The development of new HER2-directed therapies has resulted in a significant prolongation of survival for women with metastatic HER2-positive breast cancer. Discoveries in the laboratory inform clinical trials which are the basis for improving the standard of care and are also the backbone for quality improvement. Clinical trials can be completed more rapidly by expanding trial enrollment to community sites. In this article we review some of the challenges in treating metastatic breast cancer with HER2-directed therapies and our strategies for incorporating our community partners into the research network.  Although breast cancers arise from a single organ, the biology and natural history of the disease can be extremely variable. Gene expression profiling allows us to subcategorize breast cancer into four “intrinsic subtypes”, each with a unique natural history and response to therapy—Luminal A, Luminal B, Basal-like, and HER2  “enriched” ,2. HowevFour membrane tyrosine kinase receptors constitute the HER family. HER1 , HER3, and HER4 bind to almost a dozen different ligands, while HER2 has no known ligands. Rather, HER2 is activated through homo- and heterodimerization with other HER family members, and these complexes activate intracellular signaling pathways such as MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) that results in tumor growth, invasion, migration, and survival . Gene amThe humanized IgG monoclonal antibody, trastuzumab, targets the extracellular domain of HER2 effectively preventing intracellular signaling. While trastuzumab has minimal activity as a single agent, when combined with chemotherapy there is a prolongation in survival for women with metastatic breast cancer ,7,8. TraCandidacy for HER2-directed therapies is based on a pathologic determination from the primary tumor or metastatic focus of disease. It is generally agreed that HER2 positivity is defined as 3+ staining by immunohistochemical staining (IHC) of the protein or gene amplification of HER2 by fluorescence in situ hybridization (FISH). Recently, companion studies described using in situ hybridization to report gene copy number per cell. This resulted in a HER2 “equivocal” status and required modification of the ASCO/CAP  guidelines for HER2 assessment .The standard of care dictates that a tumor biopsy be performed on all patients suspected of having recurrent or metastatic breast cancer in order to document the disease as well as to determine the hormone receptor and HER2 status, which guide the choice of systemic therapy. The estrogen receptor (ER), progesterone receptor (PR), or HER2 status on a recurrence may differ from the original primary in 20–30% of instances. Furthermore, although it is not feasible to biopsy all sites of metastatic disease, discordance of receptors within an individual patient occurs in 15–20% of lesions ,12,13. UHER2-directed therapies are effective only in patients whose cancers are HER2-positive. The NSABP B47 randomized over 3000 women with early stage breast cancer whose HER2 status was negative  to receive conventional adjuvant chemotherapy with or without 12 months of trastuzumab. No benefit was seen with the addition of trastuzumab and cardiac toxicity was observed in 2.3% receiving trastuzumab . It is p64Cu-labeled trastuzumab as a PET imaging agent to study women with recurrent/metastatic breast cancer. In our experience uptake on 64Cu-trastuzumab PET/CT correlates with the qualitative assessment of HER2 by IHC. Higher uptake of 64Cu-trastuzumab is seen in women with IHC levels of 3+ compared to 1+ [64Cu-trastuzumab PET/CT does not assess HER2 status, but demonstrates that trastuzumab is effectively delivered to the cancer. This pharmacodynamics data is clinically more relevant than knowing the intensity of uptake by IHC or degree of gene amplification. The current standard of care for patients with newly diagnosed metastatic HER2-positive cancer utilizes a combination of chemotherapy, trastuzumab, and pertuzumab. It is, therefore, not possible for functional imaging with radiolabeled trastuzumab to predict impact of trastuzumab on treatment efficacy when response to therapy is confounded by the concurrent administration of chemotherapy.We have developed ed to 1+ ,16. Func64Cu-trastuzumab PET/CT is able to predict for response to therapy. In our experience, pretreatment 64Cu-trastuzumab PET/CT in women with recurrent/metastatic disease is able to identify which individuals are unlikely to benefit from TDM1. Additionally, 64Cu-trastuzumab PET/CT has identified disease within the CNS and has also demonstrated that uptake of 64Cu-trastuzumab can be heterogeneous within a single patient. If that uptake in individual sites falls below a certain level, TDM1 is not effective in those areas and the response is mixed [Ado-trastuzumab emtansine (TDM1) binds to the extracellular domain of HER2 and undergoes receptor-mediated internalization inhibiting intracellular signaling pathways . Becauseis mixed .Like trastuzumab, pertuzumab is also a monoclonal antibody that binds to the extracellular domain of HER2, but at a different epitope from that bound by trastuzumab. Pertuzumab prevents dimerization with other HER family members  . NeitherTwo antibody drug conjugates (ADC) have utilized trastuzumab to deliver a cytotoxic payload. Ado-trastuzumab emtansine (TDM1) utilizes maytansine as the cytotoxic component and has been found to be superior to other regimens containing chemotherapy and HER2-directed agents. It is currently considered second or third line therapy ,23. Fam-The two ADCs differ in other ways. Fam-trastuzumab-deruxtecan has a higher drug-to-antibody ratio than TDM1  and fam-trastuzumab-deruxtecan is selectively cleaved by cathepsins that are up regulated in tumor cells. Deruxtecan is also highly cell-membrane permeable and is readily released across cell membranes. A potential benefit for this mechanism is that release of the cytotoxic component may have cytotoxic effects on adjacent tumor cells regardless of the tumor’s HER2 dependency . Since tp < 0.001) [The oral tyrosine kinase inhibitor, tucatinib, is a selective inhibitor of HER2. In combination with capecitabine and trastuzumab, objective tumor responses have been demonstrated in heavily pretreated patients with metastatic HER2-positive breast cancer including women with brain metastases. The HER2CLIMB study randomized women with metastatic HER2-positive breast cancer who had received prior trastuzumab, pertuzumab, and TDM1 to receive trastuzumab and capecitabine with or without tucatinib. Progression-free survival (PFS) favored the addition of tucatinib (7.8 months compared with 5.4 months). The overall survival also favored the tucatinib arm with a survival prolongation of 4.5 months; 21.9 months vs. 17.4 months. For the 291 women with brain metastases, the median PFS was 7.6 months in the tucatinib arm and 5.4 months in the control arm (< 0.001) . In the < 0.001) .There are additional active agents that also deserve mention. Lapatinib was the first dual inhibitor of HER2 and HER1. Until the development of TDM1, lapatinib and capecitabine were considered to be second-line therapy . CurrentThe explosion in new and highly effective therapies has transformed metastatic HER2-positive breast cancer into a chronic disease. Conventional chemotherapy still has a role as salvage treatment and is more effective when combined with trastuzumab . New druBose used DNA sequencing on samples obtained from ACOSOG (American College of Surgeons Oncology Groupz) 1031 and data from other sequencing studies to identify somatic mutations in tumors that were pathologically determined to be HER2-negative. Seven of the 13 mutations were classified as activating mutations. Cell line studies showed a unique dependence on EGFR phosphorylation and led to an assessment of the therapeutic efficacy of the dual kinase inhibitors of HER2 and EGFR, lapatinib and neratinib. Neratinib demonstrated a stronger inhibition of cell growth than lapatinib and was effective in all activating mutations . It is eCity of Hope recently acquired 30 community practice sites and in doing so dramatically expanded the number of colleagues in surgical, medical, and radiation oncology