-2 (A72/A72) transgenic mice (harboring a mutated form of ets-2 at phosphorylation site threonine-72) and ets-2 (wild-type/wild-type [WT/WT]) control mice with sequential intraperitoneal injections of bleomycin, followed by quantitative measurements of lung fibrosis and inflammation and primary cell in vitro assays. Concentrations of phosphorylated ets-2 were detected via the single and dual immunohistochemical staining of murine lungs and lung sections from patients with idiopathic pulmonary fibrosis. Ets-2 (A72/A72) mice were protected from bleomycin-induced pulmonary fibrosis, compared with ets-2 (WT/WT) mice. This protection was characterized by decreased lung pathological abnormalities and the fibrotic gene expression of Type I collagen, Type III collagen, α-smooth muscle actin, and connective tissue growth factor. Immunohistochemical staining of lung sections from bleomycin-treated ets-2 (WT/WT) mice and from patients with idiopathic pulmonary fibrosis demonstrated increased staining of phosphorylated ets-2 that colocalized with Type I collagen expression and to fibroblastic foci. Lastly, primary lung fibroblasts from ets-2 (A72/A72) mice exhibited decreased expression of Type I collagen in response to stimulation with TGF-β, compared with fibroblasts from ets-2 (WT/WT) mice. These data indicate the importance of phosphorylated ets-2 in the pathogenesis of pulmonary fibrosis through the expression of Type I collagen and (myo)fibroblast activation.

High Lymphatic Vessel Density and Lymphatic Invasion Underlie the Adverse Prognostic Effect of Radial Growth Phase Regression in Melanoma

Regression in the radial growth phase (RGP) of primary cutaneous melanomas is common and has been shown to be an adverse prognostic factor. However, the underlying mechanism is unclear. We performed dual immunohistochemical staining of podoplanin and S-100 on paraffin tissues from 321 patients with vertical growth phase primary melanomas, who had 10 years or more of follow-up. Lymphatic vessel density (LD) and lymphatic invasion (LI) were quantified and documented. The time to first metastasis and melanoma-specific death (MSD) from the date of definite treatment were analyzed using univariate and multivariate Cox models. Among the 116 vertical growth phase melanomas that had regression in the adjacent RGP, 75 (23%) were classified as complete and 41 (13%) were classified as partial. LD was significantly higher (P<0.001) in the 75 lesions with complete regression (mean±SD, 23.7±12.3/mm²) compared with the 41 lesions with partial regression (15.5±7.1/mm²) and was lower in 155 areas of the adjacent normal dermis (7.3±3.5/mm²) and 69 areas of the distant normal dermis (5.5±2.6/mm²). Patients whose lesions had areas of complete regression with LI and either high or low LD or had no LI with high LD, had shorter time to first metastasis (hazard ratio=2.5, 3.8, and 2.5, respectively) and increased risk of melanoma-specific death (hazard ratio=3.1, 1.3 and 3.0, respectively) than those with no LI, and low LD or those without areas of complete regression. These data indicate that complete RGP regression is associated with significantly increased LD. In addition, the adverse prognostic effect of RGP regression is at least partially mediated through lymphangiogenesis and LI in this area.

Senescent Fibroblasts Promote Neoplastic Transformation of Partially Transformed Ovarian Epithelial Cells in a Three-dimensional Model of Early Stage Ovarian Cancer

Most epithelial ovarian cancers are diagnosed postmenopausally, although the well-established epidemiological risk factors (parity, oral contraceptive use) are premenopausal. We hypothesized that accumulation of senescent fibroblasts, together with concomitant loss of presenescent fibroblasts within the ovarian cortex, promotes initiation and early development of ovarian cancer from ovarian surface epithelial (OSE) cells. To test this, we established immortalized OSE (IOSE) cell lines that mimic early neoplastic transformation by overexpressing the CMYC oncogene (IOSECMYC) and normal ovarian presenescent (PSN) and senescent (SEN) fibroblast cell lines. We then evaluated the ability of PSN and SEN fibroblasts to transform IOSE and IOSECMYC after coculture. SEN fibroblasts significantly enhanced neoplastic development of IOSECMYC cells; there was an up to 15-fold increase in migration of IOSECMYC cells cocultured with SEN fibroblasts compared with PSN fibroblasts. Conditioned medium from SEN fibroblasts promoted anchorage-independent growth of IOSECMYC cells. We studied fibroblast-epithelial cell interactions in heterotypic three-dimensional spheroid models. Dual immunohistochemical staining of spheroids for a proliferation marker (MIB-1) and cytokeratin-18 indicated that SEN fibroblasts induce approximately a five-fold increase in proliferation of IOSECMYC cells relative to cocultures with PSN fibroblasts. SEN, but not PSN fibroblasts, also induced nuclear atypia in epithelial cells in three-dimensional spheroids. These data suggest for the first time that the accumulation of senescent, or loss of presenescent fibroblasts, can promote neoplastic development of partially transformed OSE cells in vitro and illustrates the power of using three-dimensional heterotypic modeling to gain better insights into the etiology underlying the development of epithelial ovarian cancer.

