Corrigendum: Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

This corrects the article DOI: 10.1038/nature22364.

Fat mitigation strategies to improve image quality of radial 4D flow MRI in obese subjects.

PURPOSE: This study addresses the challenges in obtaining abdominal 4D flow MRI of obese patients. We aimed to evaluate spectral saturation and inner volume excitation as methods to mitigating artifacts originating from adipose signals, with the goal of enhancing image quality and improving quantification. METHODS: Radial 4D flow MRI acquisitions with fat mitigation (inner volume excitation [IVE] and intermittent fat saturation [FS]) were compared to a standard slab selective excitation (SSE) in a test-retest study of 15 obese participants. IVE selectively excited a cylindrical region of interest, avoiding contamination from peripheral adipose tissue, while FS globally suppressed fat based on spectral selection. Acquisitions were evaluated qualitatively based on expert ratings and quantitatively based on conservation of mass, test-retest repeatability, and a divergence free quality metric. Errors were evaluated statistically using the absolute and relative errors, regression, and Bland-Altman analysis. RESULTS: IVE demonstrated superior performance quantitatively in the conservation of mass analysis in the portal vein, with higher correlation and lower bias in regression analysis. IVE also produced flow fields with the lowest divergence error and was rated best in overall image quality, delineating small vessels, and producing the least streaking artifacts. Evaluation results did not differ significantly between FS and SSE. Test-retest reproducibility was similarly high for all sequences, with data suggesting biological variations dominate the technical variability. CONCLUSION: IVE improved hemodynamic assessment of radial 4D flow MRI in the abdomen of obese participants while FS did not lead to significant improvements in image quality or flow metrics.

Automated MR Image Prescription of the Liver Using Deep Learning: Development, Evaluation, and Prospective Implementation.

BACKGROUND: There is an unmet need for fully automated image prescription of the liver to enable efficient, reproducible MRI. PURPOSE: To develop and evaluate artificial intelligence (AI)-based liver image prescription. STUDY TYPE: Prospective. POPULATION: A total of 570 female/469 male patients (age: 56 ± 17 years) with 72%/8%/20% assigned randomly for training/validation/testing; two female/four male healthy volunteers (age: 31 ± 6 years). FIELD STRENGTH/SEQUENCE: 1.5 T, 3.0 T; spin echo, gradient echo, bSSFP. ASSESSMENT: A total of 1039 three-plane localizer acquisitions (26,929 slices) from consecutive clinical liver MRI examinations were retrieved retrospectively and annotated by six radiologists. The localizer images and manual annotations were used to train an object-detection convolutional neural network (YOLOv3) to detect multiple object classes (liver, torso, and arms) across localizer image orientations and to output corresponding 2D bounding boxes. Whole-liver image prescription in standard orientations was obtained based on these bounding boxes. 2D detection performance was evaluated on test datasets by calculating intersection over union (IoU) between manual and automated labeling. 3D prescription accuracy was calculated by measuring the boundary mismatch in each dimension and percentage of manual volume covered by AI prescription. The automated prescription was implemented on a 3 T MR system and evaluated prospectively on healthy volunteers. STATISTICAL TESTS: Paired t-tests (threshold = 0.05) were conducted to evaluate significance of performance difference between trained networks. RESULTS: In 208 testing datasets, the proposed method with full network had excellent agreement with manual annotations, with median IoU > 0.91 (interquartile range < 0.09) across all seven classes. The automated 3D prescription was accurate, with shifts <2.3 cm in superior/inferior dimension for 3D axial prescription for 99.5% of test datasets, comparable to radiologists' interreader reproducibility. The full network had significantly superior performance than the tiny network for 3D axial prescription in patients. Automated prescription performed well across single-shot fast spin-echo, gradient-echo, and balanced steady-state free-precession sequences in the prospective study. DATA CONCLUSION: AI-based automated liver image prescription demonstrated promising performance across the patients, pathologies, and field strengths studied. EVIDENCE LEVEL: 4. TECHNICAL EFFICACY: Stage 1.

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies.

Addressing concomitant gradient phase errors in time-interleaved chemical shift-encoded MRI fat fraction and R2 * mapping with a pass-specific phase fitting method.