and almost tripled the number of new breast patients. With such a rapid expansion, our goal was to develop a common culture of research and quality clinical care that is at the heart of City of Hope’s values. This initiative was led by a steering committee of interdisciplinary subspecialty physicians and business leaders who met regularly. This team identified the individual community physicians who had an interest in treating breast cancer and in conducting clinical research and worked with all the stakeholders to define quality of care. We convened a meeting of all clinical faculty with an interest in breast cancer, compiled a brief resource inventory , and identified barriers to achieving our predefined quality of care. The sharing of our unique differences in practice and the challenges that each individual experienced, resulted in a respect for what each partner brought to the program and how we could work together to solve these problems. Through a culture of respect and trust we were able to define quality metrics for the clinical care of patients with breast cancer and created treatment guidelines which included eligibility for clinical trial participation see . ClinicaAll research decision-making is made by the breast cancer research team (Breast Disease Team), composed of medical, surgical, and radiation oncologists, basic scientists, statisticians, pharmacists, research nurses, clinical research associates, and the Executive Director for Affiliate sites. Breast Disease Team meetings are available on a remote platform so that our community partners can participate, and meeting minutes are submitted to all. The Breast Disease Team meets on a weekly basis to review proposed study concepts, endorse the written protocols developed from an approved concept, troubleshoot operational problems, and review active patients on study. Our current clinical research portfolio in HER2-positive breast cancer focuses on two main areas: 1. Defining new therapies and new roles for these systemic therapies (an area largely driven by the cooperative groups and Pharma), and 2. Identifying which patients are likely to benefit from HER2-directed therapies using functional imaging, which is a City of Hope research initiative.The Executive Director for Affiliates oversees research at the community sites and is aware of the physicians’ interests, patient population, and clinical trial infrastructure at the individual offices. The full clinical trial portfolio is not activated at every site and phase I clinical trials are generally restricted to the main campus. The most successful clinical trials to conduct in the community are those therapeutic trials that focus on improving disease outcomes through the use of new agents or testing approved agents used in different settings. For example, mutations in PIK3A have been associated with worse outcomes in adjuvant therapy trials and in CLEOPATRA ,40. A phOur radiolabeled trastuzumab imaging studies require a radiopharmacy and expertise by a committed team of radiologists and physicist who interpret the images. Our community oncologists play an important role in these studies by referring patients, thereby allowing us to complete accrual expeditiously. They are authors on our publications and are recognized by their local communities as true experts who participate in innovative research. The Breast Disease Team has a number of initiatives to ensure inclusion of our community partners in research, and the weekly Breast Disease Team meeting is at the heart of these collaborations. Discussions at weekly Tumor Boards and a bi-weekly treatment planning meeting frequently highlight on-going studies and identify study candidates. City of Hope is a founding member of the NCCN  and has a long history of participating in guideline panels. While some of the guidelines for treating breast cancer are definitive, others offer acceptable treatment options. The breast cancer team on the main campus meets on an annual basis to compile our guideline preferences for treatment, including, not only standard of care but the open studies for clinical trial participation as well see .We continue to activate investigator-initiated, cooperative group, and Pharma trials in the community that focus on improving the outcomes for women with metastatic HER2-positive disease. Our imaging research will utilize radiolabeled trastuzumab to predict for response to new agents such as fam-trastuzumab-deruxtecan and tucatinib ,29. ThesNational Cancer Institute-designated Comprehensive Cancer Centers (NCI-CCC) such as City of Hope strive to provide expert interdisciplinary clinical care and cutting-edge treatments and technologies. Between 1998 and 2008, 6.4% of adults between 22 and 65 years of age who were diagnosed with cancer in Los Angeles County received treatment at an NCI-CCC. For all primary tumor sites, the survival of patients treated at an NCI-CCC was superior to patients treated at a non-NCI-CCC. In this study, 6.5% of the 31,764 breast cancer patients included in the analysis were treated at an NCI-CCC and their survival was superior to those treated at non NCI-CCC sites . Realist


We are reporting a case of Acute Post-Infectious Flaccid paralysis also commonly known as Guillain-Barré Syndrome (GBS) in a patient with confirmed COVID-19 infection. GBS often occurs following an infectious trigger which induces autoimmune reaction causing damage to peripheral nerves. So far, only 8 cases have been described in association with COVID-19. This is the first to be described in Tanzania in an African Child, and probably the first in the continent. This report is presented for clinicians to be aware and for the medical fraternity to look into this unusual presentation which may shed some more light on possible pathways of the pathogenesis and clinical manifestations. We recommend that the presentation of GBS with acute respiratory distress should warrant extra precaution and a testing for COVID-19 especially when the symptoms of COVID-19 are protean. A novel Corona virus termed COVID-19 was first reported in Wuhan, China as early as December 2019 . ChildreMao et al. reported 36.4% of the 214 study participants hospitalized with COVID 19 had neurological manifestations . Virani LB, 12 year old young man presented to the Emergency Department with symptoms of acute progressive symmetric ascending quadriparesis with bilateral facial paresis. He was asymptomatic a week prior and then he developed a low grade fever and a dry cough. He was managed conservatively and treated symptomatically. He complained of lower back pain 5 days prior to arrival to the ED which was rapidly followed by weakness of both lower limbs. He was able to walk initially and was taken to a local dispensary where he was administered an intramuscular analgesics and discharged. The next morning, he lost function of both lower limbs with associated weakness of bilateral upper limbs as well and could not get out of bed and this prompted the family to seek further medical attention. He was taken to the same facility again and advised to start physiotherapy with a provisional diagnosis of lumbar spondylitis. He was referred when he developed facial paresis bilaterally and an altered level of consciousness associated with an increased respiratory rate. In the emergency department, the mother provided the additional information of the low grade fever and cough from the week before which they had managed conservatively at home. There was no history of recent travel or contact with a positive case of COVID 19. There was no significant past history or any known co-morbidities.Vital signs on presentation were remarkable with a respiratory rate of 24 breaths per minute with oxygen saturation of 88% [hypoxic] on room air with a heart rate of 150 beats per minute and a blood pressure of 150/100 mmHg. The patient was barely conscious with a Glasgow Coma score of 6/15 and had signs of shock with cold extremities and diaphoresis. The child was placed on nasal prongs at 6L of high flow oxygen and connected to a cardiac monitor. Blood was drawn for lab investigations, an ECG was done and a chest X-ray ordered. Physical examination revealed decreased lower extremity strength and