We investigate the difference between surface matching and target matching for pelvic radiation image guidance. The uniqueness of our study is that all patients have multiple CT‐on‐rails (CTOR) scans to compare to corresponding AlignRT images. Ten patients receiving pelvic radiation were enrolled in this study. Two simulation CT scans were performed in supine and prone positions for each patient. Body surface contours were generated in treatment planning system and exported to AlignRT to serve as reference images. During treatment day, the patient was aligned to treatment isocenter with room lasers, and then scanned with both CTOR and AlignRT. Image‐guidance shifts were calculated for both modalities by comparison to the simulation CT and the differences between them were analyzed for both supine and prone positions, respectively. These procedures were performed for each patient once per week for five weeks. The difference of patient displacement between AlignRT and CTOR was analyzed. For supine position, five patients had an average difference of displacement between AlignRT and CTOR along any direction  greater than 0.5 cm, and one patient greater than 1 cm. Four patients had a maximum difference greater than 1 cm. For prone position, seven patients had an average difference greater than 0.5 cm, and three patients greater than 1 cm. Nine patients had a maximum difference greater than 1 cm. The difference of displacement between AlignRT and CTOR was greater for the prone position than for the supine position. For the patients studied here, surface matching does not appear to be an advisable image‐guidance approach for pelvic radiation therapy for patients with either supine or prone position. There appears to be a potential for large alignment discrepancies (up to 2.25 cm) between surface matching and target matching.PACS number(s): 87.55.‐x Accurate target localization and verification for pelvic radiation therapy prior to IMRT delivery is essential, due to the interfractional change of target position that is common in the pelvis second to day‐to‐day variations of bladder, bowel, and rectal filling. There are numerous imaging modalities available for image‐guided pelvic radiation therapy, including MV and kV portal imaging, MV and kV cone‐beam CT, in‐room CT‐on‐rails (CTOR), ultrasound, and Calypso  RF tracking . Most of these modalities entail the delivery of extra radiation dose to patients. Ultrasound guidance may suffer from user subjective interpretation and Calypso requires invasive implantation of beacons.AlignRT  is a 3D surface imaging system for patient localizing, tracking, and monitoring during radiation therapy.However, it remains a question as to whether or not AlignRT surface matching can be effectively used for image‐guided pelvic radiation therapy. In this study, we investigate the difference between surface matching and target matching for pelvic radiation image guidance by comparing AlignRT with in‐room CT‐on‐rails. The uniqueness of our study is that all patients have multiple, same‐day CTOR scans to compare to the corresponding AlignRT 3D surface images. Our findings will be useful in providing guidance for future decisions concerning use of surface matching for image‐guided pelvic radiation therapy.Ten patients with gynecologic or gastrointestinal malignancies were enrolled and analyzed in this study. Data acquisition and patient treatment were conducted according to institutional review board (IRB) protocol #39913. The median age was 60 (range 28–85), median height was 165.6 cm (range 150.0–185.0 cm), and median weight was 153 lbs (range 99–211 lbs). At our center, the AlignRT system is installed in a vault that includes a Siemens Artiste linear accelerator and a Somatom Sensation 40 slice CTOR scanner . The AlignRT system is a two‐camera system (non‐HD cameras), and software version is 4.5. The two cameras are mounted on the ceiling, and the CTOR is oriented at 90° to the linac treatment couch.The standard work flow for image‐guided radiation therapy using CTOR at our center is to align the patient with linac isocenter lasers first, then rotate the couch 90° to align the couch for CTOR image acquisition. After CTOR scan, the patient is rotated back to treatment position. Image fusion between treatment CTOR and simulation CT is performed and the offset between the two image sets is calculated. The patient is then shifted in three dimensions based on the CTOR‐derived fusion offset before treatment.The image‐guidance workflow for this study entailed two initial simulation CT scans performed in supine and prone positions for each patient on a LightSpeed RT CT scanner . In the supine position, patients were immobilized in alpha cradles; for the prone position, patients were immobilized in prone belly boards . After the two CT datasets were imported into the treatment planning system, body surface contours were generated by the Eclipse treatment planning system  and exported to the AlignRT in‐room system to serve as reference images, according to the normal AlignRT workflow. The region of interest (ROI) was defined over the pelvic region without leg involvement to eliminate day‐to‐day leg position variation, and the side of pelvis above the alpha cradle or the prone belly board was also included for more accurate vertical alignment. It is noted that two separate CTs and treatment plans were acquired/produced for each patient — one for the supine and one for the prone position. In the treatment vault, once patients were aligned at their simulation‐defined treatment positions using linac isocenter lasers , AlignRT was initiated to record continuous patient displacement tracking for 1.5 to 2 min. The AlignRT topographic surface was compared with the body surface contour from the initial simulation CT within ROI, and the AlignRT shifts were calculated by averaging the tracking record . Note thAfter treatment, patients were then set up in the alternate position orientation . AlignRT and CTOR image‐guidance shifts were again determined (as previously described) and the difference between the two systems' image‐guidance solutions were again recorded for the alternative position orientation. This process of determining the difference between AlignRT and CTOR image‐guidance shifts for both supine and prone patient orientations was performed for each of the 10 patients, once per week, for five weeks. This resulted in five “weekly” supine comparisons and five “weekly” prone comparisons for each patient, leading to a total of 50 total supine comparisons and 50 total prone comparisons for this study.AlignRT and CTOR shifts from patient's tattoo aligned with linac isocenter lasers were analyzed for all patients. The difference of image‐guidance shifts between AlignRT and CTOR were calculated for each week's comparison in vertical, longitudinal, and lateral directions for both supine and prone positions of each patient. The average and maximum differences of patient displacement between AlignRT and CTOR over the five‐week treatment were calculated for each patient in the three cardinal directions.As seen from t‐test showed differences between AlignRT and CTOR between patient supine and prone positions along vertical and lateral directions were statistically significant .As seen from We observed large AlignRT and CTOR shifts from patient's tattoo aligned with linac isocenter lasers for both supine and prone positions. This is reasonable since the three tattoo body marks at pelvic area would not be able to accurately represent the whole pelvic surface position and the internal target position. The shifts for prone position are larger than for supine position for both AlignRT and CTOR. This is mainly due to the difficulty of setup reproducibility for the prone position on the belly board.In our study, large differences of patient displacement between AlignRT and CTOR were observed (up to 2.25 cm). This is not unexpected since, in our workflow, the registration between CTOR and simulation CT was based primarily on soft‐tissue target volumes (GTV) and adjacent bony anatomy; in contrast, patient shifts determined by AlignRT were based on patient skin surface alignment. The large differences of patient displacement between AlignRT and CTOR suggest large day‐to‐day variations of relative position between deep‐seated target volumes and patient surface. There are other reasons for the large difference, including different patient pelvic rotations, belly‐size and belly‐position changes at treatment days. We identified another important factor affecting patient setup reproducibility, which is significant to pelvic radiation therapy, from this study. Consistent and accurate reproduction of patient setup for pelvic radiation therapy for both supine and prone positions can be extremely challenging. In addition to nonsetup‐related changes, such as bladder and bowel filling variations, and subsequent interfractional movement of the treatment target, we observed significant setup‐related variations such as patient pelvic rotation, pelvic tilt, and back shape changes. The inability to precisely reproduce the patient's simulation setup geometry can invalidate the relative skin to target geometric relationship that is required when using the skin surface as a surrogate for target position as is required for AlignRT guidance.Additionally, we observed greater differences of patient displacement between AlignRT and CTOR for the patient prone position than for the patient supine position. This is primarily caused by greater day‐to‐day patient setup variations in patient prone position than in patient supine position. Most patients in our study had large bellies. Patients with large bellies are harder to consistently reposition for the prone setup orientation with prone belly board, than for the supine setup position in alpha cradles. The large day‐to‐day prone belly board position variation can cause a large change of relative position between GTV and patient surface.In our study, we observed that the surface‐imaging approach to image guidance struggled to adapt to the common, day‐to‐day geometric variations in pelvic region anatomy for both patient supine and patient prone positions. On the contrary, CTOR's direct focus on treatment target (GTV) alignment eliminated the vulnerability associated with use of a skin surface surrogate for alignment and, thus, provided for more accurate target alignment.To our knowledge, this study is the first study comparing AlignRT and CTOR for image‐guided pelvic radiation therapy. While there have been previously published studies of image‐guided prostate radiation therapy using AlignRTFor the patients studied here, the daily setup variations typical to pelvic RT patients, coupled with normal interfractional variations in relative soft tissue target location within the patient, rendered the skin‐surface surrogate approach of AlignRT to be potentially inaccurate for pelvic radiation therapy. We believe that for this specific application surface matching does not appear to be an advisable image‐guidance approach, as we saw potential for errors as large as 2.25 cm compared to target matching.This work is licensed under a Creative Commons Attribution 4.0 International License.Supplementary MaterialClick here for additional data file.


