Daily Archives: August 23, 2022

Among the four elements required to establish liability for the tort of negligence is causation. In addition to proving that the defendant was careless or otherwise breached the legal standard of care, the plaintiff must establish that the wrongful conduct caused the harm the plaintiff suffers. This 1941 New Hampshire Supreme Court decision explains:

Necessary elements of a cause of action based on negligence are the causal negligence of the defendant plus the resultant harm to the plaintiff. Putting it another way, there must be negligence and harm and they must have a causal connection.

At the height of asbestos litigation, mesothelioma, a cancer of the lining of the pleural (lung) cavity or the abdominal cavity (peritoneal mesothelioma) was believed to be pathognomonic of asbestos exposure, meaning that the disease was a signal cancer of asbestos exposure, and only asbestos exposure. A diagnosis of the disease was tantamount to legal recovery (and virtually certain and swift death). No longer.

Today, it is recognized that mesothelioma has other causes, including erionite, another naturally occurring mineral used in pet litter, soil conditioners, animal feed, wastewater treatment, and gas absorbents. Radiation is also a recognized cause of mesothelioma, either from employment-related exposures, or older therapeutic uses which are now known to cause disease. And then there are causes we dont even yet know about as well as those arising spontaneously. These are known as idiopathic causes.

Asbestos exposure significantly declined beginning in the 1970s when the first OSHA asbestos laws were enacted. However, the incidence of mesothelioma, even with its notoriously long latency or delay period, is increasing in women, and has remaining constant overall. This lends credence to the fact that mesotheliomas arises from causes other than asbestos or even arises spontaneously, meaning the body needs no help from asbestos or anything else in generating them.

Of course, spontaneously does not mean magically or out of the blue. Rather, of the estimates that perhaps twenty to fifty percent of mesotheliomas dont arise from asbestos, it appears some are attributable to genetic misfirings, or mutations, that arise over time, some occurring as we age, and our defenses or repair mechanisms are no longer functioning optimally. (Think how slow your older computer worked compared to the new one you bought when old-trusty melted down.)

These genetic mutations are not necessarily inherited (although some are), but rather occur in clusters - or more rarely, singly due to natural causes or DNA breakage. The mutations can cause mesothelioma, either alone or by increasing susceptibility to minute exposures. The difference in the causal mechanism, however, has a huge impact on legal liability. And so it is that attention to genetics is now finding its way into the courtroom.

Courts are becoming more attentive to the need to establish that a potential carcinogen is responsible for causing the disease in a particular person, rather than based on its statistical or historical significance for a general population. Recently, in toxic tort cases, some courts are requiring a two-stage level of proof:

Specific causation requires showing that the plaintiff was exposed to a sufficient dose which is capable of causing the disease in that person. The application of this requirement is derailing some talc-mesothelioma cases.

But attention to the plaintiffs burden of proving specific causation is not the only monkey-wrench appearing in toxic tort cases these days. And not all courts are requiring specific quantification of plaintiffs exposure to prove causal-negligence, i.e, that the defendants asbestos caused the particular plaintiffs disease.

This situation invites another option for defense lawyers to protect their clients: genetics. And so we find defense lawyers turning to genetics to establish the defense that it wasnt exposure to asbestos per se, that caused the plaintiffs mesothelioma. Rather it was something else notably, something in their genes.

The genetic defense has found itself into other types of cases as well. And as we learn more about the genetic components of disease, we can expect to find its use burgeoning. Take the case of Bowen v. EI Dupont, in which the plaintiff alleged her retarded fetal growth and birth defects were caused by exposure to the fungicide Benlate during her gestation, when her mother was spraying houseplants.

The defendant obtained a court order to genetically test the plaintiff for a specific genetic variation (CHD7) that causes a specific condition (the CHARGE syndrome, a rare condition that causes birth defects), that resembled the plaintiffs condition. The defense argued that the mutated CHD7 was the sole and proximate cause of the plaintiffs condition a CHARGE syndrome-driven birth defect, a theory with substantial support in the record and substantiated in peer-reviewed literature. After the testing and finding the mutation present, the court dismissed the case.

In a similar case, Naomi Guzman v. ExxonMobil Corp, the plaintiff claimed that she was exposed to radioactive material as a bystander to her fathers work as an oil pipe cleaner. The court granted the defendants request for genetic testing of plaintiffs preserved thyroid tissue.