muscle tone compared to upper extremities with a score of 1/5 in lower extremities versus a score of 2/5 in upper limbs as per Medical Research Council [MRC] scale. Deep tendon reflexes were absent in all four limbs. Meningeal irritation signs and upper motor neuron disorder signswere absent. The bedside blood glucose was 11.0 mmol/L. His lab results showed a white blood cell count of 17.3 x103 cells/mL, hemoglobin of 14.6g/dL and platelet count of 361 x 103 cells/mL with an otherwise normal differential. Electrolytes and renal profile were within normal ranges with a blood urea nitrogen of 5.6 mmol/L, creatinine of 54.4µmol/L, sodium of 142, potassium of 4.6 and chloride 100. Chest X-ray revealed bilateral basilar opacifications suggestive of consolidation with diffuse infiltrates bilaterally. ECG showed a sinus arrhythmia with slightly tall and tented T waves . BedsideGiven the existing epidemiological scenario and preceding symptoms of fever and cough, COVID-19 infection was suspected. Appropriate isolation precautions were implemented, and a respiratory viral panel testing [nasopharyngeal PCR] was sent. Antibiotic coverage was done empirically and other treatment modalities done as required. Just prior to transfer to Pediatric Intensive Care [PICU], child lost pulse and Cardio-Pulmonary Resuscitation [CPR] was initiated with return of spontaneous circulation [ROSC] achieved within 1 cycle of the CPR. Child was intubated and ventilated and started on positive inotrope infusion. A diagnosis of GBS was made and patient was immediately started on 400mg/kg of intravenous immune globulin [IVIG] therapy for a planned 5-day course according to clinical manifestations related to GBS. The reverse transcription-polymerase chain reaction [RT-PCR] for COVID-19 was positive and further evaluation and management was continued in the ICU. Given the classic clinical picture of GBS in absence of other identifiable etiology for his neurologic disease and strict infection control measures, additional supportive testing was not pursued. His clinical course showed improvement in his respiratory status, neurological status as well as his consciousness level with an improvement of the GCS to 14/15 within 5 days. Neurologically, his upper extremity weakness resolved after completion of the course of IVIG. Lower extremity power and tone improved significantly. He was planned for weaning off ventilation and extubation on day 6, and eventual rehabilitation and discharge. During the night preceding the planned weaning, the child self extubated accidentally and rapidly decompensated. Cardiac arrest ensued and attempts to resuscitate after CPR were futile and child died.Despite having first been reported in Wuhan, China; at the time of preparing this paper, the SARS-CoV-2 has infected over 3.2 million people worldwide with over 229,000 deaths attributed to COVID-19 directly . In TanzAlthough classically associated with Campylobacter jejuni, viruses like EBV, CMV, HIV and recently even Zika virus have also been implicated in some cases. The pathophysiology suggested is an autoimmune reaction secondary to molecular mimicry between the surface glycoproteins of the offending pathogen and the structures on peripheral nerves causing the antibodies to attack the nerves and cause neurologic symptom . We believe the offending agent in our case which triggered this neurological process was COVID 19 which also caused the respiratory symptoms. There is no direct evidence of a causative relationship between GBS and COVID 19 but due to various reports of similar cases; inclusive of the 5 cases reported in Italy , the oneAlso evidence has shown spike glycoprotein involvement with murine infection of the neurologic system with support from the hemagglutination proteins, making betacoronavirus a more likely strain of corona virus to infect neurologic cell lines . NeuromuIt is known that GBS is often triggered by a preceding bacterial or viral infection and hence COVID 19 must be considered a trigger to rule out by clinicians in such a case as the GBS maybe a neurological complication of the viral illness. Also, it raises the index of suspicion in patients whose presenting symptoms are neurological, especially when acute, and a thorough and detailed review of all systems is crucial to identify other features possibly suggestive of COVID-19. It is important to identify the GBS and start IVIG early for optimal outcomes. This report also adds stress to the fact that not all cases of COVID 19 will present with typical features or vital signs on presentation to the ED. Lastly, appropriate infection prevention measures and personal protective equipment are crucial in any and every case and in approaching any patient coming to the emergency department due to the variation in presentation of patients with COVID 19.


Information on the associations between pre-diagnosis health behavior and post-diagnosis survival time in esophageal cancer could assist in planning health services but can be difficult to obtain using established study designs. We postulated that, with a large data set, using estimated probability for a behavior as a predictor of survival times could provide useful insight as to the impact of actual behavior.Data from a national health survey and logistic regression were used to calculate the probability of selected health behaviors from participant’s demographic characteristics for each esophageal cancer case within a large cancer registry data base. The associations between survival time and the probability of the health behaviors were investigated using Cox regression.Observed associations include: a 0.1 increase in the probability of smoking 1 year prior to diagnosis was detrimental to survival (Hazard Ratio (HR) 1.21, 95% CI 1.19,1.23); a 0.1 increase in the probability of hazardous alcohol consumption 10 years prior to diagnosis was associated with decreased survival in squamous cell cancer  but not adenocarcinoma ; a 0.1 increase in the probability of physical activity outside the workplace is protective .We conclude that probability for health behavior estimated from demographic characteristics can provide an initial assessment of the association between pre-diagnosis health behavior and post-diagnosis health outcomes, allowing some sharing of information across otherwise unrelated data collections. With an incidence of 9.3/100,000 males and 3.5/100,000 females per year, esophageal cancer led to more than half a million deaths worldwide in 2018 [While there is considerable documentation of associations between health behavior and onset of esophageal cancer , the impHealth behavior prior to a cancer diagnosis is often different from health behavior post-diagnosis. Behavior prior to diagnosis can be influenced by public health activity but post-diagnosis behavior is strongly influenced by the diagnosis itself  and by tUnfortunately, investigating the effect of pre-diagnosis behavior on post-diagnosis survival can be difficult and expensive. As the disease is relatively rare, a prospective cohort study would be inefficient . Retrospective studies which enroll newly diagnosed cancer patients and ask them to recall their prior health behavior still involve considerable expense and are fraught with recall and survivor biases. In one example, an Australian study enrolling newly diagnosed esophageal cancer patients reported that patients with late-stage disease were difficult to enroll and under-represented [Secondary analyses of already existing data can provide alternate, cost-effective opportunities. It is now common for governments to sponsor both regular health behavior surveys and mandatory cancer registries. For those cancer cases who contributed to a survey prior to diagnosis, their health behavior and cancer outcomes can be linked to produce a retrospective cohort. Data linkage avoids recall and survivor biases and is cost efficient .But data linkage may not be feasible either. Confidentiality is one issue. But more fundamentally, as esophageal cancer is relatively rare, the number of cancer cases who happened to have previously participated in the health survey is likely to be very small. If data linkage cannot be applied, is there any other way