The biology and prognostic value of lymphatic vessel density (LD) and lymphatic invasion (LI) in regression in melanoma

9017 Background: Regression in melanoma is characterized by increased vascularity, lymphocytic infiltrate and fibroplasia in the papillary dermis, accompanied by the absence (complete regression, CoR) or presence (partial regression, PaR) of melanoma cells in the epidermis. The prognostic value of regression is controversial. We noticed that LD and LI were increased in the areas of regression (AR) or areas with brisk lymphocytic infiltration (AB). Our goal was to clarify the prognostic value of regression in melanoma. Methods: Dual immunohistochemical staining was done using antibodies to podoplanin (lymphatic vessels) and S100 (melanoma cells) on paraffin tissues from 321 patients with vertical growth phase (VGP) primary melanomas who had 10 years or more of follow-up. LD in AR (both CoR and PaR) was compared with that of normal dermis adjacent and distant, as well as LD in the AB. LI in these areas was also scored. Unadjusted and adjusted hazard rates were obtained from univariate and multivariate Cox models for time to melanoma-specific death using established melanoma prognostic factors. Results: 116 patients (36%) had regression: 75 CoR (23%) and 41 PaR (13%). LD significantly decreased stepwise from CoR (mean ± se, 23.7 ± 2.7) to PaR (15.5 ± 1.1), adjacent normal dermis (7.3 ± 0.28) and distant normal dermis (5.4±0.31) and it was significantly elevated in the AB (18.5±0.78). Melanomas with CoR had the highest percentage of LI in both AR and AB. In addition, the percentage of LI in AB was highest for men and for those with VGP tumor infiltrating lymphocytes (TILs). Both high LD in AR and more LI in AB were associated with poor prognosis (p=0.004 and p=0.002, respectively). Six factors were significant in the final multivariate model: LI in AB (HR=2.3), LD in AR (HR=1.04), thickness (HR=1.44), axial (HR=7.7), ulceration (HR=2.5) and no VGP TILs (HR=2.8). Conclusions: AR and AB were associated with increased LD and higher incidence of LI in primary melanomas. LD and LI in AR or AB are independent prognostic factors. Our data suggest that the effects of regression on prognosis are mediated at least in part through lymphangiogenesis and LI. No significant financial relationships to disclose.

Biclonal (CD34+ CD42b+) Myeloblastic and Megakaryoblastic Transformation of Chronic Myeloproliferative/Myelodysplastic Disorders

Despite the widespread use of molecular and genetic assays (i.e. JAK2V617F, FLT-3, bcr-abl, PML-RARalpha) for diagnoses of myeloid neoplasms, some disease entities remain difficult to define. For instance, distinguishing between blastic phase of bcr-abl negative chronic myeloproliferative disease, unclassifiable (CMPD-U), transformed myelodysplastic/myeloproliferative disease-unclassifiable (MDS/MPD-U), acute megakaryocytic leukemia (AML FAB-M7), and idiopathic myelofibrosis (IMF) per the 2001 WHO classification system remains challenging. Common to all of these diagnoses are marrow dysplasia, presence of immature myelocytic and megakaryocytic cells, and reticulin myelofibrosis. Here we describe our institute experience with 11 patients (pts) who presented between 2004–2008 with acute myeloid neoplasms characterized by prominent myeloblastic and megakaryoblastic cell populations with myelofibrosis. Morphological diagnoses were: MDS-refractory anemia with excess blasts (n=1), blastic phase of MPD/MDS with myelofibrosis (n=5), atypical or mixed acute myelocytic and megakaryoblastic leukemia (n=4), and acute megakaryoblastic leukemia (n=1). Median age at diagnosis was 69 years (range 20–89). Six pts were male (55%) and five female. The majority presented with pancytopenia with a median white blood cell count of 2.55 × 109/l (range<0.01–237.5), median hemoglobin of 9.6gm/dl (range 5.6–10.6), and median platelet count of 57 × 109/l (range 14–296). The median percentage of peripheral blood blasts was 7% (range 0–52%) with a median of 28.5% (range 18.5–73%) blasts in the bone marrow. Organomegaly was present in 4/11 pts (1 hepatomegaly, 3 splenomegaly) at diagnosis. Only two pts reported a known antecedent MDS diagnosis (7 and 9 months prior to AML). One pt had relapsed disease with a prior diagnosis of AML FAB M1. Bcr-abl was negative in all cases; FLT-3 TKD/ITD was negative in all 9/9 cases tested (2 not available). 10/11 marrows underwent correlative cytogenetic tests showing normal karyotype in 2 pts and complex in 4 pts. Four pts had chromosome 7 aberrations (−7/del (7), t(7;17), der (7). One AML had inv(3)(q21q26). Dual immunohistochemical staining for CD34 and CD42b expression confirmed the presence of separate abnormal myeloblastic (CD34+) and megakaryoblastic cells (CD42b+) in all samples with a minority of cells expressing both antigens. All patients underwent therapy for AML/MDS: 8 received cytarabine/anthracyclines/etoposide (ADE) or Hidac-based induction regimens, 1 received subcutaneous cytarabine, 1 received Zarnestra therapy, and 1 Vidaza only. 7/11 pts were refractory to or died during chemotherapy. Three patients achieved complete remission, and two underwent allogeneic stem cell transplantation with overall survival (OS) of 18 and 8.5 months. One relapsed prior to autologous transplant. Median OS for 10 pts was 6.2months (range 0.5–18.5 months) with one pt lost to follow up.Conclusions: Acute bcr-abl negative myeloid disorders displaying biclonal CD34+ myeloblastic and CD42b+ megakaryoblastic cell populations with myelofibrosis most likely represent leukemic evolution from prior chronic (undiagnosed) MPD or MDS/MPD. Dual immunohistochemical staining for CD34/CD42b is invaluable in distinguishing between the two immature blastic populations with coexpression of both markers on some cells suggestive of a common leukemia stem cell origin. Clinical outcomes of these patients are poor, and allogeneic stem cell transplantation remains the best long-term treatment option if remission can be achieved.