Genetic tests revealed both that the plaintiff did not have the genetic signature for radiation-induced cancer. It also revealed that plaintiff had several hereditary gene mutations predisposing her to thyroid cancer, leading the defenses expert-toxicologist to conclude her thyroid cancer was caused by her genetic predisposition and not exposure to her fathers drilling pipe. The jury agreed and sided with the defense.

Genetic defenses, however, must be used with care. In some cases, exposure to a chemical overwhelms multiple natural genetic repair mechanisms, leading to the cancer or disease. In those cases, the genetic defense may not work.

Thus, the failure of a genetic-defense mechanism to activate (or activation of a susceptibility gene) may not absolve the defendant, since it was the impact of exposure (to their asbestos or whatever chemical) that caused these genes to malfunction, and which in turn began the chain-reaction leading to the disease.

Instead, focusing on point mutations, where cancers occur as the direct result of a consequential change in a particular genetic set of alleles (subparts of a gene responsible for expression of a particular trait), independent of exposure, present the genetic alterations generating the most success in legal cases.

So far, two genes have been implicated as causally-related to mesothelioma in the absence of asbestos exposure. One, is a mutation on the AKL gene. While the discovery may not help plaintiffs in a legal setting, such discoveries do bode well for enhanced therapeutics and treatment.

Other studies have identified mutations on the BAP1 gene, discoveries that again bode well for possible treatment, although not necessarily for success in the legal setting. The BAPI gene is also related to several other cancers, including melanoma, so the causal connection is not one-on-one. But the BAP1 gene also has an additional effect- not only has it been related to direct causation of mesothelioma, it is also known to enhance susceptibility to prior exposures to asbestos. And that poses a legal problem.

The published findings do not establish a synergistic gene/environment interaction as a causal factor for the development of mesothelioma [and Bap1].,,, At present, it is estimated that between 1 and 8 percent of all spontaneous mesothelioma cases involve BAP1 germline mutations.

Bernier et al.

In law, the defendant is said to take the plaintiff as one finds him/her. That means if a plaintiff, by virtue of his or her individual make-up, is unusually susceptible to a condition, the defendant is still liable for all incident harms of their negligence. These include even those that might not have happened if the same wrongdoing was inflicted on a hardier plaintiff. (The term got its name from a case about running over a plaintiff with a skull as thin as an eggshell).

That maxim makes finding a BAP1 gene problematic. In some cases, the gene will render the plaintiff unusually susceptible to a small amount of exposure; in others it can cause mesothelioma, outright. Where the gene acts as a susceptibility gene, the defendant is still liable. Where it causes the disease outright, the plaintiffs case fails.

At the end of the day, the answer becomes a matter of fact for the jury to decide after listening to a battle of experts. But recent research shows that up to one-third of all cancers are produced by the bodys own mutation errors, and not by environmental causes. The complete genetic-cause defense would likely work best in cases of teenagers or women who were unlikely to have been exposed to significant levels of exposure.

To maintain a genetic defense, the plaintiff would have to petition the court to compel genetic tests of the plaintiff and sometimes the family to show a hereditary defect. Courts usually direct the plaintiff to submit to genetic testing (via blood test or tissue swipe), if the requested tests are particularized enough, although they will often reject requests to compel family-testing.

Is this forced test legal? Yes. First because such tests are not usually invasive; and second, by bringing the lawsuit, the plaintiff has put his condition into controversy and hence waives some rights.

To date, a few dozen personal injury cases have involved court-ordered genetic testing. Most courts have allowed it, especially as the tests are not painful, protracted, or intrusive. (Although, when the genetic tests are to be used for prospective or predictive purposes, such as medical monitoring or gauging the likelihood of future disease severity or longevity, the courts may not agree. In these cases courts often conclude that DNA tests are more speculative in prediction than identifying causal genes in those already suffering a disease).

Most important, however, a defendant who wants to compel a test must seekprecise information or evidence. In other words, like in any litigation discovery procedure, neither party is given largesse to go on a fishing expedition, for example by seeking reems of data, hoping to uncover an unanticipated smoking gun.

This objection wouldnt be typical in the asbestos cases, since specific and responsible genetic culprits have been identified. But we can see the abuse in other cases, notably birth-related negligence cases which often result in a surfeit of issues, such as brain damage, developmental issues, and physical disabilities. To be sure, such conditions can be caused by environmental exposures, poor pre-natal care, or even the birth process itself, especially if negligently performed.

In one birth-related negligence case, Fisher for XSF v. Winding Waters Clinic, the defendant sought a court order compelling the plaintiff to submit to genetic testing to confirm whether their impairments are more likely than not the result of a genetic condition unrelated to prenatal care

The problem was that the defendants had no specific condition in mind. They sought a WES (Whole Exome Sequencing) array meaning they wanted to look through the plaintiffs entire genomic vocabulary in the hopes of finding one or more errors to explain the condition. The court rejected the request.