in which these rich (and expensive) data sets can be used to help provide insights into the association between pre-diagnosis behavior and post-diagnosis survival times?Often the only measures in common between cancer registries and national health surveys are the demographic characteristics of participants. It is known that demographically similar people are more likely to display similar health behavior than people from different demographic groups . That isThis study investigated whether or not useful information on the association between pre-diagnosis health behaviors and post-diagnosis survival times could be obtained by analyzing cancer cases estimated probability of engaging in these behaviors. The analyses used US data and focused mainly on the three modifiable lifestyle factors identified above: cigarette smoking, alcohol consumption and excess body weight.Unit record data on esophageal cancer cases and their outcomes was extracted from the Surveillance, Epidemiology, and End Results Program (SEER) cancer registry . The SEEData on health behavior was extracted from the Behavioral Risk Factor Surveillance System (BRFSS) health survey . The BRFBoth collections provided access to cleaned, de-identified unit record data at no cost to the researcher. Although both data collections are large, with less than 0.2% of American adults participating in BRFSS and around 4000 esophageal cancer cases being recorded in the SEER data set each year, we could only expect about eight new esophageal cancer cases each year to have participated in the previous BRFSS survey.This analysis focusses on the 15-year period from 2001 to 2015. Data prior to 2001 are excluded due to changes in the definitions of some health behaviors variables and because earlier data may be less relevant to current behavior and outcomes. 2015 was the most recent year of SEER cancer registry data.As esophageal cancer is rare in young ages, all cancer cases who were less than 35 years of age are excluded as being atypical. Two hundred one of 57,025 (0.3%) cases are excluded. For the BRFSS health survey, all data records from respondents 25 or more years of age who lived in one of the 13 US States represented in the SEER cancer registries are included. Including the younger respondents allows information on health behavior up to 10 years prior to cancer diagnosis to be retained.The outcome of interest is post-diagnosis survival time in months as recorded in the SEER cancer registry data set. That is, all cases with survival less than 30.4 days after diagnosis (including cancers detected post-mortem) have a survival time of 0 months, those who died between 30.4 and 60.8 days have a survival time of 1 month, etc. The maximum possible survival time is 179 months. For those who are still alive and those who are lost to follow-up, survival time is censored at the date of last follow-up.Current smoker (yes/no) which includes those who smoke daily or less than daily;Alcohol - heavy drinking (yes or no), which is defined as more than two standard drinks per day for men and more than one standard drink per day for women in the month prior to survey;Alcohol - binge drinking (yes or no), which is defined as males reporting having five or more standard drinks or females reporting 4 or more standard drinks on one occasion in the month prior to survey;Current smoking and alcohol consumption (yes/no), which is defined as both current smoker and an average consumption of ≥1 standard drink of alcohol per day in the past month.2Obese (yes/no) which is BMI ≥ 30 kg/mUndertook physical activity or exercise in the past 30 days other than regular job (yes or no)The research focused mainly on measures relating to cigarette smoking, alcohol consumption and excess body weight. The choice of variables was restricted to measures available through the BRFSS health survey. The following variables, all recording self-reported behavior, were included:As the cancer registry data did not include information on pre-diagnosis health behavior we estimated the probability of each pre-diagnosis health behavior for each cancer case using the available demographic variables.Of the variables in common between the SEER cancer registry and the BRFSS health surveys we hypothesized that year, age, sex, race, marital status and State of residence could be helpful for predicting health behavior. For example, race is known to be associated with smoking  and alcon = 44,670, 3.1% of total) were omitted as there was no corresponding code in the SEER cancer registry data set. Marital status was categorized as married or living as married; divorced or separated; widowed; and single.As age was recorded in 5-year age groups in the SEER cancer registry data, we applied the same categories to the BRFSS health survey data. Race was categorized as White; Black; Asian or Pacific Islander; and American Indian or Alaskan native. Participants in the BRFSS health survey who self-reported as mixed race  from 2004 onwards.Other potential confounders of the association between behavior and survival were considered to be of lesser impact or potentially on the disease pathway. For example, while the relationship between smoking history and post-diagnosis survival may differ by gender, the effect may be small. In contrast, the choice between curative or palliative treatment is a strong predictor of survival time but may partially lie on the association pathway.  Adjustment for variables on the association pathway may remove some of the true association between health behavior and survival time.Fifty-six thousand eight hundred twenty-four SEER esophageal cancer cases and 1,450,775 BRFSS health survey respondents met the eligibility criteria. Additional file The characteristics of eligible cancer registry cases and health survey respondents are summarized using counts and percentages, with the exception of survival time which is summarized using medians, quartiles and maximums.The main analysis involves three discrete steps. Firstly, the probability of engaging in each health behavior were estimated from the BRFSS health survey data using logistic models; with a separate model for each behavior. Each modelled the probability of having the behavior of interest based on year of survey, age, sex, race, marital status and State of residence. We also allowed for differences in the probability of health behaviors between sexes and between marital statuses at different ages by including age by sex, age by marital status and marital status by sex interaction terms in each logistic model.i represent an eligible individual from the BRFSS data set and i is a smoker, then the logistic model has the formFor example, if we let To correct for the complexities in the BRFSS health survey sampling and non-response we weighted the logistic models by the sampling weights provided. In 2011, the BRFSS introduced a new method of calculating sampling weights which improved the weighting of some variables including race and marital status. However, as both systems weight to the State totals, we do not differentiate between the different type of weights in this analysis. We excluded data records with extreme sampling weights: those which fell in either the top or bottom 0.5% of the distribution. To assist the models to converge we use Firth’s bias reduced penalized-likelihood when fitting the models; using the logistf package (version 1.23) in R software (version 3.5.2). The fitted models are summarized in Additional file Year and age category were fitted as numeric variables while sex, race, marital status and State of residence are categorical. Preliminary investigations (not reported) confirmed that a linear model was reasonable for both year and age category. Year is coded as 0 for 2001 through to 14 for 2015 for analysis.We confirmed that the chosen risk profiling variables were indeed predictors of each health behavior by visual inspection of odds ratios from logistic regression models. To help gauge the predictive ability of each demographic variable we present areas under the curve (AUC) of the receiver operating characteristic (ROC) curve for each predictor alone and for the full logistic model using the pROC package (version 1.13.0) in R