Malte Renz, MD, PhD

Abstract

Vulvar cancers account for 5% of all gynecologic malignancies; only 1%-3% of those vulvar cancers are primary vulvar sarcomas. Given the rarity of vulvar sarcomas, outcome data specific to histopathologic subtypes are sparse. The aim of this study was to identify clinical and pathologic factors of primary vulvar sarcomas that are associated with survival and may inform treatment decisions.The Surveillance, Epidemiology, and End Results (SEER) database was searched for women diagnosed with vulvar sarcoma between 1973 and 2018. We identified 315 patients and reviewed their demographic, clinicopathologic, surgical, and survival information. Statistical analyses included 2 and t-tests, Kaplan-Meier survival, and Cox regression analyses.The most common histopathologies of vulvar sarcomas were dermatofibrosarcomas (85/315, 27%) and leiomyosarcomas (72/315, 22.9%). Rhabdomyosarcomas (18/315, 5.7%), liposarcomas (16/315, 5.1%), and malignant fibrous histiocytomas (16/315, 5.1%) were less frequent. The majority of patients underwent surgery (292/315, 92.7%), which included lymph node dissections in 21.6% (63/292). Survival and lymph node involvement varied significantly with histologic subtype. The 5-year disease-specific survival for dermatofibrosarcomas, liposarcomas, and fibrosarcomas was 100% and only 60.3% and 62.5% for malignant fibrous histiocytomas and rhabdomyosarcomas, respectively. None of the patients with (dermato)fibrosarcomas, liposarcomas, or leiomyosarcomas had positive lymph nodes, in contrast to rhabdomyosarcomas and malignant fibrous histiocytomas with 77.8% and 40% positive lymph nodes, respectively. The 5-year disease-specific survival for women with positive lymph nodes was 0%.Vulvar sarcomas are heterogeneous with survival highly dependent on the histopathologic subtype. While surgical excision is the mainstay of treatment for all vulvar sarcomas, staging lymphadenectomy should be deferred for (dermato)fibrosarcomas, liposarcomas, and leiomyosarcomas as there were no cases of lymph nodes metastases.

View details for DOI 10.1136/ijgc-2020-001516

View details for PubMedID 32641392

Abstract

Proliferative endometrium has been reported in 15% of endometrial biopsies of women aged 50 years and older. Contrary to endometrial hyperplasia, proliferative endometrium has not been associated with the risk of endometrial cancer.This study aimed to report on the long-term outcome of postmenopausal women who received a diagnosis of proliferative endometrium.This is a retrospective cohort study of 1808 women aged 55 years and older who underwent endometrial sampling between January 1997 and December 2008. Outcome data were available through February 2018. Women with a proliferative endometrium were compared with those with an atrophic endometrium for future development of endometrial hyperplasia or cancer. A subanalysis was performed for those who presented with postmenopausal bleeding. Uni- and multivariable logistic regression analyses were used to assess for confounders.In this study, 297 women (16.4%) received a diagnosis of proliferative endometrium. Furthermore, 962 women met the inclusion criteria. Among those women, 278 had a proliferative endometrium, and 684 had an atrophic endometrium. Women with a proliferative endometrium were younger (61.2 vs 64.5 years; P<.0001) and had a higher body mass index (33.9 vs 30.6 kg/m2; P<.0001). More African American women had a proliferative endometrium. Both groups had a similar length of surveillance (11.9 vs 11.5 years; P=.27). Women with a proliferative endometrium had a higher risk of developing endometrial hyperplasia or cancer (11.9% vs 2.9%; P<.0001), any endometrial cancer (5.8% vs 1.8%; P=.002), atypical endometrial hyperplasia (2.2% vs 0.4%; P=.02), and nonatypical endometrial hyperplasia (2.0% vs 0.7%; P=.001). The risk of developing endometrial cancer and endometrial hyperplasia remained similar after excluding cases on hormonal replacement therapy (12.2% vs 3%; P=.001). On logistic regression analysis, proliferative endometrium histology (odds ratio, 3.89; 95% confidence interval, 2.03-7.49; P<.0001), age >60 years (odds ratio, 1.98; 95% confidence interval, 1.03-3.82; P=.04), and body mass index >35 kg/m2 (odds ratio, 2.3; 95% confidence interval, 1.09-4.83; P<.0001) remained significant risk factors for progression to cancer.One of the 6 postmenopausal women who underwent endometrial sampling had a proliferative endometrium. Furthermore, 11.9% of women developed endometrial hyperplasia or cancer, a 4-fold greater incidence than women with an atrophic endometrium. The findings of this study suggest that long-term monitoring is warranted for women with postmenopausal bleeding and a proliferative endometrium histology. Further studies are needed to examine if a treatment is required to negate the risk of unopposed estrogen.