At higher baseline screening coverage levels , screening three times in a lifetime yielded greater health benefits than expanding screening once in a lifetime to 90% coverage, and would have a cost‐effectiveness ratio (I$590 per YLS) below Uganda's per capita GDP. Given very low baseline coverage at present, we conclude that a policy focus on increasing access for previously unscreened women appears to be more compatible with improving both equity and efficiency than a focus on increasing frequency for a small subset of women.Cervical cancer is a leading cause of cancer death worldwide, with 85% of the disease burden residing in less developed regions. To inform evidence‐based decision‐making as cervical cancer screening programs are planned, implemented, and scaled in low‐ and middle‐income countries, we used cost and test performance data from the START‐UP demonstration project in Uganda and a microsimulation model of HPV infection and cervical carcinogenesis to quantify the health benefits, distributional equity, cost‐effectiveness, and financial impact of either (1) improving access to cervical cancer screening or (2) increasing the number of lifetime screening opportunities for women who already have access. We found that when baseline screening coverage was low ( What's new?Most cervical cancer cases and deaths occur in less‐developed countries, where resource constraints challenge the planning and implementation of screening programs. The present report examines tradeoffs between equity and efficiency in cervical cancer screening approaches specifically in Uganda, where current baseline screening coverage is low. Analyses indicate that the expansion of access to once‐in‐a lifetime cervical cancer screening in areas with initially low baseline coverage is likely to yield greater benefits for health, distributional equity and cost‐effectiveness than increasing the number of screening opportunities per woman in low‐resource settings. Improving access for previously unscreened women should be a priority in such areas. Cervical cancer is a leading cause of cancer death worldwide, with 85% of the disease burden residing in less developed regions.Where resources are available, the World Health Organization (WHO) recommends a “screen‐and‐treat” strategy for women aged 30 to 49 years, screening with HPV testing and treating eligible HPV‐positive women with timely cryotherapy.careHPV test  has minimal laboratory requirements, but the testing system is designed to be run in batch mode,Implementing HPV‐based “screen‐and‐treat” programs in low‐resource settings is logistically difficult because of constraints associated with laboratory processing and availability of cryotherapy. The i.e., two or three screenings in a lifetime) to women who already have access. The health outcomes and cost‐effectiveness of a screening program may depend on whether the program prioritizes greater access versus higher frequency of screening. To inform evidence‐based decision‐making, we integrated cost and test performance data from the START‐UP demonstration project in Uganda into a microsimulation model of HPV infection and cervical carcinogenesis. Our objective was to quantify the health benefits, distributional equity, cost‐effectiveness and financial impact of expanding screening coverage to more women versus increasing screening frequency for a select population.As cervical cancer screening programs are planned, implemented, and scaled in low‐ and middle‐income countries, decision‐makers will face tradeoffs as they seek to maximize population health or equitably distribute health benefits given resource constraints. One tradeoff is whether to concentrate resources on (1) expanding coverage of a single lifetime screen in the general population of screening‐eligible women, or (2) offering multiple screening opportunities , and stage of invasive cancer. Individual girls enter the model at age 9 with a healthy cervix, and are simulated until death. Transition probabilities may vary by age, HPV type, duration of infection or precancerous lesion status, and prior HPV infection. Cancer detection can occur through symptoms or via screening. Each month, death can occur from non‐cervical causes or from cervical cancer after its onset. The model tracks disease progression and regression, clinical events, and economic outcomes over the lifetime of each individual woman, which are then aggregated for analysis.An individual woman is represented as a sequence of monthly transitions between mutually exclusive health states, including type‐specific HPV infection status, grade of precancer . We selected the top 50 input parameter sets that produced a good fit to the epidemiologic data to use in analyses as a form of probabilistic sensitivity analysis.For natural history transitions, we estimated baseline “prior” input parameter values and set plausible ranges using epidemiologic data..We assumed screening with HPV DNA testing took place either once in a woman's lifetime at age 30 years, twice in a lifetime at ages 30 and 40 years, or three times in a lifetime at ages 30, 40 and 50 years. We varied baseline screening coverage— which we defined as existing coverage with once in a lifetime screening, ranging from 30% to 80%— and compared health and cost outcomes as either 1) screening coverage associated with onetime screening increased from baseline to 90% in 10% increments, or (2) screening frequency increased to two or three times in a lifetime at the baseline screening coverage level (30% to 80%). For screening two or three times in a lifetime, we made the simplifying assumption that the same women received each screening, while the remaining proportion of the population was never screened. We assumed women were screened by a provider during an initial screening visit, and returned for a second visit to obtain results unless they were lost to follow‐up. If a woman screened positive and was eligible, most received same‐day cryotherapy at the results visit. Treatment protocols for women who were not eligible for immediate cryotherapy and management following treatment, were based on current practice in Uganda and are documented in the Supporting Information Appendix. Test performance, treatment, and compliance parameters are displayed in Table  screenincareHPV test kit was a tradable good valued at US$5, including the test, sampling brush, and container; for tradable goods such as the careHPV test, one US dollar is equivalent to one I$.Cost data have been published elsewhere and are summarized in Table Women's transportation costs and the cost of women's time spent traveling, waiting for, and receiving care were dependent upon the facility level and were derived from START‐UP data and the published literature, as previously described.i.e., having an ICER below Uganda's per capita GDP).To measure the added value of shifting from screening once in a lifetime at the baseline coverage level to screening with either greater frequency or greater coverage, we calculated the incremental net monetary benefit (INMB) for 1) screening three times in a lifetime (at baseline coverage) and 2) screening once in a lifetime (at higher coverage), relative to once in a lifetime (at baseline coverage). The INMB translates the incremental health benefit  into monetary terms for a specified cost‐effectiveness threshold. To calculate the INMB, the life‐years gained are multiplied by the threshold; then the incremental cost (the change in the expected lifetime cost per woman from shifting to a strategy with greater frequency or greater coverage) is subtracted. Thus, the INMB represents the maximum dollar amount per woman by which the cost of an intervention can be increased to achieve an improvement (in either frequency or coverage) while remaining “very cost‐effective”  given a specified coverage level and frequency of screening, we used the individual‐based simulation model to estimate the expected direct medical cost per woman of each screening strategy, including the costs of screening and any relevant diagnostic testing and treatment of precancer. The financial impact analysis did not consider cost offsets from future cancer cases prevented or women's time and transportation costs that were considered in the cost‐effectiveness analysis. We report the cost per 100,000 women for each coverage level and frequency in 2013 US$ instead of I$to provide a meaningful estimate to the international and donor communities.Figure As baseline coverage level rises, greater coverage increases are required for once in a lifetime screening to achieve comparable reductions in cancer risk relative to screening two or three times in a lifetime at baseline coverage. At a baseline coverage level of 40%, screening once, twice or three times in a lifetime reduces cancer risk by 14.5%, 22.8% or 27.2%, respectively. Expanding coverage of once in a lifetime screening to 70% of the target population reduces cancer risk by 25.4%, exceeding the health benefits associated with screening twice in a lifetime at baseline coverage. Expanding coverage of onetime screening to 80% reduces cancer risk by 29.1%, exceeding the health benefits associated with screening three times in a lifetime at baseline coverage.At a baseline coverage level of 50%, screening once, twice or three times in a lifetime reduces cancer risk by 18.0%, 28.4% and 33.9%, respectively. Expanding coverage of once in a lifetime screening to 80% reduces cancer risk by 29.1%, and increasing coverage to 90% reduces cancer risk by 32.9%, exceeding the health benefits of screening twice in a lifetime at baseline coverage but yielding slightly lower cancer risk reductions than screening three times in a lifetime at baseline coverage. As the baseline coverage level reaches 60%, screening two or three times in a lifetime yields greater risk reduction than screening once in a lifetime at 90% coverage. Cancer risk reductions for baseline coverage levels of 70% and 80% are shown in the Supporting Information Appendix.i.e., the difference between the life expectancy for women with access to screening and the population average life expectancy). While each 10% increase in coverage for screening two or three times in a lifetime screening yields slightly greater increases in population average life expectancy  than comparable coverage gains with screening once in a lifetime, improvements in distributional equity are more modest. For instance, assuming baseline coverage of 60%, the life expectancy associated with screening twice in a lifetime is similar to the life expectancy associated with screening once in a lifetime with coverage at 90% (approximately 53.39 years), but the difference between the population average life expectancy and the life expectancy of screened women is 0.23 years when 60% of women have access to screening