software. The higher above 0.5 the AUC, the greater the ability of the model to predict the health behavior.In the second step of the analysis, for each esophageal cancer case in the SEER cancer registry, we estimated their probability of participating in each health behavior by substituting their demographic characteristics into the logistic predictive model for that behavior.j represent an eligible cancer case from the SEER data set and xj the set of observed values of the demographic variables for individual j and j being a smoker asFor example, if we let mentclass2pt{minimAs we were specifically interested in health behavior prior to diagnosis we trialed three pre-diagnosis time points: 1, 5 and 10 years prior to diagnosis. This entailed substituting diagnosis year minus 1, 5 or 10 as the year variable of the logistic model and 5-age group minus 0, 1 or 2. To avoid extrapolating earlier than the observed data, the 5-year lag analysis was restricted to esophageal cancer cases from 2006 to 2015 and the 10-year lag model was restricted to cases from 2011 to 2015.p-values. Models were fitted with and without correction for age and cancer stage at diagnosis.In the third step of the analysis, the relationship between the estimated probability of each behavior and survival was investigated using Cox regression models using the survival package (version 2.43–3) in R software. Separate models were fitted for each behavior. Results are presented as hazard ratios (HRs) with associated 95% confidence intervals (CIs) and j relative to their estimated probability of smoking, adjusting for age and disease stage, could be writtenβ ’s are different to the β ’s listed in Eq.  appeared most accurate and the model for predicting obesity (AUC 0.59) appeared least accurate.Table Smoking 1 year prior to diagnosis appears to be unrelated to survival until adjustment for age and disease stage at diagnosis. In the adjusted model, each 0.1 increase in the probability of pre-diagnosis smoking is associated with a 20%  increase in post-diagnosis hazard with no discernible difference in results for ESCC and EAC subgroups.Results for alcohol consumption are mixed. When using behavior 1 year prior to diagnosis as the predictor, a 0.1 increase in the probability of heavy drinking appears to be protective of survival even after adjustment for age and cancer stage at diagnosis . However, when looking at behavior 10 years prior to diagnosis, the adjusted model finds heavy drinking to be detrimental to post-diagnosis survival in ESCC  and with no discernable association in EAC . The pattern of results for binge drinking is quite similar.A 0.1 increase in the probability of concurrently smoking and drinking ≥1 standard drink per day in the year prior to diagnosis is associated with double the risk of death , after adjustment for age and cancer stage with no difference between ESCC and EAC.After adjustment, a 0.1 increase in probability of obese 1 year prior to diagnosis is associated with an apparently trivial increase in post-diagnosis hazard . A slightly larger hazard  was recorded for a 0.1 increase in the probability of obese 10 years prior to diagnosis. A 0.1 increase in the probability of exercise outside employment 1 year prior to diagnosis is associated with improved survival  with little difference between ESCC and EAC.The results above appear to support of the proposition that demographic-derived estimates of the probability of health behaviors can assist in identifying association between pre-diagnosis health behavior and post-diagnosis survival time in esophageal cancer. The hazard ratios quoted in this paper show the increased hazard of death associated with each additional 0.1 probability of the health behavior of interest. That is, we are reporting the association between the estimated likelihood of engaging in a particular behavior and survival time. This is quite different from the association between the actual health behavior and survival time and more difficult to interpret. Never-the-less, there is consistency between the results of the present study and previously published results: especially in the presence and direction of associations.We have found that a 0.1 increase in the probability of smoking 1 year prior to diagnosis, adjusted for age and cancer stage at diagnosis, had an estimated HR of 1.20 (95% CI 1.18–1.22) in esophageal cancer survival. This association is consistent with findings from previous meta analyses such as HR 1.41   for smokThe current analyses found that increased probability for ‘at risk’ alcohol consumption in the year prior to diagnosis were generally protective of survival but that a 0.1 increase in ‘at risk’ alcohol behavior 10 years prior to diagnosis was detrimental to survival in ESCC . The 10 year results are consistent with a previous meta-analysis  which foThe unexpectedly protective result for alcohol consumption one-year prior to diagnosis could indicate insufficient adjustment for confounding  or weaknesses in the measurement tool .Previous authors have found that pre-diagnosis smoking and alcohol consumption combined produce a disproportionately high risk to post-diagnosis survival . We havWe observed that a 0.1 increase in the probability of obese 1 year prior to diagnosis was associated with slightly higher risk of death adjusted HR = 1.04  mainly associated with ESCC . The association seems small and the literature on obesity is sparse with mixed findings. One review found pre-diagnosis obesity could be associated with higher risks of death in cancer   but a laWe found that a 0.1 increase in probability of pre-diagnosis physical activity outside of the workplace was associated with improved survival . This is consistent with a recent review  which foOur analyses using estimated probability for health behaviors has produced results which have some face validity. A strength of this example is that the data sets used are large, public domain and well understood. Any interested researcher can reproduce, refine and/or extend these analyses using the same data sets.Both the data sets and the analysis technique used have some limitations and weaknesses. In relation to the data sets, there are response biases within the BRFSS  which thFor the model, estimating the probability of a behavior is less accurate than a direct measure of behavior and conveys less information about that behavior: so will have less power for detecting associations. There may be residual confounding from unmeasured variables . Finally, omitting interactions with year may have contributed to the apparent lack of difference in outcomes between behavior one, five and 10 years prior to diagnosis.The rarer the disease, the less feasible it is to conduct either prospective cohort studies or record linkage (retrospective cohort) studies. Retrospective data collection (including case-control studies) are fraught with recall and survivor biases. Exploiting existing data provides cost-effective opportunities for investigations but may require different methodologies.Analyses of the associations between estimated probability for pre-diagnosis health behavior (based on demographic characteristics) and survival time in esophageal cancer produced results with some face validity. Expressing associations in units of changes in the probability of the health behavior was cumbersome. However, the required data are already available, allowing relatively quick and inexpensive investigations of possible associations between pre-diagnosis behavior and post-diagnosis outcomes for relatively rare diseases. And of course, most diseases are relatively rare.Additional file 1:Table S1. Disease Characteristics and Outcomes of Eligible SEER Cancer Registry Esophagael Cancer Cases and BRFSS Health Survey Respondents 2001–2015.Additional file 2:Table S2. Associations Between the Selected Demographic Variables and Health Behaviors in the BRFSS Health Survey Data Set.Additional file 3:Table S3. Relationship Between Adjusted Variables and Survival Time.Additional file 4:Table S4. Association Between Survival Time and 5-Year Pre-Diagnosis Health Behavior.Additional file 5:Table S5. Logistic Regression Models Predicting Health Behaviors from Demographic Variables.