View details for DOI 10.1016/j.ajog.2020.06.045

View details for PubMedID 32640199

Abstract

Although photoactivated localization microscopy offers the potential to interrogate protein interactions in the physiological environment of a cell, uncertainties in the detection efficiency of photoactivatable proteins lead to complications with data interpretation. Here, we present a numerical model that provides probabilities to detect neighboring molecules dependent on their oligomerization status, density, detection efficiency, and radius, and can be used to assess oligomeric states or detection efficiencies of two molecular species. 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

View details for DOI 10.1002/cyto.a.24167

View details for PubMedID 32558006

Abstract

To investigate the utility of asymptomatic screening, including CA-125, imaging, and pelvic exam, in the diagnosis and management of recurrent ovarian cancer.Women with ovarian cancer whose cancer recurred after remission were categorized by first method that their provider suspected disease recurrence: CA-125, imaging, symptoms, or physical exam. Differences in clinicopathologic, primary treatment characteristics, and outcomes data including secondary cytoreductive surgery (SCS) outcome and overall survival (OS) were collected.102 patients were identified at our institution from 2003 to 2015. 20 recurrences were detected by symptoms, while 62 recurrences were diagnosed first by asymptomatic rise in CA-125, 5 by pelvic exam, and 15 by imaging in the absence of known exam abnormality or rise in CA-125.Mean time to recurrence was 18.9months, and median survival was 45.8months. These did not vary by recurrence detection method (all p>0.4). Patients whose disease was detected by CA-125 were less likely to undergo SCS than those detected by other means (21.7% vs. 35.0%, p=0.007). In addition to the 5 patients whose recurrence was detected primarily by pelvic exam, an additional 10 (total n=15) patients had an abnormal pelvic exam at time of diagnosis of recurrence.Recurrence detection method was not associated with differing rates of survival or optimal SCS, however those patients detected by CA-125 were less likely to undergo SCS. The pelvic exam was a useful tool for detecting a significant proportion of recurrences.

View details for DOI 10.1016/j.gore.2020.100595

View details for PubMedID 32548232

View details for PubMedCentralID PMC7286959

Abstract

Sentinel lymph nodes sampling (SLN) in endometrial cancer is being evaluated as a means to gather prognostic information about lymphatic metastasis while avoiding the morbidity associated with complete lymphadenectomy. SLN ultrastaging has been advocated to identify low-volume metastases, but its value remains uncertain. This study aims to evaluate a pathological protocol for the immediate intraoperative SLN work-up using H&E staining alone. In this retrospective single-center study, patients received standardized cervical injection of indocyanine green, SLN mapping followed by pelvic lymphadenectomy with or without para-aortic lymphadenectomy. SLNs were entirely frozen, multiple H&E stained sections prepared and evaluated intraoperatively. No immunohistochemistry was performed. SLN results were compared with the complete lymphadenectomy specimen. Over 3.5 years, 90 patients were identified who underwent SLN mapping and subsequent complete pelvic lymphadenectomy. At least one SLN was detected in 79 (88%) patients. The median number of SLNs removed was 2.0. Para-aortic SLNs were detected in 7%. Final pathology showed 67% Type I tumors, 76% locally confined. The mean number of lymph nodes removed during complete lymphadenectomy was 21. In this series, only 6 patients had lymph node metastases. 5/6 were identified by the described SLN approach resulting in 83.3% sensitivity and a negative predictive value of 98.7%. Our approach permits immediate intraoperative results and helps guide the primary surgery. The immediate SLN work-up using frozen sections showed both high accuracy and negative predictive value. The comparably lower sensitivity may be related to the low number of patients with positive lymph nodes (7.6%).