PURPOSE: Concomitant gradients induce phase errors that increase quadratically with distance from isocenter. This work proposes a complex-based fitting method that addresses concomitant gradient phase errors in chemical shift encoded (CSE) MRI estimation of proton density fat fraction (PDFF) and R2 * through joint estimation of pass-specific phase terms. This method is applicable to time-interleaved multi-echo gradient-echo acquisitions (i.e., multi-pass acquisitions) and does not require prior knowledge of gradient waveforms typically needed to address concomitant gradient phase errors. THEORY AND METHODS: A CSE-MRI spoiled gradient echo signal model, with pass-specific phase terms, is introduced for non-linear least squares estimation of PDFF and R2 * in the presence of concomitant gradient phase errors. Cramér-Rao lower bound analysis was used to determine noise performance tradeoffs of the proposed fitting method, which was then validated in both phantom and in vivo experiments. RESULTS: The proposed fitting method removed PDFF and R2 * estimation errors up to 12% and 10 s-1 , respectively, at ±12 cm off isocenter (S/I) in a water phantom. In healthy volunteers, PDFF and R2 * bias was reduced by ~10% (12 cm off-isocenter) and ~30 s-1 (16 cm off-isocenter), respectively. An evaluation in 29 clinical liver datasets demonstrated reduced PDFF bias and variability (8.4% improvement in the coefficient of variation), even with the imaging volume centered at isocenter. CONCLUSION: Concomitant gradient induced phase errors in multi-pass CSE-MRI acquisitions can result in PDFF and R2 * estimation biases away from isocenter. The proposed fitting method enables accurate PDFF and R2 * quantification in the presence of concomitant gradient phase errors without knowledge of imaging gradient waveforms.

Transmission dynamics of COVID-19 in household and community settings in the United Kingdom, January to March 2020.

BackgroundHouseholds appear to be the highest risk setting for COVID-19 transmission. Large household transmission studies in the early stages of the pandemic in Asia reported secondary attack rates ranging from 5 to 30%.AimWe aimed to investigate the transmission dynamics of COVID-19 in household and community settings in the UK.MethodsA prospective case-ascertained study design based on the World Health Organization FFX protocol was undertaken in the UK following the detection of the first case in late January 2020. Household contacts of cases were followed using enhanced surveillance forms to establish whether they developed symptoms of COVID-19, became confirmed cases and their outcomes. We estimated household secondary attack rates (SAR), serial intervals and individual and household basic reproduction numbers. The incubation period was estimated using known point source exposures that resulted in secondary cases.ResultsWe included 233 households with two or more people with 472 contacts. The overall household SAR was 37% (95% CI: 31-43%) with a mean serial interval of 4.67 days, an R0 of 1.85 and a household reproduction number of 2.33. SAR were lower in larger households and highest when the primary case was younger than 18 years. We estimated a mean incubation period of around 4.5 days.ConclusionsRates of COVID-19 household transmission were high in the UK for ages above and under 18 years, emphasising the need for preventative measures in this setting. This study highlights the importance of the FFX protocol in providing early insights on transmission dynamics.

Pneumomediastinum in the COVID-19 era: to drain or not to drain?

Pneumomediastinum (PNM) is a rare clinical finding, usually with a benign course, which is managed conservatively in the majority of cases. However, during the COVID-19 pandemic, an increased incidence of PNM has been observed. Several reports of PNM cases in COVID-19 have been reported in the literature and were managed either conservatively or surgically. In this study, we present our institutional experience of COVID-19 associated PNM, propose a management algorithm, and review the current literature. In total, 43 Case Series were identified, including a total of 747 patients, of whom 374/747 (50.1%) were intubated at the time of diagnosis, 168/747 (22.5%) underwent surgical drain insertion at admission, 562/747 (75.2%) received conservative treatment (observation or mechanical ventilation. Inpatient mortality was 51.8% (387/747), while 45.1% of the population recovered and/or was discharged (337/747). In conclusion, with increased incidence of PNM in COVID-19 patients reported in the literature, it is still difficult to assign a true causal relationship between PNM and mortality. We can, however, see that PMN plays an important role in disease prognosis.  Due to increased complexity, high mortality, and associated complications, conservative management may not be sufficient, and a surgical approach is needed.

Epidemiological and clinical characteristics of early COVID-19 cases, United Kingdom of Great Britain and Northern Ireland.