twice in a lifetime, and only 0.039 years when 90% women have access to screening once in a lifetime.Figure e.g., once in a lifetime screening at 40% coverage) were compared against improvements in number of screens per lifetime , screening twice in a lifetime was no longer an efficient strategy. When once in a lifetime screening was available at 50% coverage or higher, it was less costly and more effective than screening two or three times in a lifetime at baseline coverage, with a stable ICER of I$140 to I$150 per YLS.The cost‐effectiveness of screening two or three times in a lifetime at baseline coverage relative to screening once in a lifetime at baseline coverage or greater is presented in Figure When baseline screening coverage was 50% for all screening frequencies, screening once, twice, or three times in a lifetime cost I$140 per YLS, I$270 per YLS, or I$530 per YLS, respectively. When once in a lifetime screening coverage was also assumed to be available at a higher coverage level of 60%, all strategies remained attractive, with ICERs below Uganda's per capita GDP. However, when once in a lifetime screening was assumed to be available at 70% coverage (I$140 per YLS), it was less costly and more effective than screening twice in a lifetime at baseline coverage levels; the ICER associated with screening three times in a lifetime at baseline coverage rose to I$1,010 per YLS. As coverage of once in a lifetime screening expanded to 80%, it was less costly and more effective than screening two or three times in a lifetime at baseline coverage, and its ICER remained stable at I$140 per YLS.At a higher baseline screening coverage of 70%, screening once, twice or three times in a lifetime cost I$140 per YLS, I$270 per YLS, or I$540 per YLS, respectively. As screening coverage of once in a lifetime screening increased to 80%, screening two or three times in a lifetime remained attractive strategies. When coverage of once in a lifetime screening was assumed to be available at 90%, screening twice in a lifetime at baseline coverage was no longer cost‐effective. Screening three times in a lifetime remained the most effective strategy, and would be considered very cost‐effective with an ICER of I$590 per YLS.In a sensitivity analysis, we assumed higher loss to follow‐up between visits; results are presented in the Supporting Information Appendix. Findings were qualitatively similar to the main analysis, although the value of screening more frequently in a woman's lifetime increased as women who had previously been lost to follow‐up had more opportunities for successful linkage to treatment. At low baseline coverage levels, once in a lifetime screening needed to reach higher coverage levels relative to the main analysis in order to achieve reductions in cancer risk that were comparable to screening two or three times in a lifetime.The INMB values for shifting from once in a lifetime screening at baseline coverage to screening either three times in a lifetime (at baseline coverage) or once in a lifetime (at greater than baseline coverage) are presented in Figure The INMB of increasing coverage of screening once in a lifetime was dependent on the absolute coverage gain, and was fairly stable as baseline coverage increased. As baseline coverage varied from 30% to 70%, the INMB of an absolute gain in coverage of 10% ranged from I$12 to I$13; the INMB of a 20% gain in coverage ranged from I$25 to I$26; and the INMB of a 30% gain in coverage ranged from I$38 to I$39. While absolute coverage gains of 40% to 60% were only relevant when baseline screening coverage was 50% or less, the INMB values for these gains were high, ranging from approximately I$51 (for coverage gains of 40%) to I$77 (for coverage gains of 60%).The financial impact of a screening program for 100,000 women in a single birth cohort, juxtaposed to the projected number of cervical cancer cases averted, is presented in Figure i.e., 30%) in Uganda, expanding coverage of screening once in a lifetime to 50% or more can yield comparable health benefits to screening two or three times in a lifetime at baseline coverage, and can be very cost‐effective. As the baseline coverage level rises to 50% and higher, extending the number of screens in women already with access becomes more attractive, and increasingly greater expansions in coverage for once in a lifetime screening are necessary to achieve comparable reductions in cancer risk. When baseline coverage reaches 70%, screening two or three times in a lifetime yielded greater health benefits than screening once in a lifetime at 90% coverage, and screening three times in a lifetime at baseline coverage of 70% was very cost‐effective. Thus, the relative efficiency of increasing screening coverage versus increasing frequency will depend on baseline coverage levels and achievable coverage gains. Importantly, we found that increasing coverage of once in a lifetime screening is more likely to reduce health disparities and improve distributional equity than screening a smaller proportion of the target population with greater frequency. For a specified payer's budget, more cases may be averted from achieving higher coverage with once in a lifetime screening than from increasing screening frequency.Our aim was to inform decision‐makers in low‐resource settings as they make programmatic choices to concentrate limited funds on (1) improving access to cervical cancer screening or (2) increasing the number of screening opportunities for women who already have access. We present both health and economic outcomes for a wide range of baseline coverage levels— which we define as existing coverage with once in a lifetime screening— as well as a range of coverage gains for once in a lifetime screening. We found that at low levels of baseline coverage  are needed for laboratory processing and treatment. In the absence of data on programmatic costs, we determined the maximum dollar amount per woman by which the cost of screening once in a lifetime (at baseline coverage) could be increased to achieve either higher coverage with once in a lifetime screening or greater screening frequency while remaining “very cost‐effective.” We calculated the INMB for each of these potential program improvements. At a low baseline coverage level of 30%, the INMB value for shifting from screening once in a lifetime to three times in a lifetime was I$18. This implies that the total per‐woman lifetime costs  of incorporating two additional screening episodes would need to be I$18 or less in order for screening three times in a lifetime to have an ICER below per capita GDP. As baseline coverage increased to 70%, the INMB for shifting from screening once in a lifetime to three times in a lifetime was much higher (I$41), reflecting the greater value of increasing screening frequency when most women already have access to screening at least once in a lifetime. The INMB values for increasing coverage of once in a lifetime screening steadily increased along with coverage gains, and indicate that total per‐woman lifetime costs could increase by I$12 to I$13 in order for a 10% absolute coverage gain to remain very cost‐effective, while costs could increase by I$38 to I$39 for a 30% coverage gain, regardless of baseline coverage. The presentation of INMB findings allows decision‐makers to assess the value of different screening program improvements, depending on baseline coverage and expected costs of either increasing frequency of screening or expanding coverage of once in a lifetime screening.In Uganda, screening with HPV testing is currently limited to demonstration projects, and thus baseline coverage is low.There are limitations to this analysis. Because we did not have costing data to reflect expansion of screening coverage with self‐collection in a community setting, our estimates of the cost‐effectiveness of expanding coverage are based on provider‐collection at the clinic. However, a previous analysis suggests that differences in test performance and effectiveness between collection methods are small.Our assumption of equivalent loss to follow‐up rates for expanding coverage versus increasing screening frequency may be an oversimplification. For instance, if coverage expansion efforts focus on outreach to rural areas while programmatic increases in screening frequency focus on urban areas where women may already have greater access to care, linkage to treatment may differ between strategies. Gas‐based cryotherapy relies on consistent resupply of gas, which is expensive to transport and not always available. In remote areas, women may need to travel farther to reach a facility with cryotherapy equipment, increasing the risk of loss to follow‐up. However, new ablative technologies currently undergoing testing are smaller, portable, and do not require gas,We did not consider the potential impact of differential risk of disease among women who might benefit from increases in programmatic coverage versus those who might benefit from increased frequency of screening. If, for instance, program efforts focus on providing multiple screening opportunities for HIV‐infected women— who have a greater prevalence of oncogenic HPVBoth the cost‐effectiveness profile and recurrent financial costs must be favorable to implement a sustainable screening program. Here we present information on both the cost‐effectiveness and affordability of screening in Uganda, at varying frequencies and screening coverage levels, to convey both the relative value and the costs of different programs. Our findings suggest that prioritizing greater access to at least one screening over number of screenings per woman may yield greater population health benefits, improve distributional equity, and provide greater value for money when baseline screening coverage is low. As coverage extends to a greater proportion of the target population, increasing the frequency of screening may yield additional health benefits while remaining very cost‐effective. We have quantified this programmatic tradeoff between expanding coverage and increasing frequency in order to inform evidence‐based decision‐making by those formulating screening guidelines and implementing new programs in low‐resource settings. Harnessing scarce resources to prioritize expansion of screening coverage may provide an opportunity to strengthen primary health care systems. For the millions of women in low‐ and middle‐income countries who are past the target age for HPV vaccination, secondary prevention with cervical cancer screening is the only way to prevent a leading cause of cancer death in women. Given very low baseline coverage at present, a policy focus on increasing access to screening appears to be compatible with improving both equity and efficiency.Supporting InformationClick here for additional data file.