View details for PubMedID 30687881

Abstract

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce. Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

View details for PubMedID 30663670

View details for DOI 10.1201/9780429200199

View details for DOI 10.1101/506436

Abstract

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. 2017 International Society for Advancement of Cytometry.

View details for PubMedID 29286574

Abstract

For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Recent developments improved both visualization and quantification by fluorescence microscopy imaging and established a methodology of fluorescence microscopy. By outlining basic principles and their historical development, I seek to provide insight into and understanding of the ever-growing tools of fluorescence microscopy. Thereby, this synopsis may help the interested researcher to choose a fluorescence microscopic method capable of addressing a specific scientific question.

View details for DOI 10.1002/cyto.a.22295

View details for Web of Science ID 000323480200003

View details for PubMedID 23585290

Abstract

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

View details for DOI 10.1073/pnas.1211753109

View details for Web of Science ID 000311149900008

View details for PubMedID 23043115

Abstract

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

View details for DOI 10.1073/pnas.1204270109

View details for Web of Science ID 000304881700050

View details for PubMedID 22592793

Abstract

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

View details for DOI 10.1038/NMETH.1704

View details for Web of Science ID 000296891800022

View details for PubMedID 21926998

Abstract

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

View details for DOI 10.1021/ja100906g

View details for Web of Science ID 000277445400039

View details for PubMedID 20394363

Abstract

FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation.

View details for DOI 10.1007/s10577-008-1234-6

View details for Web of Science ID 000255680800007

View details for PubMedID 18461482

Abstract

The CapG protein, a Gelsolin-related actin-binding protein, is expressed at higher levels in breast cancer, especially in metastasizing breast cancer, than in normal breast epithelium. Furthermore, it is known that an increased expression of the CapG protein triggers an increase in cell motility. According to in vitro experiments, it was supposed that it is the nuclear fraction of the protein, which causes the increase in cell motility. Here, we examined the dynamical distribution of the CapG protein within the living cell, i.e. the import of the CapG protein into the nucleus. The nuclear import kinetics of invasive, metastasizing breast cancer cells were compared to the import kinetics of non-neoplastic cells similar to normal breast epithelium. FRAP kinetics showed a highly significant increase in the recovery of photobleached CapG-eGFP in the cancer cells, so that a differentiation of invasive, metastasizing cells and non-invasive, non-metastasizing cells on the basis of transport processes of the CapG protein between the nucleus and the cytoplasm seems to be possible. Comprehension of the mobility and compartmentalization of the CapG protein in normal and in cancer cells in vivo could constitute a new basis to characterize the invasiveness and metastasizing potential of breast cancer.

View details for DOI 10.1002/ijc.23215

View details for Web of Science ID 000253441100004

View details for PubMedID 18059028

Abstract

The CapG protein, a Gelsolin related actin-binding protein, is overexpressed in breast cancer, especially in metastasizing breast cancer. The CapG protein is known to be involved in the control of motility of non-muscle cells. Its increased expression is described to trigger an increase in cell motility of benign cells. Thus it can be expected that in cancer cells, which overexpress the CapG protein, motility, invasiveness and metastasizing are especially promoted. In this work it was achieved via fluorescence labeling of the CapG protein to visualize the dynamics of cellular processes as migration and invasiveness and to analyze the kinetics of the intracellular distribution of the CapG protein. Differences of the intracellular mobility of CapG between non-neoplastic and neoplastic breast epithelial cells could be demonstrated, correlations of increased tumor cell motility and accelerated intracellular CapG mobility revealed and a causal context of CapG expression and distribution on one hand and invasiveness and metastasizing on the other could be made probable, so that a differentiation of tumor cells applying kinetic analysis of single live cells seems to be possible.