Recognizing that overbroad requests promote the possibilities for abuse, including privacy violations or the potential to uncover genetic predispositions to numerous unrelated conditions, the court noted that the defendants request was particularly troubling.

Nevertheless, we can expect an onslaught of compelled genetic testing arising out of litigation to continue. As to where the results take us, thats an interesting and open- question. Perhaps the most curious case is the pending matter of Lohmann v. Aaon Inc. There, the defendants experts sought evidence of the BAP-1 disorder. Not only did they want to use it for the litigation, they wanted it for their own research purposes, as well.

Seems the value of these tests is greater than we might imagine. No word yet, on the legal resolution. But Id bet the judge denies the request.

Dr. Barbara Pfeffer Billauer, JD MA (Occ. Health) Ph.D. is Professor of Law and Bioethics in the International Program in Bioethics of the University of Porto and Research Professor of Scientific Statecraft at the Institute ofWorld Politics in Washington DC. Find Barbara on Twitter@BBillauer

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A legal defense grounded in genetics: Is DNA testing the magic wand to winning (or losing) a negligence case? - Genetic Literacy Project

Water cannons being used to disperse Central Teacher Eligibility Test (CTET) and Bihar Teacher Eligibility Test (BTET) qualified candidates during a protest demanding permanent jobs, in Patna on Monday | Photo: ANI

A video has surfaced from Patna, the capital of Bihar, where an unemployed youth of Bihar is lying on the road helpless. The one who is beating this young man with a stick is KK Singh, Additional District Magistrate, Patna. In the intoxication of the power of his post, the magistrate is raining his lathi. The youth was holding the tricolour in his hand.

This young man had crossed the barricading with the tricolor in his hand and sat on the road. He started accusing the government of cheating the unemployed. Seeing the young man waving the tricolor, the cameras of the media turned towards him, then the eyes of the ADM also fell on that young man. Seeing the young man talking to the media, his anger exploded.

All this happened when a protest was being held at Dak Bungalow crossroads in Patna regarding the recruitment of teachers for the seventh phase, where about 5,000 unemployed youth who have passed STET and BTET were demonstrating. For the last three years, these unemployed youth have already demonstrated many times for their appointment.

After 8 years in Bihar, in January 2020, the State Teachers Eligibility Test examinations were held, which were conducted in offline mode. But it was canceled after reports of fraud surfaced at some centres. After this, there were re-exams in September 2020, then these exams were online. The youths who passed the examination allege that they have not yet received the appointment letters. For the last two years, only assurance is being given from the government.

The disease of unemployment appears to have become incurable in Bihar. Here are some statistics.

- The unemployment rate in Bihar was 18.8 percent in July, which is more than double the country's unemployment rate of 7.7 percent.

- Data from the National Career Service Portal shows that the number of unemployed youth in Bihar has tripled in the last one year.

- There are 14,15,914 registered unemployed in Bihar, these are the youths who have registered themselves for jobs.

- In Bihar, the number of unemployed youth is more than two and a half lakhs.

However, there is no dearth of vacant posts in government departments of Bihar.

- According to the National Career Service Portal, 2,75,255 posts are vacant in Bihar.

- The maximum number of vacant posts in Bihar is in the Education Department where 1,80,000 posts are lying vacant for teachers.

- A total of 47,099 posts are vacant in the police department, this figure is 34 percent of the total sanctioned posts.

- More than fifty percent of government doctors in Bihar, about 6565 posts are vacant.

- Apart from this, more than 50,000 posts are vacant in the health department.

- Bihar government figures that more than 1 lakh posts are lying vacant in secretariat and district offices.

That is, there is no dearth of employment opportunities in Bihar. The shortfall is in the intention of the government.

READ | JNU: Students clash with security staff over fellowship funds, several injured

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DNA Special: Is the problem of unemployment incurable in Bihar? - DNA India

The death of 6-year-old JonBenet Ramsey on Christmas day 1996 is perhaps one of the countrys greatest and most high-profile unsolved mysteries.

The Ramsey family, once wrapped up in suspicions they were responsible for JonBenets death, has continued to fight for justice. JonBenets father, John Ramsey, has fought to try to find the answers behind his daughters death.

Most recently, the 78-year-old has called on the state of Colorado to independently test DNA from the case using new technology not available at the time of JonBenets death.