CRC) is the second most common cause of cancer death in USA. We analyzed CRC disparities in African Americans, Hispanics, Asians/Pacific Islanders, and American Indians/Alaska Natives compared to non‐Hispanic Whites. Current guidelines recommend screening for CRC beginning at age 50. Using SEER  database 1973–2009 and North American Association of Central Cancer Registries (NAACCR) 1995–2009 dataset, we performed frequency and rate analysis on colorectal cancer demographics and incidence based on race/ethnicity. We also used the SEER database to analyze stage, grade, and survival based on race/ethnicity. Utilizing SEER database, the median age of CRC diagnosis is significantly less in Hispanics (66 years), Asians/Pacific Islanders (68 years), American Indians/Alaska Natives (64 years), and African Americans (64 years) compared to non‐Hispanic whites (72 years). Twelve percent of Asians/Pacific Islanders, 15.4% Hispanics, 16.5% American Indians/Alaska Natives, and 11.9% African Americans with CRC are diagnosed at age <50 years compared to only 6.7% in non‐Hispanic Whites (P < 0.0001). Minority groups have more advanced stages at diagnosis compared to non‐Hispanic Whites. Trend analysis showed age‐adjusted incidence rates of CRC diagnosed under the age of 50 years have significantly increased in all racial and ethnic groups but are stable in African Americans. These results were confirmed through analysis of NAACCR 1995–2009 dataset covering nearly the entire USA. A significantly higher proportion of minority groups in USA with CRC are diagnosed before age 50 compared to non‐Hispanic Whites, documenting that these minority groups are at higher risk for early CRC. Further studies are needed to identify the causes and risk factors responsible for young onset CRC among minority groups and to develop intervention strategies including earlier CRC screening, among others.Colorectal cancer ( Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer death in the United States in both males and females. CRC is a common cause of death among racial and ethnic minorities. For example, African Americans have been reported to have the highest over‐all incidence, highest incidence of advanced stage at disease presentation, highest attributable mortality, and lowest survival rates after diagnosis compared to any other ethnic or racial group Colorectal cancer incidence and mortality are known to increase with age, however, recent studies showed a significant increase in the CRC onset and incidence in individuals <50 years of age The primary aim of the study was to provide a complete and up‐to‐date evaluation of incidence and mortality trends of CRC among racial/ethnic minorities when compared with NHW using SEER  database 1973–2009 and North American Association of Central Cancer Registries (NAACCR) 1995–2009 dataset, including analysis of data in subjects with CRC diagnosed at age <50 years.The SEER program began collecting data on cancer incidence for cases diagnosed in 1973 for seven state and metropolitan registries with an additional two added for 1974 and 1975; these are collectively referred to as the SEER nine Registries. Four more registries began contributing cases diagnosed on and after 1992 (SEER 13). A total of 18 registries (SEER 18) have reported since 2000. Race is categorized in the groupings of NHW, African American, AI/AN, and A/PI for all years. Hispanic ethnicity information is available for all years and is the result of manually collected Hispanic ethnicity and the North American Association of Central Cancer Registries (NAACCR) Hispanic Identification Algorithm (NHIA), which corrects and imputes ethnicity based on last name, birthplace, and other factors NAACCR is the professional organization that certifies population‐based cancer registries in the United States and Canada. We obtained appropriate Institutional Review Board (IRB) approval to receive data on CRC reported to NAACCR by 54 population‐based cancer registries in the United States. The NAACCR Cancer in North America (CINA) Deluxe Dataset contains data from 48 states, the District of Columbia, and five metropolitan areas. This dataset contains data from population‐based cancer registries funded by the Centers for Disease Control and Prevention's (CDC) National Program of Cancer Registries (NPCR) as well as SEER data and contains nearly all the cases of CRC reported in the entire US We used SEER*Stat Software We compared the distribution of CRC incident cases of all ages by age‐, gender‐, and cancer‐specific variables such as stage and grade across race/ethnicity groups . We repeated this analysis for subjects diagnosed with CRC at age <50 years. Missing values were assumed to be missing at random in all groups. Age‐adjusted incidence rates of CRC were calculated based on incidence data from the SEER 13 Registries only from 1992 through 2009.Population data used to calculate rates in this report was obtained from SEER based on the National Center for Health Statistics (NCHS) bridged single‐race modification to the estimates released by the Census Population Estimates Program; rates were age‐adjusted to the 2000 US Population (Census P25‐1130) standard in 19 groups.From 1973 to 2009, approximately 580,000 cases of CRC were diagnosed among NHW in the SEER registries and about 160,000 among racial/ethnic minorities. CRC was the second leading cause of cancer in Hispanics and the third leading cause of cancer in A/PI, AI/AN, AA, and NHW comprising 9.9%, 13.1%, 12.1%, 11.5%, and 11.4% of all cancers in these groups, respectively. Descriptive statistics of CRC cases diagnosed in the above racial/ethnic groups compared to NHW are presented in Table We also analyzed the data utilizing a more remote period 1973–1990) and a more modern period (1991–2009); overall, the observed patterns were consistent in both time periods  and a more modern period 1991–2009); overall, the observed patterns were consistent in both time periods  of the age‐adjusted incidence rates for various racial/ethnic groups were calculated according to age groups as reported in the SEER 13 registries for the period 1992–2009 . Analysis utilizing all ages revealed that minority groups were diagnosed with CRC at more advanced stages compared to NHW  recommends screening for CRC using FOBT, sigmoidoscopy, or colonoscopy beginning at age 50 years; people at higher risk of developing CRC, such as those with family history of CRC, should begin screening at a younger age and may need to be tested more frequently The SEER Program collects and publishes cancer incidence and survival data from population‐based cancer registries that currently cover more than 28% of the US population, providing a large high‐quality database for assessing differences among racial/ethnic groups. The population covered by SEER is comparable to the general US population; however, it tends to be somewhat more urban and has a higher proportion of foreign‐born persons than the general US population In summary, we analyzed disparities of CRC among racial/ethnic groups at a national level utilizing large population‐based data extracted from the SEER database with confirmation of results utilizing the NAACCR CINA data virtually covering the entire United States. Large disparities continue to persist among racial/ethnic minorities compared to NHW. Minority groups are at higher risk for early CRC. These findings suggest a need for further studies to address this alarming trend and examine the causes and risk factors involved. In addition, studies are needed to develop and test intervention strategies including a consideration for lowering the screening age in minority groups, among other strategies.None declared.