OBJECTIVE: To describe the clinical presentation, course of disease and health-care seeking behaviour of the first few hundred cases of coronavirus disease 2019 (COVID-19) in the United Kingdom of Great Britain and Northern Ireland. METHODS: We implemented the World Health Organization's First Few X cases and contacts investigation protocol for COVID-19. Trained public health professionals collected information on 381 virologically confirmed COVID-19 cases from 31 January 2020 to 9 April 2020. We actively followed up cases to identify exposure to infection, symptoms and outcomes. We also collected limited data on 752 symptomatic people testing negative for COVID-19, as a control group for analyses of the sensitivity, specificity and predictive value of symptoms. FINDINGS: Approximately half of the COVID-19 cases were imported (196 cases; 51.4%), of whom the majority had recent travel to Italy (140 cases; 71.4%). Of the 94 (24.7%) secondary cases, almost all reported close contact with a confirmed case (93 cases; 98.9%), many through household contact (37 cases; 39.8%). By age, a lower proportion of children had COVID-19. Most cases presented with cough, fever and fatigue. The sensitivity and specificity of symptoms varied by age, with nonlinear relationships with age. Although the proportion of COVID-19 cases with fever increased with age, for those with other respiratory infections the occurrence of fever decreased with age. The occurrence of shortness of breath also increased with age in a greater proportion of COVID-19 cases. CONCLUSION: The study has provided useful evidence for generating case definitions and has informed modelling studies of the likely burden of COVID-19.

Aorto-innominate artery bypass for migrated stent.

We report a case of a 64-year-old female who first presented with a transient ischemic attack in 2007 due to an innominate artery stenosis, which indicated an endovascular stent placement. In 2008, she presented with recurrence of symptoms and was diagnosed with in-stent restenosis alongside an unusual occurrence of retrograde migration into the ascending aortic arch. We performed an aorto-innominate bypass through a median sternotomy. The patient was discharged without any complications thereafter, and the graft has shown excellent patency. As of 2019, the patient remains well.

The challenging management of a giant intrathoracic desmoid tumour: a case report.

Desmoid tumours are rare, locally aggressive neoplasms exhibiting high tendency for recurrence, even after complete resection. Only 1 in 5 of them originates from the chest wall, usually measuring less than 10 cm at diagnosis. Herein, we report the case of a woman presenting with symptoms of gradual lung compression by a giant desmoid tumour occupying the entire hemithorax. She underwent complete surgical resection of the tumour and chest wall reconstruction. She had disease recurrence 15 months later and currently remains under regular follow-up. The management of intrathoracic desmoid tumours is challenging because they are usually not diagnosed until they become large enough to cause compression symptoms. While medical management is the primary modality of treatment, surgery could be considered in selected cases where significant symptoms arise, and the functional status is impaired secondary to the tumour. Adjuvant radiotherapy to minimise the risk of local recurrence should also be considered.

Metastatic mandibular ameloblastoma of the lung ten years after primary resection: A rare case report.

Ameloblastoma is a rare odontogenic neoplasm of the jaw. It usually behaves as a benign, slow growing tumour of the oral cavity with a high recurrence rate, especially when it is inadequately resected. A small proportion of ameloblastomas metastasize to distant organs, with lungs representing the most common site of metastatic spread. In this report, we present the case of a middle-aged man with two pulmonary nodules and a history of mandibular ameloblastoma excised 10 years prior to this radiological finding. Following resection and histopathological analysis of the lung lesion, a diagnosis of metastatic ameloblastoma was confirmed. No local recurrence of the primary tumour was identified. At 1-year follow-up, the patient had no evidence of local or metastatic disease.

Tracking the Evolution of Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. METHODS: In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. RESULTS: We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10-4), which remained significant in multivariate analysis. CONCLUSIONS: Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).

Forecasting the 2017/2018 seasonal influenza epidemic in England using multiple dynamic transmission models: a case study.

BACKGROUND: Since the 2009 A/H1N1 pandemic, Public Health England have developed a suite of real-time statistical models utilising enhanced pandemic surveillance data to nowcast and forecast a future pandemic. Their ability to track seasonal influenza and predict heightened winter healthcare burden in the light of high activity in Australia in 2017 was untested. METHODS: Four transmission models were used in forecasting the 2017/2018 seasonal influenza epidemic in England: a stratified primary care model using daily, region-specific, counts and virological swab positivity of influenza-like illness consultations in general practice (GP); a strain-specific (SS) model using weekly, national GP ILI and virological data; an intensive care model (ICU) using reports of ICU influenza admissions; and a synthesis model that included all data sources. For the first 12 weeks of 2018, each model was applied to the latest data to provide estimates of epidemic parameters and short-term influenza forecasts. The added value of pre-season population susceptibility data was explored. RESULTS: The combined results provided valuable nowcasts of the state of the epidemic. Short-term predictions of burden on primary and secondary health services were initially highly variable before reaching consensus beyond the observed peaks in activity between weeks 3-4 of 2018. Estimates for R0 were consistent over time for three of the four models until week 12 of 2018, and there was consistency in the estimation of R0 across the SPC and SS models, and in the ICU attack rates estimated by the ICU and the synthesis model. Estimation and predictions varied according to the assumed levels of pre-season immunity. CONCLUSIONS: This exercise successfully applied a range of pandemic models to seasonal influenza. Forecasting early in the season remains challenging but represents a crucially important activity to inform planning. Improved knowledge of pre-existing levels of immunity would be valuable.