Unidentified DNA was found in JonBents underwear and touch DNA was discovered on the waistband of her long johns.

RELATED: JonBent Ramsey's Brother Tells Police 'It's Time To Talk' As DNA Evidence Revives Hope Of Solving Her Murder

KDVR reported that as of December 2021, the Boulder Police Department said they processed more than 1,500 pieces of evidence related to the murder of JonBenet.

At that time, the Boulder Police Department said it was actively reviewing genetic DNA testing processes to see if those can be applied to this case moving forward, the outlet noted.

They also shared BPDs claim they have analyzed nearly 1,000 DNA samples in the case to date.

Among the DNA evidence in the case is evidence collected from the Ramsey home and from JonBenets body and belongings, as well as DNA samples collected from the family, family friends who came to the home on the day of JonBenets disappearance, friends they saw at the Christmas party the night before, and household employees.

ABC News reports that all members of the Ramsey family, as well as 200 other potential suspects, were excluded as the possible murderer in this case as a result of then-newly discovered touch DNA found on JonBenet's long johns.

The touch DNA was traced to an unknown male, the male also responsible for DNA found in two spots of blood found in the 6-year-olds underwear.

The testing John has in mind is investigative genetic genealogy (IGG) research. According to the Oxford Sciences Journal of Law and the Biosciences, the technique involves uploading a crime scene DNA profile to one or more genetic genealogy databases with the intention of identifying a criminal offenders genetic relatives and, eventually, locating the offender within the family tree.

RELATED: 6 Secret Details About The JonBent Ramsey Case 25 Years Later

The technique was famously employed in capturing the Golden State Killer in 2018 after four decades. John believes the case can deliver a breakthrough in JonBenets case 25 years in the making.

The Boulder Police Department released a statement in response to Johns public pleas.

The Boulder Police Department is aware of the recent request involving the homicide investigation of JonBenet Ramsey and wants the community to know that it has never wavered in its pursuit to bring justice to everyone affected by the murder of this little girl, the statement begins.BPD note that detectives have followed up on every lead that has come into the department, to include more than 21,016 tips, letters and emails and traveling to 19 states to interview or speak with more than 1,000 individuals in connection to this crime.

As recently as March 2022, the Boulder Police Department hosted another meeting with federal, state, and local agencies working on this case and in consultation with DNA experts from around the country, they continued, adding. That collaboration will continue.

John is prepared to take legal action against the state of Colorado if the cases designation is not changed to cold so that further investigations can take place, FOX News reports.

The bereaved father has also started a Change.org petition with over 23,000 signatures calling on Colorado Governor Jared Pollis to allow DNA evidence to be released by the Boulder Police Department to an independent agency for testing.

JonBenets parents, John and late wife Patricia Ramsey, put JonBenet and older brother Burke to bed on Christmas Eve 1996 after attending a party at a neighbors home. The following morning, Patricia called 911 frantically after finding a ransom note demanding $118,000 the amount of Johns annual bonus in exchange for their daughter.

Boulder Police responded to the call and began working their investigation at the Ramseys home.

In a sweep of the home hours later, John found his daughters body in a back room of the basement of their home. Her mouth was duct taped shut, and she was wrapped in a blanket.

An autopsy would reveal that JonBenet died of strangulation and an 8.5-inch skull fracture.

RELATED: Inside The Conspiracy Theory That Katy Perry And JonBenet Ramsey Are Actually The Same Person

Angela Andaloro is a writer who passionately explores all things entertainment, parenting, and true crime. Follow her on Twitter here.

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What New DNA Testing Could Reveal About The Murder Of JonBent Ramsey 26 Years Later - YourTango

Patient cohort, clinical characteristics, and recurrence patterns

We investigated 821 patients with advanced-stage EBV-associated NPC enrolled between 2009 and 2015, who consistently received cisplatin-based NAC followed by CRT. The diagram of the study population is shown in Fig. 1. The baseline clinical characteristics are presented in Table 1. Blood samples were collected at baseline and after the completion of NAC (post-NAC) and CRT (post-CRT). On-treatment imaging evaluation was conducted post-NAC using magnetic resonance imaging (MRI). The collection schema of cfEBV DNA and MRI is presented in Additional file 1: Fig. S1. The median follow-up was 64.9 months (interquartile range [IQR]: 58.172.5 months). We recorded 109 locoregional recurrences, 143 distant metastases, and 28 synchronous locoregional and metastatic recurrences. The 5-year rates of disease-free survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS), and locoregional relapse-free survival (LRFS) were 72.5%, 82.9%, 83.1%, and 87.1%, respectively.