Thalassoma, with abundances up to 15 times higher than any other labrid. A key locomotor modification—a winged pectoral fin that facilitates efficient underwater flight in high‐flow environments—is likely to have underpinned this global success, as numerical dominance by Thalassoma was contingent upon the presence of high‐intensity wave energy. The ecological success of the most abundant species also varied with species richness and the presence of congeneric competitors. While several fish taxa have independently evolved winged pectoral fins, Thalassoma appears to have combined efficient high‐speed swimming (to relax abiotic selection) with trophic versatility (to maximize exploitation of rich resources) to exploit and dominate extreme coral reef habitats around the world.Phenotypic adaptations can allow organisms to relax abiotic selection and facilitate their ecological success in challenging habitats, yet we have relatively little data for the prevalence of this phenomenon at macroecological scales. Using data on the relative abundance of coral reef wrasses and parrotfishes (f. Labridae) spread across three ocean basins and the Red Sea, we reveal the consistent global dominance of extreme wave‐swept habitats by fishes in the genus  In identifying the dominant taxa among locations and wave exposures, we explored the macroecological consequences arising from an ecologically relevant adaptation  that has independently evolved among several lineages of ray‐finned fishes Fulton, . We focu2−2), and the total number of reef crest labrid species (richness) tallied for each location. To explore the relationship between species richness and relative abundance, we divided the density of the most abundant species by the density of the second most abundant species as a conservative measure of numerical dominance. We then explored the extent to which numerical dominance of the most abundant species varied across a sixfold gradient in labrid species richness, which can represent a gradient of competitive pressure from confamiliar fishes that tend to have very similar resource requirements  wave‐swept crest habitats were investigated for eleven coral reef locations across three ocean basins and the Red Sea Table . We usedThalassoma (the most abundant labrid genus) and all other genera, we quantified the abundance of all labrid fishes within shallow reef habitats (3 m depth) that are either sheltered (back reef) or directly exposed (crest) to incident trade wind‐driven waves across a 40‐km cline on the northern Great Barrier Reef (GBR), Australia ‐transformed data to explore differences in the relative density of Thalassoma versus other genera with the fixed factors of continental shelf position and habitat zone (crest/back), and random reef replicates nested within shelf position. A minimum of 9,500 permutations of residuals under a reduced model were performed using type III sums of squares within the PERMANOVA+ (v1.0.6) module of Primer (v6.1.16). We then fitted least‐squares linear and nonlinear (quadratic) regressions between fish density and mean habitat flow velocities to explore trends in the abundance of the two fish groups. All regression were fitted using Sigmaplot (v9.01). To examine links with fin morphology, we plotted mean density against the pectoral fin aspect ratio for each species present within the most exposed reef crests of the outer Great Barrier Reef, and documented the pectoral fin aspect ratio for the top three species present in each of our global locations. Pectoral fin aspect ratios were mostly taken from published sources  were calculated from calibrated images taken by the first author or by J. E. Randall  of all labrid individuals within exposed coral reef crests  had a density that was 5.3 ± 1.1 times higher than the next most abundant labrid species. The extent of numerical dominance by the top species increased along a gradient in species richness, with the greatest dominance evident in low‐diversity systems  was seven to eight times more abundant than their confamiliars, versus two to three times more abundant in Indonesia and Papua New Guinea , and over ten times more abundant than species of all the other labrid genera  pectoral fins have emerged as a significant adaptation in the biology and ecology of several independent reef fish lineages , possibly because the next most abundant species was often another Thalassoma. Presumably, this pattern is influenced by the tendency for close relatives to be most ecologically similar and compete most strongly for the same resources , Thalassoma fishes are known for their flexible trophic morphology and broad dietary preferences relative to their confamiliars, and generalized foraging among a wide spectrum of coral reef microhabitats  with broad trophic preferences, but rounded pectoral fins, is one of the most common labrids within wave‐sheltered and deeper reef habitats across tropical and temperate latitudes (Fulton & Bellwood Thalassoma exhibit a range of traits that predispose them to success within a range of habitat types to which they gain access according to their locomotor capabilities.Trophic versatility may be one such trait that helps explain the global success of Thalassoma may have been a key component of this invasion, which coincided with a wave of phenotypic innovations among the Labridae, and speciation of Thalassoma (Bernardi et al., Thalassoma to gain a competitive edge in exploiting challenging high‐flow environments around the world. For Thalassoma, it appears the energy savings afforded by a highly tapered pectoral fin have allowed the relaxation of environmental selection and an ecological release in shallow wave‐swept habitats, where the ecological success of Thalassoma has manifested in the global domination of these extreme coral reef habitats.Colonization of wave‐swept habitats by fishes appears to have been a relatively recent phenomenon in the evolution of coral reef ecosystems, with an initial invasion in the early Miocene about 23–20 Ma (Bellwood, Goatley, & Bellwood, We have no conflict of interests to declare.10.5061/dryad.24q8p.Supporting data is available in Table


Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.Long non‐coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell‐specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single‐molecule RNA  In the post‐genomic era, the ‘junk’ moniker is giving way to the understanding that most of the genome is fully functional 6 adenosine methylation (m6A) Most lncRNAs are transcribed by RNA polymerase II LncRNAs play a diversity of regulatory functions due to their modular structures and RNA–RNA or RNA–DNA base pairing ability HOXD gene by binding two enzymes (PRC2 and LSD1) involved in chromatin modification HOXD gene, where PRC2 deposits the H3K27me3 modification needed for repression and LSD1–CoREST–REST erases the H3K4me2 modification involved in activation By combining their modularity and base‐pairing ability, lncRNAs act as guiding molecules, directing proteins to other RNA or DNA molecules PCDH10, PCDHB5 and JAM2) LncRNAs, now being emerged as master regulators of gene expression, have been shown to be involved in a variety of malignancies In addition, lncRNAs have been proven as important diagnostic and prognostic biomarkers of cancer and other disorders Thanks to advances in increasing the depth of RNA sequencing techniques, thousands of novel lncRNAs have been discovered in recent years. However, the task of characterizing newly discovered lncRNAs is an arduous one, contingent on the power of technologies at our disposal.In this review, we strive to shed light on the ever‐expanding toolkit of techniques employed in lncRNA research. The methods are classified based on methodology as techniques used to visualize lncRNAs, determine their structure, map their interactions with other cellular components and elucidate their functions. Each method is described briefly, with their attributed pros and cons being discussed and summarized.Most of the genomic sequence is transcribed, and the majority of such transcripts do not code for proteins As with other molecules, our knowledge of the subcellular localization of lncRNAs opens up new vistas to their potential cellular roles et al., made use of two pools of probes labelled with different fluorophores and co‐localizing the signals obtained using the two probe sets. If the signals co‐localize, then they are construed as true ones indicating the lncRNA localizations, otherwise at least some of the signals are considered to be due to off‐target hybridizations A common obstacle in revealing the subcellular localization of lncRNA molecules is the off‐target hybridization of probes to nuclear aggregations, generating blob‐like signals like that of true lncRNA signals. Cabili RNA FISH and immunofluorescence have been used in order to show the co‐localization of Urothelial carcinoma associated 1 (UCA1(and phosphorylated hnRNP1 in the perinuclear regions to