Significance: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use.Aim: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue Approach: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at Results: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy.Conclusions: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel–probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns. The tum33.1First, we evaluated the rate of leaching of PtG4 from the agarose gel into the surrounding solution . Initialsolution . The leasolution . The hal3.2The CELI images of agarose-PtG4 sensor pellets in high- and low-oxygen environments were acquired at different delay times as shown in ectively . The pO2solution . Briefly3.3Phosphorescence images of the sensor pellets at different depths in liquid tissue-simulating media were used to investigate the potential to carry out oxygen imaging at depth . Quantif3.4To test the ability to sense oxygen in living tissue, the gel sensors were implanted into subcutaneous MD-MBA-231 tumors at the flank of the mice. Phosphorescence images at different delay times were acquired before and 30 min after sacrificing the mice by cervical dislocation . Phosphogenation . The pO2stograms .Oxygen levels in subcutaneous MD-MBA-231 tumors and in normal tissues were further investigated by CELI using the implanted sensors. The lifetime maps for a tumor and normal tissue (muscle) are shown in 3.5To monitor oxygen changes in tumors undergoing multiple fractions of radiotherapy, gel sensors were implanted into three mice with subcutaneous MDA-MB-231 tumors on day 1 and imaged by CELI during each fraction of radiotherapy. A 6 MV photon beam was delivered to each tumor with 5 Gy/fraction for 4 daily fractions, for a total of 20 Gy. The otherapy . The medotherapy .4The ability to assess PtG4 has been designed to sense oxygen in aqueous solutions, and its accumulation in tissue is governed by extravascular diffusion and retention kinetics. The toxicology, inflammatory, and immunogenicity properties of PtG4 have not been fully elucidated, so it cannot be translated to clinic at present, although this could be achieved in the future. However, the delivery of an oxygen-sensing probe encapsulated in a gel, e.g., by a direct intratumoral injection, should allow local oxygen sensing, while avoiding systemic circulation and associated toxicity issues. Local injection is a well-known way to provide microdose levels to a tissue region. Sufficiently long retention time of PtG4 within the agarose gel allowed us to perform Almost all optical imaging technologies are critically limited by the depth at which they can sense in tissue. If an optical method is used in clinical oxygen monitoring, the tissue absorption and scattering characteristics are the factors that limit the depth of imaging the most. One advantage of CELI is that Cherenkov emission is produced within the tissue, providing an inner excitation source generated along the pathways of the MV x-ray beams. Thus, PtG4 can be excited at all depths where the radiation dose is deposited. The only major limitation is the modest intensity of Cherenkov emission, which results in a very low-phosphorescence signal. The biosensor produced in this study was directly injected into tumor, and that allowed us to effectively enhance the phosphorescent signals by increasing local concentration of PtG4 inside the tumor.in vivo studies. Nevertheless, our experiments suggest that with proper chemical design, gel-based materials may be useful as local probes for CELI of oxygen. And in some cases, gels that are able to release the probe into the nearby tissue may in fact be advantageous for sensing over a larger tumor volume. In this regard, agarose-PtG4 gel pellets described in this study will be further evaluated as local probe delivery vehicles.Clinical imaging of 5In summary, an implantable oxygen sensor has been developed for CELI of oxygen by encapsulating water-soluble phosphorescent probe PtG4 into agarose gel. The agarose-PtG4 solution could be injected into the tumor prior to gelation, which provided an opportunity to monitor tumor oxygen pressure during radiotherapy. This type of sensor design and delivery could significantly decrease concerns related to the toxicity of oxygen probes in clinical setting. The approach overall can provide a method for monitoring tumor hypoxia in clinical radiotherapy and thus help to identify hypoxic areas in tumors for planning and optimizing therapeutic intervention.


VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells. Vps34 are embryonic lethal arresting at gastrulation stage , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this manuscript, the authors identify a cancer cell line (RKO cells) as a VPS34-dependent cell line through RAB7A. They try to propose that VPS34 induces RAB7A-dependent lysosomal degradation of transferrin receptors, which led to impaired iron uptake. Upon several experiments, they suggest that iron limitation is a primary driver of VPS34-dependency in RKO cells. Unfortunately, this paper is plenty of correlations that are not supported at all by direct experimental evidence supporting the author’s claims. Also, what is the biological interest of demonstrating that in an in-vitro system, using only one cell line these findings are relevant for cancer? Below I have listed some major concerns:MAJOR CONCERNS• The authors use the shRNA of VPS34 and different members of C1 and CII to claim that CII is responsible for RKO sensitivity. However, they did not show direct evidence excluding CI from this phenotype. ATG14 depletion is not enough since all the common members of CI and CII are involved. Block autophagy from so early steps would have some effects, I guess.• RAB7A is just one  of the potential downstream effectors of VPS34. Why the authors focus on it. As expected, and showed by the authors, RAB7A KO partially mimics VPS34 inhibitions. What is the reason to use very sophisticated screening methods to focus on one protein that even does not fully recapitulate the phenotype? This is a mix of results linked together without a strong rationale….• There are no evidences in the manuscript indicating that VPS34 induces RAB7A!!• Then, the authors come back to omics, using a hit that is not strongly supported by experimental evidence, performing transcriptomics analysis. From this analysis, GO indicates that cholesterol pathway and HIF1 are significantly modulated in RAB7A. Cholesterol pathway is discarded without check cholesterol levels in cells? What is the rationale to add cholesterol to the cells, why not deplete cholesterol? Again, no clear rationale …• Finally, the authors decide to focus on iron homeostasis. IRP2 levels and aconitase activity are an indirect approach to evaluate the intracellular iron availability... why they didn’t look at the quantification of the labile iron pool (LIP) and ferritin protein levels ?• The staining of the Transferrin Alexa Fluor 488 probe in WT and RAB7A KO cells is strange. In particular, the group RAB7A KO treated with a VPS34 inhibitor. Does that mean the positive vacuoles positive (in their limiting membranes) for the probe in this group?Comments:• Moreover, to me, it is necessary to check at ferritin because the inhibition of VPS34 by PIKIII also caused impairment in ferritinophagy, as described in the first paper that they cited. They only observed that RCO cells are not suffering by depletion of ferritinophagy (fig. S3), but they didn’t show the ferritin levels in Ctrl and RAB7a KO cells during PIKIII treatment (i.e. in figure 6F-G)• As VPS34 inhibition provokes the shift from oxidative phosphorylation towards glycolysis, the luciferase assay (CellTiter-Glo) is a reliable readout of cell viability?• How do the authors explain that at a higher dose of PIKIII the increment of HIF1 appears also in RAB7 KO cells ?Reviewer #2: The manuscript by Kobylarz and colleaguesexplores the dependency of RKO cancer cells on the protein Vps34. The experiments are well conducted and presented, and the story line flows logically.The authors show, using a KO screen, that in the absence of Vps34 activity, removal of Rab7a is sufficient for viability. They then search for the underlying mechanism using a transcriptome approach to identify the underlying mechanism. There are, however, a few key experiments that are required in order to support the conclusions drawn by the authors.