Flowchart showing the study design and patient selection process. The medical records of 10,126 patients with non-metastatic NPC were screened, and 821 patients with LA-NPC who received NAC plus concurrent CRT and had detectable pretreatment cfEBV DNA with on-treatment circulating cfEBV DNA surveillance were selected stepwise. Abbreviations: AC, adjuvant chemotherapy; CCRT, concurrent chemotherapy; cfEBV DNA, cell-free Epstein-Barr virus DNA; IC, induction chemotherapy; LA-NPC, locally advanced nasopharyngeal carcinoma; MRI, magnetic resonance imaging

All patients (n = 821) had detectable cfEBV DNA at baseline. The distribution of pretreatment cfEBV DNA titers (median, 12.50 103 copies/mL; IQR, 2.9652.50 103 copies/mL) are shown in Additional file 1: Fig. S2A, with 661 (80.5%) patients having pretreatment cfEBV DNA higher than 2000 copies/mL. Correlation analyses revealed that pretreatment cfEBV DNA was positively associated with node (N) stage (P < 0.05, Wilcoxon test; Fig. 2A), but not tumor (T) stage, age, sex, and smoking status (P > 0.05). Additionally, in line with our previous observations [17], higher baseline cfEBV DNA load (cut-off value, 2000 copies/mL) was preferentially associated with worse survival outcomes, especially with the occurrence of distant metastasis (hazard ratio [HR] = 2.88, 95% confidence interval [CI] = 1.595.20, P < 0.01; Fig. 2B). It remained significant after correcting for clinically important covariates using the inverse probability weighting (IPW) algorithm (HRDMFS = 2.51; 95% CI = 1.464.32; P < 0.01; Additional file 2: Table S1), suggesting that in addition to their well-acknowledged reflection on tumor burden, higher pretreatment cfEBV DNA levels may also be related to tumoral biological features (i.e., sensitivity to treatment and/or tumor microenvironmental heterogeneity as were referred in previously published researches [18,19,20]). Comparisons of the baseline covariates in the unadjusted and IPW-adjusted cohorts are shown in Table 2, demonstrating that the IPW succeeded in generating balanced distributions of covariates across subgroups.

Biological responses to NAC and their correlations with radiological responses. A Comparison of pretreatment cfEBV DNA levels across N categories. B Kaplan-Meier survival plot of DMFS in patients with pretreatment cfEBV DNA 2000 copies/mL versus <2000 copies/mL. C Scatter plot showing circulating cfEBV DNA levels before treatment initiation, at NAC completion (post-NAC), and at CRT completion (post-CRT). D Changes in cfEBV DNA from baseline in patients with increased cfEBV DNA levels post-NAC (n = 33). E Kaplan-Meier survival plot of DMFS in patients with cBR post-NAC versus decreased/increased cfEBV DNA in patients with non-cBR. F RECIST groupings (columns) and cfEBV DNA biological responses (rows) of 821 patients with matched treatment-nave and post-NAC surveillance data. G Kaplan-Meier survival plot of DFS in patients with cBR versus non-cBR post-NAC. H Kaplan-Meier survival plot of DFS in patients achieving cBR at the end of CRT stratified by biological responses to NAC. Abbreviations: cBR, complete biological response; cfEBV DNA, cell-free Epstein-Barr virus DNA; CI, confidence interval; CR, complete response; CRT, chemoradiotherapy; DFS, disease-free survival; DMFS, distant metastasis-free survival; HR, hazard ratio; IC, induction chemotherapy; N, node; NAC, neoadjuvant chemotherapy; non-cBR, non-complete biological response; PD, progression disease; PR, partial responses; PreEBV, pretreatment cfEBV DNA; SD, stable disease

Upon the initiation of NAC, 586 patients (71.4%) achieved complete biological response (cBR; defined as undetectable cfEBV DNA) during the NAC phase (Fig. 2C); the distributions of post-NAC cfEBV DNA titers (median, 0 copies/mL; IQR, 00.20 103 copies/mL) are shown in Additional file 1: Fig. S2B. Among 235 patients with non-complete biological response post-NAC (non-cBR; defined as detectable cfEBV DNA; median, 1.55 103 copies/mL; IQR, 0.417.84 103 copies/mL), 33 (14.0%) had increased cfEBV DNA levels from baseline, which demonstrated worse prognosis (Fig. 2D, E).