confirm that the phosphorylated form of hnRNP1 is able to interact with UCA1 RNA tracking methods have been developed that take advantage of green fluorescent protein fused to MS2 coat protein (MCP) and the MS2 binding sequence inserted into the RNA molecule to be analysed. The RNA molecule containing the MS2 binding site and the GFP‐MCP fusion protein are expressed inside the cell. The localization of RNA molecules is revealed through GFP‐MCP binding to the MS2 site The GFP fluorophore, 4‐hydroxybenzylidene imidazolinone (HBI) does not show fluorescence in the absence or denaturation of the GFP protein. RNA mimics of GFP are aptamer molecules that can bind HBI or other GFP like fluorophores and enable them to fluoresce The CRISPR/Cas9 system is considered as a type of adaptive immunity in bacteria and archaea The RNA‐binding capability of Cas9 protein has spurred the development of technologies for tracking the subcellular localization of RNA molecules Despite being tested on few mRNA molecules, the CRSPR‐based RNA tracking technology has the potential to be utilized in studies aimed at revealing the subcellular localization of RNAs including lncRNAs.Unlike RNA detection technologies based on RNA–protein interactions, the CRSPR‐based RNA tracking technique does not require laborious genetic engineering procedures, as target recognition is based on RNA–RNA interactions LncRNAs are capable of folding on themselves and therefore taking a wide variety of secondary and tertiary structures, enabling them to perform their functions SHAPE is among the key techniques commonly used to unravel the secondary structure of lncRNAs in vivoin vitro to in vivo in icSHAPE(in vivo click SHAPE) Despite being a powerful technique in unravelling the single and double‐stranded regions of lncRNA molecules, SHAPE is not able to determine the range of such base paring interactions An array of chemical probing techniques including SHAPE has been employed to characterize HOTAIR in structural terms in vitro, subjected to RNase treatment, ligated to adaptors and sequenced. Finally, a PARS score is obtained as the log ratio of in a double‐ or single‐stranded conformation. The log ratio between the number of sequence reads obtained for each nucleotide in the V1 (cleaving double‐stranded RNA) and S1 (cleaving single‐stranded RNA) experiments. A nucleotide with a higher log ratio, or PARS score, thus indicates a higher probability of being in a double‐stranded conformation  is a modification of ChIRP (described in 4.1.1) that allows simultaneous mapping of RNA–RNA, RNA–DNA and RNA–protein interactions at the level of individual domains In this technique, the cells are subjected to cross‐linking agents to preserve protein–nucleic acid interactions and then sonicated to shear the nucleic acids. Specific biotinylated oligonucleotides are used to tile individual domains. Streptavidin magnetic beads are then added to capture specific interactions and non‐specific interactions are washed away. The material recovered from dChIRP is finally fractioned into protein, DNA and RNA parts for extraction and analysis. The parts can be subjected to real‐time PCR using primers for the lncRNA domain of interest and other RNAs suspected to be in its interaction. The protein content can be analysed by Western blotting or mass spectrometry to reveal any protein interacting with different domains. Recovered DNA can be also sequenced, revealing any binding sites on genome that may become occupied by a given lncRNA domain Unlike other techniques used to map lncRNA interactome, dChIRP is capable of revealing lncRNA multivalent interactions with other RNA, DNA and protein molecules in a single reaction Using dChIRP, Rox lncRNA was indicated to have interaction with MSL proteins through its D1, D2 and D3 domains. The Rox binding sites on X‐chromosome  were also clearly determined. dChIRP has the potential to be applied to other lncRNAs with important implications in cancer, on the way to revealing their interactions in a domain by domain manner The traditional approach for gene discovery called forward genetics relies on finding aberrant phenotypes in a model organism and then unravelling the genes behind such phenotypes. In reverse genetics, however, a given gene of interest is perturbed and the resulting phenotypic change is observed, helping to find the gene function. Reverse genetics are particularly important in delineating the important roles of lncRNAs in cancer, as gene ablations can clearly depict their contributions to cancer phenotype or transformation of normal cells. Here, we look at the techniques utilized in lncRNA reverse genetics Several technologies for genome editing based on programable proteins including Zinc‐finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs) and Cas9 have emerged over the past 10 years Making use of two gRNAs, the dual CRISPR system is suited for perturbing lncRNAs in contrast to conventional methods, which may lead to insertions or deletions, not enough to eliminate the function of lncRNAs The applicability of the all‐in‐one system strategy in perturbing lncRNAs was illustrated by ablating urothelial carcinoma‐associated 1 (UCA1), lncRNA‐21A and AK023948 in various human cell lines While the knockout techniques eliminate gene expression by deleting the genes, RNAi suppresses expression by sequestering and then cleaving RNA molecules Any RNAi experiment has to be accompanied by standard controls Involved in aggressive prostate cancer, second chromosome locus associated with prostate‐1 (SChLAP1), is an lncRNA that inhibits the tumour‐suppressive activity of the SWI/SNF complex. The role of SChLAP1 in contributing to aggressiveness of prostate cancer was assayed by performing siRNA‐mediated knock‐down experiments. Remarkably, the SChLAP1 knock‐down dramatically decreased cell proliferation and invasiveness.RNAi has been successfully applied to many loss‐of‐function studies involving lncRNAs GFP in HEK293 cells and endogenous genes including CD71, CXCR4 in HeLa cells The versatility of CRISPR/Cas9 has enabled repurposing of its utility from genome editing to genome regulation Acting at the DNA level, CRISPRi can be a useful tool to target non‐coding transcripts including lncRNAs as well as microRNAs and products of RNA polymerase III in vitro transcription in the presence of labelled UTPs can be performed for the generation of RNA FISH probes. In vitro transcription followed by RNase III digestion can be implemented for the production of esi‐RNAs. c‐KLAN combines the power of RNA FISH and RNAi for characterizing lncRNAs through loss‐of‐function studies and subcellular visualization RNA‐FISH is utilized to localize RNA transcripts inside the cell c‐KLAN has been efficiently utilized to reveal the lncRNAs involved in pluripotency Despite being highly effective devices in loss‐of‐function studies, siRNA and shRNAs cannot be used effectively in depleting nuclear lncRNAs, although the RNAi machinery also exists in the nucleus 2′‐O‐methoxyethylribose (2′‐MOE)) on their termini, intervened by a central ‘gap’ of unmodified DNA, 8‐10 bases in size As effective tools for silencing nuclear lncRNAs, ASOs are modified or unmodified single‐stranded deoxyribonucleotides, with the ability to hybridize to their targets among a population of transcripts present in the cell. Knock‐down experiments using ASOs are based on the ability of RNase H to degrade the RNA part of a DNA–RNA duplex MALAT1 was also observed in systematic MALAT1 depletion using ASOs Several groups have used ASOs successfully in loss‐of‐function studies of nuclear lncRNAs. For instance, ASOs were utilized in an attempt to knock‐down the nuclear‐retained MALAT1, systemically. Two gapmers against different parts of MALAT1 were used, and systemic knock‐downs using ASOs were shown to recapitulate the same results observed in knockout studies. Besides, the same reduction in metastasis happening upon genetic loss of Cell function is governed by a complex network of interactions involving different cellular components Conventional methods of chromatin precipitation commonly make use of antibodies against proteins associated with DNA ChIRP, which can be considered as an analogue of ChIP, has been used by different research groups hitherto to map the genomic binding sites of different lncRNAs RNA immunoprecipitation (RIP) is a technique which can be employed to unravel all lncRNAs that interact with specific proteins of interest Like other protein‐centric pull‐down assays, RIP is dependent on antibodies previously produced against the suspected counterpart proteins In a study of the role of the lncRNA, UCA1, in breast cancer promotion, RIP was employed to indicate that UCA1 interacts with hnRNP1 and suppresses the p27 protein level by competitive inhibition To investigate the role of MALAT 1 in splicing of pre‐mRNAs, antibodies against SRSF1 splicing factor, and primers specific to MALAT1 were used to reveal a specific interaction between