- The transcriptional signature of Rab7A-KO+Vps34 inhibition pointed towards activation of HIF-1a and cholesterol synthesis. This is exactly the same signature that is obtained by inhibiting lysosomal acidification . Yet, the authors never test this possibility. In my view, this experiment is required, because it raises the possibility that the Vps34-Rab7A pathway acts by a completely different mechanism than that suggested by the authors. This is further substantiated by the fact that the authors can rescue many of the observed cellular and molecular phenotypes with exogenous iron citrate.- line 229 - the statement that bioavailable iron is predominantly complexed with transferrin is not necessarily correct. In fact, upon iron deficiency, cells readily activate ferritinophagy and mitophagy, as well as up-regulation of the transferrin receptor. Therefore, it is pivotal that the autophagy branch of the pathway is also tested. I recall that the authors showed that the removal of Vps34-C1 had no effect on the viability, while C2 did, which they possibly used to discount the involvement of autophagy. However, this is not sufficient, as UVRAG, involved in C2, is also involved in autophagy. Thus, it is important that the authors test if the autophagy pathway is involved .- line 252 - the use of bafilomycin is per se sufficient to trigger iron deficiency and TFRC induction (see papers mentioned above in the context of HIF-1a). Therefore this experiment should be done in the presence of a lysosomal inhibitor that does not affect lysosomal pH - for example, leupeptin or some other caspase inhibitor.- in Suppl Fig2, it is unclear if the slight differences on OCR are significant (they don't seem to be) - it should be noted whatever the case is. Also, Rab7A should be compared to the controls, i.e., in the same plate and not just in terms of absolute OCR.- the analysis of the RNA seq data is unnecessarily conservative. Behjamin-Hochberg is known to hammer the data. That is the authors' choice, so I will not dwell on it. However, the RNA seq raw data files should be made available in a repository , so that other interested labs can reanalyze the data with statistical strategies that are more appropriate for biological data.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  29 May 2020Reviewer #1In this manuscript, the authors identify a cancer cell line (RKO cells) as a VPS34-dependent cell line through RAB7A. They try to propose that VPS34 induces RAB7A-dependent lysosomal degradation of transferrin receptors, which led to impaired iron uptake. Upon several experiments, they suggest that iron limitation is a primary driver of VPS34-dependency in RKO cells. Unfortunately, this paper is plenty of correlations that are not supported at all by direct experimental evidence supporting the author’s claims. Also, what is the biological interest of demonstrating that in an in-vitro system, using only one cell line these findings are relevant for cancer? Below I have listed some major concerns:• Inhibition of VPS34 inhibits growth of most cancer cell lines, but to various degrees as visualized in figure 1. By computationally screening for genetic or expression features, we were unable to identify general predictors of VPS34 sensitivity across all cell lines. This is the reason why we decided to focus on one highly VPS34-dependent cell line and perform an in depth analysis what drives dependency. This is acknowledged in the title of our manuscript and we do not generalize our findings.• We disagree that our paper is plenty of correlations not supported by direct experimental evidence. Using rescue experiments, we demonstrate that iron is necessary and sufficient to reverse both RKO growth defect and metabolic alterations due to VPS34 inhibition.• Furthermore, we expect the pooled CRISPR screen, transcriptional profiling and lipidomics data sets to be a valuable resource for future studies on VPS34 biology.MAJOR CONCERNSThe authors use the shRNA of VPS34 and different members of C1 and CII to claim that CII is responsible for RKO sensitivity. However, they did not show direct evidence excluding CI from this phenotype. ATG14 depletion is not enough since all the common members of CI and CII are involved. Block autophagy from so early steps would have some effects, I guess.• We have included project DRIVE data  and individual dox-inducible shRNAs  showing that knockdown of canonical autophagy genes ATG5, ATG7 and ATG16L1 do not significantly impact growth of RKO cells RAB7A is just one  of the potential downstream effectors of VPS34. Why the authors focus on it. As expected, and showed by the authors, RAB7A KO partially mimics VPS34 inhibitions. What is the reason to use very sophisticated screening methods to focus on one protein that even does not fully recapitulate the phenotype? This is a mix of results linked together without a strong rationale….• The goal of the pooled CRISPR screen was to map the cellular pathways that can suppress VPS34-dependency, without a comprehensive follow-up of all hits. We have clarified this in the manuscript. • The pooled CRISPR screen identified a strong enrichment of suppressors that regulate endolysosomal maturation. In addition to being the strongest hit, RAB7A was picked based on its well characterized role in endolysosomal maturation. We view the RAB7A KO data as validation of endolysosomal maturation being a key driver of VPS34-depenency in RKO cells. To better reflect this point, he have re-organized the manuscript and now show all RAB7A KO data in the final figure, linking it to transferrin receptor trafficking, iron uptake, metabolic alterations and cell growth.• The partial rescue in RAB7 KO cells suggests that the VPS34-RAB7A axis plays a critical role, but is likely not the only contributor to the VPS34-dependent growth defect. Involvement of other pathways such as sphingolipid metabolism will be explored in further studies.• The loss of RAB7A alone has a partial iron deprivation effect, which is reversed by PIK-III treatment suggesting a complex relationship between RAB7A and VPS34 in the endosome maturation process and regulation of iron homeostasis.There are no evidences in the manuscript indicating that VPS34 induces RAB7A!!• We acknowledge that the manuscript lacks experimental evidence that VPS34 inhibition induces RAB7A. Given the scope of this study, we focused on characterizing the iron-dependency rather than the molecular mechanism how VPS34 inhibition activates RAB7A. Unfortunately, we are unable to deliver such data now due to the COVID19-related lockdown and can only refer in the discussion to Jaber et al. who have shown that VPS34 deficient MEFs accumulate GTP bound RAB7A due to feedback inhibition of TBC1D2, the GAP for RAB7A. Then, the authors come back to omics, using a hit that is not strongly supported by experimental evidence, performing transcriptomics analysis. From this analysis, GO indicates that cholesterol pathway and HIF1 are significantly modulated in RAB7A. Cholesterol pathway is discarded without check cholesterol levels in cells? What is the rationale to add cholesterol to the cells, why not deplete cholesterol? Again, no clear rationale …• We have now included a complete analysis of the cholesterol deprivation phenotype in Figure 4. Using lipidomics and labeled LDL probe, we show that VPS34 inhibition decreases intracellular cholesteryl esters and blocks cholesterol uptake through enhanced lysosomal degradation of LDL receptors. Since PIK-III treated RKO cells display cholesterol deprivation phenotype, we added excess cholesterol to see if this could rescue the growth defect, which it did not.Finally, the authors decide to focus on iron homeostasis. IRP2 levels and aconitase activity are an indirect approach to evaluate the intracellular iron availability... why they didn’t look at the quantification of the labile iron pool (LIP) and ferritin protein levels ?• We evaluated colorimetric iron assay kits, but were unable to validate a reliable method to measure intracellular iron levels in RKO cells. Therefore, we settled on examining intracellular iron levels by indirect and established approaches such as aconitase activity assay and immunoblots of IRP2 levels.• We have now included immunoblots for ferritin (FTH1) . Ferritin levels are reduced at low doses of PIK-III, in line with cellular iron depletion. The staining of the Transferrin Alexa Fluor 488 probe in WT and RAB7A KO cells is strange. In particular, the group RAB7A KO treated with a VPS34 inhibitor. Does that mean the positive vacuoles positive (in their limiting membranes) for the probe in this group?