Regarding the RECIST-based radiological assessment, 56 patients (6.8%) achieved complete response (CR), 648 (78.9%) patients achieved partial response (PR) during the NAC phase, 116 patients (14.1%) had SD post-NAC, and one patient had progressive disease (PD) after receiving three cycles of docetaxel plus cisplatin (TP) NAC. Survival analysis demonstrated that patients with radiological PR had significantly worse survival compared to those with CR (HRDFS = 2.40, 95% CI = 1.135.11, P = 0.019, Additional file 1: Fig. S2C), and patients with SD/PD demonstrated the worst survival outcome. Based on this finding, we classified radiological responses into 3 subgroups: CR, PR, and SD/PD. Notably, patients with tumor stage I-II (T1-2) and tumor stage III-IV (T3-4) did not show significant differences in CR and PR rates (T1-2: 8 [7.7%] CR vs. 96 [92.3%] PR; T3-4: 48 [8.0%] CR vs. 552 [92.0%] PR; P = 0.91). The possible explanation for the comparable distribution of CR/PR in T1-2 versus T3-4 was that only locally advanced NPC (LA-NPC) (stage III-IV) patients were included in this study, thus patients with T1-2 would have more advanced N stages.

Next, we explored the relationships between biological and radiological responses and identified that they were positively correlated, with ~95% CR patients and ~75% PR patients having their cfEBV DNA dropped to zero after NAC, respectively (P < 0.01; Fig. 2F, and Additional file 1: Fig. S2D). Intriguingly, we observed an inconsistency between the biological and radiological responses in a subset of patients: of 56 and 648 patients with radiological CR and PR (radiological response) after NAC, 3 (5.3%) and 160 (24.7%) patients had detectable post-NAC cfEBV DNA (non-cBR), respectively (Fig. 2F). Moreover, across 117 patients with SD/PD (radiological responses), about 45 patients (38.5%) achieved cBR after 24 cycles of chemotherapy (Fig. 2F). These results prompted us to hypothesize that therapeutic responses evaluated by MRI and ctDNA may reflect distinct aspects of tumor biology and sensitivity to systemic treatment.

To further understand the clinical implications of cfEBV DNA-based biological responses. We first examined the correlations between post-NAC cfEBV DNA and post-CRT ctDNA. A total of 690 (84.0%) patients with matched post-NAC and post-CRT cfEBV DNA tests were included in the analysis. Among these, 51 patients had detectable post-CRT cfEBV DNA (median, 0.81 103 copies/mL; IQR, 0.334.79 103 copies/mL). The results demonstrated that detectable post-NAC DNA had 83.6% prediction sensitivity for detectable post-CRT ctDNA (95% CI = 78.088.1%). The probabilities of detectable post-CRT cfEBV DNA were 14 of 464 (3.0%) and 37 of 226 (16.4%), respectively, for patients with and without cBR after NAC (P < 0.01, 2 test; Additional file 1: Fig. S3A), suggesting that early cfEBV DNA kinetics was an informative indicator of whole-course treatment responses.

Next, we sought to determine the predictive value of post-NAC cfEBV DNA in long-term prognosis. Survival analysis revealed that cBR post-NAC was strongly predictive of long-term prognosis (HRDFS = 3.28; 95% CI = 2.554.23; P < 0.01; Fig. 2G and Additional file 1: Fig. S3B) and was independent of other clinically relevant prognostic factors in the IPW-adjusted survival analysis (Table 3). Interestingly, we identified that post-NAC cfEBV DNA was most prominently associated with distant metastasis after adjusting for clinically significant covariates (HRcfEBV DNA = 3.45 vs. HRMRI = 1.71, Pboth < 0.05; Table 3). In contrast, although post-NAC cfEBV DNA was also an independent predictor for locoregional recurrence, radiological response exhibited higher HRLRFS compared to post-NAC cfEBV DNA, suggesting that radiological response was a more preferential predictor for locoregional recurrence (HRcfEBV DNA = 1.89 vs. HRMRI(PR vs. CR) = 2.70 & HRMRI(SD/PD vs. CR) = 5.57; Table 3). This observation echoed with the above presumption that MRI and ctDNA reflected distinct aspects of tumor biology and sensitivity to systemic treatment.

Furthermore, we found that patients with non-cBR post-NAC that finally achieved cBR at the end of the CRT still sustained worse prognoses compared to those with cBR post-NAC (HRDFS = 2.70; 95% CI = 2.003.64; P < 0.01; Fig. 2H), suggesting that early biological responses were informative and that delayed ctDNA response conferred unfavorable outcomes. Moreover, among 242 patients with disease progression events, detectable cfEBV DNA post-NAC encompassed over half (122/242) of all failures, while detectable post-CRT ctDNA encompassed only 18% (39/211) of all failures (P < 0.05). Together, these data indicated that unfavorable biological cfEBV DNA responses at early treatment course identified an at-risk subgroup that encompassed large proportions of long-term failures.