them, using the RIP technique Further refinement of RIP has led to cross‐linking and immunoprecipitation (CLIP) technique, which is capable of revealing a more accurate picture of lncRNA–protein interfaces with single nucleotide resolution In contrast to other CLIP variants, which makes use of cross‐linking with 254 nm UV light, PAR‐CLIP takes advantage of 4‐thiouridine to facilitate the cross‐linking process, allowing it to occur at a higher UV wavelength (365 nm) irCLIP and eCLIP are newly introduced versions of CLIP techniques developed to increase reverse transcription efficiency and decrease RNA loss and sequencing backgrounds The advantages and disadvantages of each of the CLIP techniques have been reviewed in Table .In a genome‐wide analysis of lncRNA binding sites on different RBPs, CLIP‐seq was employed to show the interaction of Ezh, a PRC2 complex subunit, with 35 lncRNAs including Tsix, Meg3, Rian and Pvt1 Using PAR‐CLIP, TUG1, DLEU2 and GAS5 lncRNAs were also shown to bind at least two of the three RNA‐binding proteins HuR, Ago2 and MOV10 at identical binding sites Moreover, CLIP has been employed to show air interactions with H3K9 histone methyl‐transferase G9a Over the past 20 years important efforts have been made allowing the research to be directed at transcriptome‐wide analysis of RNAs and changes in their global patterns of expression Several methods have been developed for global or single‐molecule mapping of local primary and secondary structures of RNA molecules in vitro cross‐linking Previous attempts have been made to map miRNA–mRNA interactions in RNA induced silencing complexes Some lncRNAs are shown to function through interaction with other RNA molecules Deamination of Adenosine to inosine is a common RNA modification, which plays important regulatory roles and has been implicated in several disorders including cancer N1‐cyanoethylinosine adduct halts the cDNA extension process and allows identification of A‐to‐I editing points. The procedure is carried out with or without CE. In CE− conditions both I and A positions recognized in sequencing. However, under CE conditions the RNAs undergoing A‐to‐I modifications stop to extend beyond the edition point. Therefore, the G band (I is read G in sequencing process) is erased, and a shaper A band is detected and that point is identified as a true modification site ICE‐seq makes use of Inosine cyanoethylation with Acrylonitrile. The resulting Caenorhabditis elegans poly (U) polymerase (PUP‐2) Despite being effective in determining RNA–protein interactions, RIP and CLIP can only unravel protein‐RNA interactions when specific antibodies against the protein of interest are already available These techniques have the potential to be extended to lncRNA research, shedding light on all the interactions a given protein of interest may have with the population of lncRNAs inside the cell and depicting probable interruptions in such interaction networks that may lead to cancer.A global understanding of genomic regions undergoing active transcription can provide valuable information for discovery and functional analysis of lncRNAs. Global run‐on assay sequencing (GRO‐seq) is a technique based on NGS technologies that provides valuable insights into of the location, orientation and density transcripts undergoing active transcription by RNA polymerase II. In this technique, the RNA polymerase is allowed to function in the presence the UTP analogue Bromodeoxyuridine (BrdU). The nascent RNAs are captured using anti‐BrdUTP antibodies and subjected to deep sequencing. The resulting sequence reads are aligned across the genome to map the genomic positions undergoing active transcription The technique, however, is limited to cell culture and other artificial systems due to its dependence on incorporation of Bromodeoxyuridine (BrdU). New initiation events that may happen and introduction of artefacts during preparation of nuclei are among other demerits of the technique Using this method, several studies have indicated that transcription by RNA pol II often occurs divergently and leads to production of lncRNAs et al. The result of many studies has shown that there is an intimate relationship between mRNA half‐life and its function, which implies an understanding the half‐life of lncRNAs can provide useful information about their functions The method involves pulse labelling of cells with 5′‐bromo‐uridine and then quantifying the decrease in labelled RNA levels that occurs over time making use of deep sequencing. The result of this study indicated that RNA molecules with half‐lives <4 hrs include the majority of regulatory and lncRNAs in situ hybridization allows simultaneous visualization of cellular location of a small number of transcripts RNA sequencing provides valuable insights into global quantitative and qualitative changes in transcripts but does not picture the spatial distribution of transcripts in vitro quantitative measurement of transcripts to in vivo and allows spatial organization of gene expression to be revealed globally FISSEQ extends the applicability of RNA‐seq beyond FISSEQ has the potential to reveal the global spatial changes in lncRNA expression in disease conditions including cancer. However, the technique needs to be refined regarding its read length, sequencing depth and coverage and library preparation The complexity of cells is better appreciated with this emerging view that most of the genome is functional and ubiquitously transcribed into a repertoire of coding and non‐coding RNAs. LncRNAs are highlighted in the new transcriptional landscape replete with transcripts of unknown function. They play vital roles in various cellular processes and disease conditions including cancer.. Therefore, considering their important regulatory roles, lncRNAs are among the missing parts of the puzzle in the long‐lasting fight against cancer and have to be scrutinized comprehensively so as to enhance our current understanding of this disease. The boundaries of our capability to understand how lncRNAs can contribute to cancer prevention or progression are contingent on power of the techniques available.in vitro. To overcome the single‐molecule limitation of SHAPE, global structural analysis of the complete RNA reservoir or structurome has been now possible by PARS and Fraq‐seq. icSHAPE has extended the applicability of lncRNA structural studies by SHAPE to the realm of in vivo. LncRNA interaction networks have been clearly delineated using RNA‐centric approaches comprising ChIRP, CHART, RAP and protein‐centric methods including CLIP. dChIRP has enabled domain by domain interrogation of lncRNA interactions. CLIP has been refined regarding its throughput and resolution to iCLIP and PAR‐CLIP. Recently, further refinement of CLIP has led to irCLIP and eCLIP which allow more accurate and rapid determination of lncRNA–protein interactions. RNAi has allowed the knock‐down experiments employed in functional studies of lncRNAs and CRISPRi can increase the precision with which we can suppress lncRNA expression in reverse genetic investigations. TRIBE‐seq enables global mapping of lncRNA–RNA interaction network, without any pre‐existing antibody for affinity purification required in CLIP techniques. ICE‐seq allows the research to be directed at depicting a global picture of lncRNA epitranscriptome and is not confounded by individual variations due to SNPs. The half‐life of lncRNAs can be screened in real time using BRIC‐seq. GRO‐seq provides a picture of transcriptionally active genomic regions as it can capture nascent RNA molecules.High‐throughput sequencing has accelerated the pace at which new lncRNAs are being discovered and allows global analysis of lncRNAs. However, our ability to characterize the expanding catalogue of unknown lncRNAs is also dependent on other techniques enabling us to map lncRNA interactions, their structure and function. For instance, the subcellular location of lncRNAs can be determined using FISH and further clarified by the combination of FISH and esi‐RNA technology (cKLAN). Compared to RNA FISH which can localize few transcripts in a single reaction, FISSEQ allows the research to be directed at quantitative global and spatial analyses of lncRNA expression. New technologies developed based on CRISPR‐Cas system can now be a complement to the current RNA visualization techniques as they may be better applied to non‐coding transcripts. Structural studies of lncRNAs have become more feasible using SHAPE. But SHAPE is limited to single‐molecule analyses carried out As aberrations in abundance, subcellular localization and interaction networks of lncRNAs are among key contributors to cancer progression, the technological toolkit described throughout this review is central to a deeper understanding of lncRNAs not yet characterized, revealing their likely widespread roles in cancer disease and opening new perspective for therapy.The authors confirm that there are no conflicts of interest.