Cystic fibrohistiocytic tumour of the lung presenting with recurrent pneumothoraces: a case report.

Cystic fibrohistiocytic tumour of the lung is a very rare pathological entity that occurs either as a primary pulmonary neoplasm or as a metastasis from skin lesions called cellular fibrous histiocytomas. Herein, we present the case of a 19-year old man with a history of recurrent pneumothoraces who was managed surgically and was eventually diagnosed with cystic fibrohistiocytic tumour of the lung. Clinicians should include this disease in the differential diagnosis of pulmonary cystic lesions and be aware of its association with cellular fibrous histiocytoma. Reporting of more cases is warranted to further elucidate the natural course of the disease and optimise its management.

A man in his sixties with acute back pain and increasing weakness in his lower limbs. / En mann i 60-årene med akutte ryggsmerter og økende kraftsvikt i underekstremitetene.

BACKGROUND: European and Norwegian consensus-based guidelines for the management of acute low back pain endorse red flag screening. Red flag symptomatology may ignore important information in the case history and clinical findings. CASE PRESENTATION: An active man in his sixties presented with acute low back pain with radiation to the left thigh. A paramedic visited the patient at home and found no serious disease. Over a period of 4 hours, progressive loss of sensation and weakness in both legs developed. He arrived at the Accident and Emergency Department with paralysis, reduced sensation distal to the hips, absent reflexes, urinary retention and reduced sphincter tone. INR was 2.6. MR scan showed an intradural haematoma compressing Th9-L1, and dislocation of medulla and conus. This was followed by urgent Th10-12 laminectomy and evacuation of subdural haematoma, and thereafter rehabilitation. Three years later, he has sequelae for spinal cord injury at level Th12 with impaired mobility and function in the legs. INTERPRETATION: This case highlights that patients with ongoing anticoagulation and acute back pain have increased risk of spinal haematoma requiring emergency treatment. Anticoagulation therapy should therefore be included in red flag screening of acute back pain.

End of season influenza vaccine effectiveness in adults and children in the United Kingdom in 2017/18.

BackgroundIn the United Kingdom (UK), in recent influenza seasons, children are offered a quadrivalent live attenuated influenza vaccine (LAIV4), and eligible adults mainly trivalent inactivated vaccine (TIV).AimTo estimate the UK end-of-season 2017/18 adjusted vaccine effectiveness (aVE) and the seroprevalence in England of antibodies against influenza viruses cultured in eggs or tissue.MethodsThis observational study employed the test-negative case-control approach to estimate aVE in primary care. The population-based seroprevalence survey used residual age-stratified samples.ResultsInfluenza viruses A(H3N2) (particularly subgroup 3C.2a2) and B (mainly B/Yamagata/16/88-lineage, similar to the quadrivalent vaccine B-virus component but mismatched to TIV) dominated. All-age aVE was 15% (95% confidence interval (CI): -6.3 to 32) against all influenza; -16.4% (95% CI: -59.3 to 14.9) against A(H3N2); 24.7% (95% CI: 1.1 to 42.7) against B and 66.3% (95% CI: 33.4 to 82.9) against A(H1N1)pdm09. For 2-17 year olds, LAIV4 aVE was 26.9% (95% CI: -32.6 to 59.7) against all influenza; -75.5% (95% CI: -289.6 to 21) against A(H3N2); 60.8% (95% CI: 8.2 to 83.3) against B and 90.3% (95% CI: 16.4 to 98.9) against A(H1N1)pdm09. For ≥ 18 year olds, TIV aVE against influenza B was 1.9% (95% CI: -63.6 to 41.2). The 2017 seroprevalence of antibody recognising tissue-grown A(H3N2) virus was significantly lower than that recognising egg-grown virus in all groups except 15-24 year olds.ConclusionsOverall aVE was low driven by no effectiveness against A(H3N2) possibly related to vaccine virus egg-adaption and a new A(H3N2) subgroup emergence. The TIV was not effective against influenza B. LAIV4 against influenza B and A(H1N1)pdm09 was effective.

Accuracy of pre-interventional computed tomography angiography post-processing software and extravascularly calibrated devices to determine vessel diameters: comparison with an intravascularly located calibrated catheter.