• VPS34 inhibition is known to cause enlargement of late endosomes and lysosomes (2), which we also observe in RKO cells treated with PIK-III. The transferrin probe binds to transferrin receptor found on the luminal side of the endosome limiting membrane hence, the positively stained limiting membranes seen in PIK-III treated RAB7A KO cells.• We have examined the PIK-III-induced vacuoles in RKO cells in more detail and included the data here for full transparency . We only included the data for the response to reviewers because we believe it does not add significant value to the manuscript. Phase-contrast microscopy confirmed that VPS34 inhibition induced enlarged vacuoles in RKO CTRL cells, and RAB7A KO caused even greater and larger vacuoles . Using confocal microscopy, we identified that the vacuoles in RKO RAB7A KO cells treated with PIK-III contain transferrin receptor in the limiting membrane . Comments:Moreover, to me, it is necessary to check at ferritin because the inhibition of VPS34 by PIKIII also caused impairment in ferritinophagy, as described in the first paper that they cited. They only observed that RCO cells are not suffering by depletion of ferritinophagy (fig. S3), but they didn’t show the ferritin levels in Ctrl and RAB7a KO cells during PIKIII treatment (i.e. in figure 6F-G)• We would like to thank the reviewer for this suggestion, which has also been raised by reviewer #2. We have added immunoblots for ferritin (FTH1) and the ferritinophagy component NCOA4 . We see minimal effects on NCOA4 and decreased ferritin levels, at low doses of PIK-III when we observe clearance of TFR. It is only at higher concentrations of PIK-III that we see inhibition of ferritinophagy, and accumulation of NCOA4 and ferritin. We have clarified this point in the manuscript starting on line 295 in the results section and on line 444 on the discussion section. As VPS34 inhibition provokes the shift from oxidative phosphorylation towards glycolysis, the luciferase assay (CellTiter-Glo) is a reliable readout of cell viability?• We have assessed cell viability using both CellTiter-Glo  and colony formation  assays, which show similar results.How do the authors explain that at a higher dose of PIKIII the increment of HIF1 appears also in RAB7 KO cells ?• At higher doses of PIK-III, knockout of RAB7A does not fully rescue iron deprivation phenotype including IRP2 levels, OCR/ECR ratio and HIF1� accumulation. This is consistent with the fact that loss of RAB7A partially rescues the growth defect due to PIK-III treatment shifting the IC50 curve ~9-fold. Reviewer #2The manuscript by Kobylarz and colleagues explores the dependency of RKO cancer cells on the protein Vps34. The experiments are well conducted and presented, and the story line flows logically.The authors show, using a KO screen, that in the absence of Vps34 activity, removal of Rab7a is sufficient for viability. They then search for the underlying mechanism using a transcriptome approach to identify the underlying mechanism. There are, however, a few key experiments that are required in order to support the conclusions drawn by the authors.Comments:The transcriptional signature of Rab7A-KO+Vps34 inhibition pointed towards activation of HIF-1a and cholesterol synthesis. This is exactly the same signature that is obtained by inhibiting lysosomal acidification . Yet, the authors never test this possibility. In my view, this experiment is required, because it raises the possibility that the Vps34-Rab7A pathway acts by a completely different mechanism than that suggested by the authors. This is further substantiated by the fact that the authors can rescue many of the observed cellular and molecular phenotypes with exogenous iron citrate.• We have now addressed impaired endosomal acidification as a possible mechanism. Using the pH-sensitive pHrodo Red and the pH-insensitive Alexa Fluor 488 transferrin probes we show that the intensities of the transferrin probes reduce with 24 hours of PIK-III treatment as expected but, the ratio of the probes do not change compared to vehicle treatment. This indicates that PIK-III does not impact the acidification of the transferrin-positive endosomes. Inhibition of endolysosomal acidification with bafilomycin A1 was used as a positive control .line 229 - the statement that bioavailable iron is predominantly complexed with transferrin is not necessarily correct. In fact, upon iron deficiency, cells readily activate ferritinophagy and mitophagy, as well as up-regulation of the transferrin receptor. Therefore, it is pivotal that the autophagy branch of the pathway is also tested. I recall that the authors showed that the removal of Vps34-C1 had no effect on the viability, while C2 did, which they possibly used to discount the involvement of autophagy. However, this is not sufficient, as UVRAG, involved in C2, is also involved in autophagy. Thus, it is important that the authors test if the autophagy pathway is involved .• We would like to thank the reviewer for these suggestions, which have been also raised by reviewer #1. • We have included project DRIVE data  and individual dox-inducible shRNAs  showing that knockdown of canonical autophagy genes ATG5, ATG7 and ATG16L1 do not significantly impact growth of RKO cells We have added immunoblots for the ferritinophagy component NCOA4 . At low doses of PIK-III when we observe clearance of TFR, we see minimal effects on NCOA4. It is only at higher concentrations of PIK-III that we see inhibition of ferritinophagy and accumulation of NCOA4. We have clarified this point in the manuscript starting on line 295 in the results section and on line 444 in the discussion section.line 252 - the use of bafilomycin is per se sufficient to trigger iron deficiency and TFRC induction (see papers mentioned above in the context of HIF-1a). Therefore this experiment should be done in the presence of a lysosomal inhibitor that does not affect lysosomal pH - for example, leupeptin or some other caspase inhibitor.• Chloroquine has a different mechanism of action to bafilomycin A1 in blocking lysosomal activity and we show that chloroquine treatment also prevents PIK-III mediated reduction of LDLR and TFR . • The pooled CRISPR screen also revealed cathepsin L (CTSL) as a PIK-III suppressor. Cathepsin L is a lysosomal endopeptidase involved in protein degradation. While we have not rigorously investigated the CTSL mechanism of action in our model, we are including additional cell viability data in this document for full transparency. Loss of CTSL (RKO CTSL KO), or inhibition with the selective CTSL inhibitor (Z-Phe-Tyr(tBu)-diazomethylketone; CTSLi) partially rescued cell proliferation in cells treated with PIK-III . This data suggests that inhibition of a single lysosomal protease is sufficient to partially rescue RKO cell growth upon VPS34 inhibition. in Suppl Fig2, it is unclear if the slight differences on OCR are significant (they don't seem to be) - it should be noted whatever the case is. Also, Rab7A should be compared to the controls, i.e., in the same plate and not just in terms of absolute OCR.• We have included OCR and ECAR data for RKO CTRL and RAB7A KO cells in Fig S5. The seahorse experiments with CTRL and RAB7A KO cells (or DMSO and FAC treatment) were performed in the same plate to allow for direct comparison. We merely presented the data separately for ease of visualization. the analysis of the RNA seq data is unnecessarily conservative. Behjamin-Hochberg is known to hammer the data. That is the authors' choice, so I will not dwell on it. However, the RNA seq raw data files should be made available in a repository , so that other interested labs can reanalyze the data with statistical strategies that are more appropriate for biological data.• RNA-seq data was uploaded to the Sequence Read Archive. The accession number is PRJNA633293. Data will be released publicly upon manuscript acceptance.AttachmentResponse to Reviewers FINAL.docxSubmitted filename: Click here for additional data file. 18 Jun 2020An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cellsPONE-D-20-03526R1Dear Dr. Menon,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.I would like to congratulate you for a very nice study that contributes greatly to the connection between membrane trafficking, cholesterol and iron biology.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Fanis Missirlis, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I appreciate the big effort the authors made to review this manuscript. They answered satisfactorily most of my major comments. ThanksReviewer #2: The authors have addressed my comments.The authors have addressed my comments.The authors have addressed my comments.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Yes: Nuno Raimundo