Given the above observations, we asked whether early ctDNA kinetics provided additional clinical utility beyond imaging response assessments. To answer this question, we first stratified patients according to their radiological response and investigated whether patients with RECIST CR or PR had an unfavorable prognosis when they had detectable post-NAC ctDNA. Interestingly, we identified that post-NAC cfEBV DNA further stratified PR subgroup, with non-cBR patients having significantly worse DFS (HRDFS = 3.17, 95% CI = 2.364.25, P < 0.01; Fig. 3A). Unfortunately, the survival outcomes for CR subgroup (non-cBR vs. cBR) were not depicted due to the limited sample size in CR+non-cBR subgroup (n = 3). In addition, across patients with RECIST SD/PD, patients who achieved cBR post-NAC had more favorable DFS compared with those who did not (HRDFS = 2.32; 95% CI = 1.284.20; P < 0.01; Fig. 3A).

Biological responses provide additional prognostic information to RECIST. A Top panel: Kaplan-Meier survival plot of DFS in patients achieving RECIST PR stratified by biological responses to NAC. Bottom panel: Kaplan-Meier survival plot of DFS in patients with RECIST PD/SD stratified by biological responses to NAC. B Top panel: Kaplan-Meier survival plot of DFS in patients achieving cBR stratified by RECIST (CR vs. PR vs. SD/PD). Bottom panel: Kaplan-Meier survival plot of DFS in patients who did not achieve cBR stratified by RECIST (PR vs. SD/PD). C Kaplan-Meier survival plot of DFS, OS, DMFS, and LRFS across response phenotypes based on biological plus radiological responses to NAC. G1: cBR+CR, G2: non-cBR+CR, G3: cBR+PR, G4: non-cBR+PR; G5: cBR+SD/PD, and G6: non-cBR+SD/PD. Abbreviations: cBR, complete biological response; cfEBV DNA, cell-free Epstein-Barr virus DNA; CR, complete response; DFS, disease-free survival; DMFS, distant metastasis-free survival; HR, hazard ratio; LRFS, locoregional relapse-free survival; non-cBR, non-complete biological response; OS, overall survival; PD, progression disease; PR, partial responses; SD, stable disease

Next, we determined whether radiological responses can further stratify patients with or without cBR. We found that radiological response further stratified patients with cBR and that patients with SD/PD had significantly worse DFS compared to those with CR (HRDFS = 4.93; 95% CI = 2.2510.82; P = 0.02; Fig. 3B) and PR (HRDFS = 2.06; 95% CI = 1.522.78; P = 0.04), whereas the difference was not significant between CR versus PR (P > 0.05), possibly attributed to the limited events, given that cBR patients had superior prognosis compared to the overall cohort (Additional file 1: Fig. S2C). In addition, across the non-cBR subgroups, although patients with PR demonstrated better prognosis compared to those with SD/PD, the differences did not reach statistical significance for DFS (P > 0.05; Fig. 3B), suggesting that patients who did not successfully achieve biological response (non-cBR) would have equally inferior long-term tumor control regardless of radiological PR or SD/PD.

Based on the above observation, we further combined the radiological and biological response subgroups and yielded 6 response phenotypes: G1 (cBR+CR, n = 53, 6.5%), G2 (non-cBR+CR, n = 3, 0.4%), G3 (cBR+PR, n = 488, 59.4%), G4 (non-cBR+PR, n = 160, 19.5%); G5 (cBR+SD/PD, n = 45, 5.5%), and G6 (non-cBR+SD/PD, n = 72, 8.8%). Across diverse phenotypes, we next mainly focused our following analysis on phenotypes with contradictory biological and radiological response evaluations (G4 [non-cBR+PR] and G5 [cBR+SD/PD]). For G4, one important issue here was whether non-cBR was potentially confounded by false-positive cfEBV DNA tests. To address this point, we further compared their baseline characteristics with G3 (cBR+PR) and identified that patients with non-cBR+PR response phenotype tended to have higher clinical stages and baseline cfEBV DNA load (Additional file 2: Table S2). Interestingly, even adjusting for clinical covariates in multivariate analysis, patients with non-cBR+PR still had significantly worse prognosis in all endpoints compared to cBR+PR (Table 4), suggesting that detectable cfEBV DNA for patients with PR was clinically informative, rather than just confounded by false-positive tests. Analogously, to further address whether cBR was potentially confounded by false-negative cfEBV DNA tests for patients with SD/PD in G5, we further compared their baseline characteristics with G6 (non-cBR+SD/PD) and observed that they had lower pretreatment cfEBV DNA compared to G6 (Additional file 2: Table S3). Interestingly, after adjusting for clinically relevant covariates, patients with cBR+SD/PD (G5) still harbored significantly better prognosis in OS, DFS, and DMFS compared to non-cBR+SD/PD (G6) (Table 5). Notably, the differences in DMFS were most prominent (HRDMFS = 5.81, 95% CI = 2.0916.18, P < 0.01), whereas the difference in LRFS did not reach statistical significance (P > 0.05). These data indicated that undetectable cfEBV DNA for patients with SD/PD was clinically informative rather than just confounded by false-negative tests, especially in forecasting better distant control across patients with SD/PD, but not for local control. Collectively, we revealed that the contradictory biological and radiological responses bred additional valuable prognostic information.