Helicosporidium and Prototheca, and the free‐living chlamydomonadalean genera Polytomella and Polytoma. Here, we examine the published data on colorless green algae and argue that investigations into the different evolutionary routes leading to their current nonphotosynthetic lifestyles provide exceptional opportunities to understand the ecological and genomic factors involved in the loss of photosynthesis.The endosymbiotic origin of plastids was a launching point for eukaryotic evolution. The autotrophic abilities bestowed by plastids are responsible for much of the eukaryotic diversity we observe today. But despite its many advantages, photosynthesis has been lost numerous times and in disparate lineages throughout eukaryote evolution. For example, among green algae, several groups have lost photosynthesis independently and in response to different selective pressures; these include the parasitic/pathogenic trebouxiophyte genera  However, ‘mixotrophic’ organisms can make use of both inorganic (via photoautotrophism) and organic (via chemoheterotrophism) carbon sources; the latter involves prey consumption through phagocytosis (phago‐mixotrophism), endocytosis, or the intake of small organic compounds via osmosis (osmo‐mixotrophism). Whether or not photoautotrophy or chemoheterotrophy is the main form of nutrient assimilation varies among mixotrophic organisms and depends on the availability of light and organic compounds in the environment  or the environment (in free‐living species). Interestingly, although there are no reported cases of phagotrophic colorless green algae, a few examples of phago‐mixotrophic prasinophytes are known  and at least three times in the order Chlamydomonadales (Chlorophyceae) Fig. b. These Prototheca and Helicosporidium  include unicellular nonflagellated parasites/pathogens that still retain vestigial plastids. Members of the genus Prototheca are ubiquitous opportunistic animal pathogens that can be found in diverse habitats, such as soil detritus, fresh and brackish water, and plant‐ and animal‐derived foods for human consumption. Prototheca is the causative agent of protothecosis, a disease that develops after Prototheca comes in contact with skin wounds, causing cutaneous lesions, bursitis, and major systemic alterations in immunosuppressed hosts  than to other Prototheca species  that ultimately harnessed their heterotrophic abilities to invade novel ecological niches.The loss of photosynthesis probably occurred in the ancestors of Polytoma and Polytomella are two nonphotosynthetic genera that belong to the Chlamydomonadales (Chlorophyceae) , alcohols (ethanol and butanol) and monosaccharides   Fig.   are closely related to the osmo‐mixotrophic Chlamydomonas leiostraca and Chlamydomonas humicola as well as to other photosynthetic species, including Chlamydomonas applanata, Chlamyomonas pulsatilla, and Chlamydomonas pumilio. The colorless Polytoma oviforme (SAG 62‐27), however, branches independently of other Polytoma species and forms a sister lineage to the photosynthetic Chamydomonas chlamydogama and Chlamydomonad sp. Itas 9/21 T‐4w  P. oviforme as sister to C. chlamydogama . For instance, the C. reinhardtii ‘white’ mutant lts1‐204, defective in phytoene synthase , and ‘yellow’ mutants, defective in Mg‐protoporphyrine IX methyltransferase (an enzyme involved in tetrapyrrole biosynthesis), produce stable phenotypes with no carotenoid pigments and a low chlorophyll concentration, respectively  associated with the evolution of the colorless green algae discussed in the previous subsection are likely to be reflected in distinct genomic evolutionary processes in these lineages. For instance, parasitism is expected to result in genome reduction  or compaction . However, the recently sequenced nuclear genome of the parasite et al., et al., V. carteri, which is c. 80% noncoding and contains c. 96 genes , is among the largest plastid chromosomes sequenced thus far  and contain 104 and 103 genes, respectively. The ptDNAs of Chlorellales also show a range of architectures: Chlorella variabilis , Chlorella sorokiniana , Chlorella vulgaris , Parachlorella kessleri , Oocystis solitaria  and Auxenochlorella protothecoides .The plastid genomes (ptDNA) of chlorophytes have revealed interesting evolutionary patterns. Comparative analyses of diverse chlamydomonadalean species have shown that ptDNA size and amount of noncoding regions correlate positively with the level of cellular organization, whereby multicellular taxa or taxa with large cells have larger ptDNAs than unicellular species or those with small cells  and genetic (e.g. key mutations) mechanisms involved in the ‘no‐return’ transition from a mixotrophic lifestyle to an obligate heterotrophic one, and the role of nonphotosynthetic plastids in colorless algae. Further comprehensive genomic and functional investigations are critical to a better understanding of how these major trophic shifts occurred several times independently in chlorophytes.C. reinhardtii (121 Mbp)    . In photosynthetic lineages, proteins encoded in the ptDNA contribute very little to the overall plastid proteome, and the vast majority (95–98%) of proteins serving plastids are nuclear encoded  is almost two‐thirds smaller and encodes about one‐third fewer genes than the genomes available from photosynthetic Chlorellales. Moreover, c. 15% (882 sequences) of the Helicosporidium nuclear genes have no recognizable orthologs in C. subellipsoidea and C. variabilis  machinery (plastid protein import), and the general secretory (SEC) pathway (protein export) are also nuclear encoded. Although proteins for many plastid‐localized pathways are still encoded in the nuclear genome, nearly 44% of the plastid‐targeted proteins present in other viridiplants  have probably been lost from the Helicosporidium metabolic repertoire  are also retained in the colorless plastid of apicomplexans, which possess a secondary, red‐algal‐derived plastid  has revealed numerous putative nuclear‐encoded, plastid‐targeted enzymes (Table Helicosporidium and Prototheca (Borza et al., There are no complete nuclear genomes available yet for free‐living colorless chlorophytes, but such data will be important to investigate the evolutionary patterns following the loss of photosynthesis under an ecological scenario that is different from the parasitic/pathogenic lifestyle underlying the evolution of es Table  shared wOverall, we argue that genomic and transcriptomic studies of colorless green algae have the potential to greatly improve our understanding of photosynthesis and its evolutionary loss. The available data are largely skewed towards pathogenic/parasitic species and, thus, are impacted by the gene‐repertoire reduction associated with both the loss of photosynthesis and parasitism. Current transcriptomic data suggest that there is a certain degree of ‘convergence’ in the plastid protein repertoire among diverse colorless algae and land plants; however, the data available for colorless Chlamydomonadales are limited.Helicosporidium and Prototheca are examples of loss of photosynthesis associated with the transition to parasitic/pathogenic lifestyles and sequencing additional nuclear genomes from Helicosporidium and Prototheca species will be important to understand the evolution of this trophic transition. For example, is the relative lack of genomic reduction in Helicosporidium sp. ATCC 50920 a common trend among pathogenic/parasitic trebouxiophytes? Unlike Helicosporidium and Prototheca, Polytomella and Polytoma are not believed to have gone through pathogenic/parasitic stages en route to losing photosynthesis. Thus, they should lack the genomic consequences typically associated with parasitism, such as high nucleotide substitution rates, gene loss, and reduced rates of recombination, and can provide a different perspective on our understanding of the loss of photosynthesis. Important insights could come from comparative genomic studies of Polytomella and Polytoma species and their close photosynthetic Chlamydomonas relatives.Mixotrophy has been an important driving force in the loss of photosynthesis across diverse green algal lineages. The genera P. uvella–C. leiostraca and P. oviforme–C. chlamydogama represent exceptional duos for investigating the consequences of the loss of photosynthesis in free‐living algae without the confounding effects associated with adopting a parasitic/pathogenic lifestyle. Our preliminary analyses of Polytoma uvella ptDNA sequence data reveal similar patterns of gene loss between the genomes of free‐living and parasitic chlorophytes. These similarities are notable given the different ecological scenarios that presumably drove the independent evolution toward heterotrophism, and suggest that the convergence in ptDNA gene content after the loss of photosynthesis has been shaped by similar constraints.For example, the colorless‐photosynthetic ‘sister taxa’ pairs Polytoma lineages and Polytomella species compare to the repertoire of their closely related photosynthetic taxa? Has the loss of photosynthesis caused expansions or contractions of particular gene families in colorless algae? Are there ‘unique’ genes, or even complete pathways, encoded in the nuclear genomes of the colorless species not present in those of their photosynthetic relatives? Has horizontal gene transfer had any role in the evolution of colorless Chlamydomonadales? How did the organelles of cyanobacterial origin recruited > 1 billion yr ago become essential for other cellular roles beyond the photosynthesis? Are there other biochemical and molecular functions, other than photosynthesis, critical for the establishment of primary plastids?Other questions to be addressed concern the nuclear genomic complements of colorless green algae. For example, how do the gene collections of different Polytomella and Polytoma nuclear genomes and plastid proteomes will be key to understand in detail the physiological roles of their colorless organelles. The very distinct evolutionary outcomes in the two free‐living chlamydomonadalean lineages that lost photosynthesis independently raise interesting questions, including: What mechanisms led to ptDNA loss in Polytomella? Did any ‘unique’ functions evolve independently in the colorless plastids of the different Chlamydomonadales? Answers to these basic questions will be critical to reveal the ecological and genomic process underlying the origin of these remarkable green algae that have evolved as ubiquitous free‐living organisms even after ‘the lights went out’.Finally, the study of the