Background Accurate vessel sizing might affect treatment outcome of endovascular therapy. Purpose To compare accuracy of peripheral vessel diameter measurements using pre-interventional computed tomography angiography post processing software (CTA-PPS) and extravascularly located calibrated devices used during digital subtraction angiography (DSA) with an intravascular scaled catheter (SC). Material and Methods In 33 patients (28 men, mean age = 72 ± 11 years) a SC was used during DSA of the femoro-popliteal territory. Simultaneously, one scaled radiopaque tape (SRT) was affixed to the lateral thigh, one scaled radiopaque ruler (SRR) was positioned on the angiography table. For each patient, diameters of five anatomic landmarks were measured on DSA images after calibration using different scaled devices and CTA-PPS. Diameters were compared to SC (reference) and between groups of non-obese (NOB) and obese (OB) patients. Results In total, 660 measurements were performed. Compared to the reference, SRT overestimated the diameter by 1.2% (range = -10-12, standard deviation [SD] = 4.1%, intraclass correlation coefficient [ICC] = 0.992, 95% confidence interval [CI] = 0.989-0.992, P = 0.01), the SRR and CTA-PPS underestimated it by 21.3% (range = 1-47, SD = 9.4%, ICC = 0.864, 95% CI = 0.11-0.963, P = 0.08) and 3.2% (range = 17-38, SD = 9.7%, ICC = 0.976, 95% CI = 0.964-0.983, P = 0.01), respectively. Underestimation using the SRR was greatest in the proximal superficial-femoral artery (31%) and lowest at the P2 level of the popliteal artery (15%). In the NOB group, diameter overestimation of the SRT was 0.8% (range = 4-7, SD = 4.2%, B = 0.071, 95% CI = 0.293-0.435, P = 0.08) compared to the OB group of 1.6% (range = -7-4, SD = 2.9%, B = 0.010, 95% CI = 0.474-0.454, P = 0.96). Diameter underestimation of the SRR was 17.3% (range = 13-21, SD = 3.1%, B = 0.946, 95% CI = 0.486-1.405, P = 0.002) in the NOB group, 23.3% (range = 11-36, SD = 6.6%, B = 0.870, 95% CI = 0.268-1.472, P = 0.007) in the OB group. Conclusion For calibrated measurements SRT and CTA-PPS prove accurate compared to the reference, while SRR does not. Obesity has a significant impact on underestimation of diameter if SRR is used.

Conservative versus Surgical Management of Iatrogenic Tracheal Rupture.

Iatrogenic tracheal rupture (ITR) represents a life-threatening condition requiring prompt diagnosis, management, and treatment. The management of ITR is challenging, and treatment options depend on tear location, size, injury extent, and the patient's respiratory status. Although this complication has been extensively reported in published literature, the best evidence practice, for the management, requires clarification. In this review, the authors focused on the establishment of a differential diagnosis and the potential mechanism of the injury, the decision-making process, and the therapeutic approaches. It is suggested that for small lacerations or stable patients, conservative management could be considered sufficient, whereas invasive surgical therapy would be more appropriate in cases of large defects with significant air leak and patient instability.

Lymphovascular infiltration in the tumor bed is a useful marker of biological behavior in breast cancer.

PURPOSE: Tumor cells can metastasize by entering existing vessels or new vessels actively recruited into the primary tumor. Invasion of the lymphatics and blood vessels in the periphery of the tumor seems to be a prerequisite step in the metastatic process. The aim of this study was to correlate peripheral lymphatic vessel infiltration (PLI) and peripheral blood vessel infiltration (PVI) in a cohort of patients with invasive ductal carcinoma of the breast with various other prognostic parameters and outcome. METHODS: The study population consisted of 236 female patients with invasive ductal breast carcinomas, who had been operated between 2011 and 2013. The registered data included age at diagnosis, histological subtype, tumor size, TNM stage, histological grade, estrogen (ER) and progesterone receptors (PR), HER-2, p53, and PLI and PVI. RESULTS: Pathological examination revealed that 22.5% of the patients had PVI and 37.3% had PLI at the tumor front. PVI correlated with younger age (p<0.05), higher histologic grade (p<0.05), advanced TNM stage (p<0.05), higher T stage (p<0.05), higher N stage (p<0.05) and positive Ki67 expression (p<0.05). Similarly, PLI correlated with higher histologic grade (p<0.05), advanced TNM stage (p<0.05), higher T stage (p<0.05) and higher N stage (p<0.05). Statistical analysis did not reveal significant correlation between the presence of tumor blood and lymphatic vessels with infiltration in overall (OS) and disease-free survival (DFS). CONCLUSIONS: PLI and PVI are important markers of worse clinical outcome as shown by their association with other established factors, but no association with recurrence and survival could be proven.