Finally, we asked whether patients with biological cBR plus radiological SD/PD (G5) would have comparable survival with patients who achieved radiological PR plus biological non-cBR (G4). To our surprise, G5 had significantly more favorable long-term prognosis compared to G4, especially in the control of distant metastasis (Pall < 0.05; Fig. 3C).

Given the above findings that cfEBV DNA harbored critical biological information and that its on-treatment clearance kinetics identified preferentially at-risk populations beyond the traditional imaging evaluations, we presumed that inclusion of ctDNA testing would refine the risk estimates across patients with similar initial risks based on clinically relevant factors; moreover, as therapy is introduced, further risk stratification considering the on-treatment ctDNA measurement, radiological response, and therapeutic information would refine personalized dynamic risk estimates. To test this hypothesis, we established five risk prediction models incorporating clinically important factors with/without ctDNA and on-treatment parameters (Fig. 4A). The models were constructed based on Cox proportional hazard regression (CpH) model.

The combinations of biological and radiological responses refine risk groupings. A Bar plot showing the C-index and 95% CI for predicting the 5-year DFS by five models incorporating pretreatment risk factors with/without ctDNA and on-treatment parameters using the CpH method. B Nomogram for predicting the 3- and 5-year DFS, which integrated conventional pretreatment risk factors with pretreatment ctDNA, radiological and ctDNA-based response phenotypes, and therapeutic information. The total point values were independently calculated and then applied to the corresponding probability scale. C Calibration plots showing the actual risk probability by decile (y-axis) over the nomogram-predicted risk probability (x-axis). Abbreviations: cBR, complete biological response; cfEBV DNA, cell-free Epstein-Barr virus DNA; CR, complete response; DFS, disease-free survival; non-cBR, non-complete biological response; PD, progression disease; PR, partial responses; SD, stable disease

In the first model, three parameters (sex, age, clinical stage) established from prior literature or datasets were initially incorporated (Model-I: pretreatment clinical [Model-I_preCLI]). We determined the performance of the model for predicting 5-year DFS, a clinically relevant milestone and standard endpoint in cancer, and identified a bias-corrected Harrells concordance index (C-index) of 0.57. Importantly, the predictive accuracy of 5-year DFS significantly improved when pretreatment cfEBV DNA was incorporated (Model-II: pretreatment clinic-biological [Model-II_preCLIBIO]), with the C-index reaching 0.60. Next, we introduced treatment information and radiological/biological response parameters into the model (Model III-V). Model-III_postMRI, incorporating treatment information and radiological responses, had a significantly improved C-index of 0.65, and the C-index of Model IV (Model-IV_postctDNA), which incorporated treatment information and biological responses, was 0.68. Finally, given the above observation that on-treatment MRI and ctDNA reflected distinct aspects of tumor biology and sensitivity to systemic treatment, we established Model-V (Model-V_INTEGR), which integrated pretreatment factors with radiological and ctDNA-based response phenotypes, and therapeutic information. The C-index of Model-V reached 0.69.

Given that Model-V_INTEGR outperformed the models using pretreatment risk factors or on-treatment radiological assessments, we further developed a nomogram for quantifying the 3- and 5-year risks of disease progression in patients with diverse pretreatment and on-treatment features (Fig. 4B). The calibration plots indicated good agreement between the models predicted and observed survival estimates (Fig. 4C).

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Improving on-treatment risk stratification of cancer patients with refined response classification and integration of circulating tumor DNA kinetics -...