The purpose of this study is to determine the relationship between maternal primary and recurrent CMV infection during pregnancy, symptoms at birth in the newborn, and long term hearing loss through18 years of age.237 mother-infant pairs in the Houston, Texas area identified through maternal CMV IgG and IgM antibody serologic screening and newborn screening using urine CMV culture to identify congenital CMV infection were enrolled in the Houston Congenital CMV Longitudinal Study. Mothers were categorized as having primary or recurrent or unknown maternal CMV infections, and newborns were categorized at birth as having symptomatic or asymptomatic congenital CMV infection, or as uninfected controls. All three newborn groups were followed longitudinally with serial hearing evaluations up to 18 years of age. The relationship between type of maternal CMV infection, newborn classification, and the occurrence of hearing loss over time was determined through Kaplan-Meier survival analysis, life table analysis, and a simulated ascertainment of maternal infection type for the unknown categories.Of 77 newborns with symptomatic congenital CMV infection, 12 (16%) of mothers had a primary CMV infection during pregnancy; 4 (5%) had a non-primary infection, and the type of infection in 48 (79%) could not be determined and were classified as unknown type of maternal infection. Fifty Seven (74%) of the 77 symptomatic children had hearing loss by 18 years of age, including 9 of the 12 (75%) who were born to mothers with primary infection and 48 (79%) of the 61 with unknown type of maternal infection. Of the 109 newborns with asymptomatic congenital CMV infection, 51 (47%) were born to mothers with a primary CMV infection during pregnancy, 18 (17%) to mothers with a recurrent infection; and 40 (37%) had unknown type of infection. Of these 109 asymptomatic cases, 22 (20%) developed hearing loss, including 14 out of 51 (28%) of those born to mothers with primary infection, two out of the 18 (11%) born to mothers with recurrent infection, and 6 out of the 40 (15%) to mothers of unknown infection type. Of the 51 uninfected newborn controls, 10 (20%) of mothers had a primary CMV infection during pregnancy, 5 (10%) had a non-primary infection, 10 (20%) were never infected, and 26 (51%) were assigned unknown type of infection. Three controls (6%) developed hearing loss, with 1 being born to a mother with primary infection and 1 to a mother never infected with CMV.Both primary and non-primary maternal CMV infections during pregnancy resulted in symptomatic and asymptomatic congenital CMV infection. Symptomatic congenital CMV infection was more likely to occur after primary maternal CMV infection. Sensorineural hearing loss occurred in children born to mothers with both primary and non-primary CMV infections, and in both asymptomatic and symptomatic congenital CMV infection, but was more common after maternal primary infection. Most, but not all, hearing loss in children with cCMV associated hearing loss was first detected within the first year of life. Maternal infection with cytomegalovirus (CMV) resulting in congenital CMV (cCMV) infection continues to be a serious public health problem, affecting cCMV-infected fetuses, newborns and children. Congenital cytomegalovirus infection is a common congenital infection, with an estimated birth prevalence of 1%, ranging from 0.2% to 2.5%, depending on the population studied, resulting in tens of thousands of newborns each year born with cCMV infection in the United States [The various health outcomes of congenital CMV infection may be apparent in the fetus and newborn, with one or more symptoms at birth; or become apparent later in infancy, childhood or adolescence in an otherwise asymptomatic, normal-appearing newborn. It is estimated that 10 to 15% of newborns with cCMV infection will be symptomatic at birth, with significant risk of sensory, neurological and motor sequelae . The majMaternal infection with CMV may be from a primary infection (first experience with the virus) or a non-primary, recurrent infection (or second experience with the virus) –6. RecurConflicting opinions exist about the influence of the type and timing of maternal CMV infection during pregnancy and the outcome in the child. Most studies stress the importance of primary maternal CMV infections, especially those occurring early in pregnancy, as having the greatest impact on the outcome of the newborn and child –14. The It was previously thought that non-primary maternal CMV infections during pregnancy had both low transmission rates to the fetus and very low rates of symptomatic cCMV infection in the newborn, with a few exceptions found in isolated case reports. However, a growing body of evidence has indicated that maternal pre-conception immunity to CMV, while conferring some protection, is imperfect, and that maternal non-primary CMV infections during pregnancy may result in both asymptomatic and symptomatic cCMV, even with severe manifestations, in a much larger percentage of newborns than previously thought –28. StilIn this study we examine the relationship of maternal primary vs. non-primary CMV infection during pregnancy to long-term sensorineural hearing loss outcomes in children born with congenital CMV infection, and compare the relationship to uninfected control subjects. This information has important implications for CMV vaccine trial development, as well as for identifying target populations for CMV vaccine administration once a licensed CMV vaccine is available for prevention of congenital CMV infection.th year. The original hypothesis was that both symptomatic and asymptomatic congenital CMV infection in the newborn was associated with long-term sequelae in vision, hearing, and development in the child and adolescent, and that these outcomes could be predicted by type of maternal CMV infection in pregnancy and by newborn characteristics at birth [The Houston Congenital CMV Longitudinal Study began in 1981 and enrolled subjects through 2005. It is a prospective, longitudinal, multidisciplinary, natural history study of cCMV now in its 38at birth –33.Fig 1).In a sub-study of CMV infection in mothers and their infants, during 1981 to 1986, 4,578 pregnant women who were also patients of a private obstetric group practice participating in The Houston Congenital CMV Longitudinal Study were enrolled and screened prospectively for CMV IgM and IgG antibodies, beginning at the first obstetrical visit, after providing informed consent. The demographics and seroprevalence results of the serologic screening have been previously published . In addiThis study was approved by the Baylor College of Medicine Institutional Review Board and all subjects were enrolled after obtaining written informed consent.Maternal sera for determination of CMV IgG and IgM antibodies were obtained as early in pregnancy as possible, and repeated at each trimester and at delivery when possible . DefinitPrimary maternal infection with CMV was defined as either seroconversion from CMV IgG negative antibody to CMV IgG positive antibody  or the presence of both CMV IgG antibody and CMV IgM antibody during pregnancy . The presumptive primary CMV infections were considered presumptive because CMV IgM antibody may last up to 3 to 6 months; therefore, some of these maternal CMV infections may have occurred in the months immediately prior to conception. In addition, mothers who were CMV IgG antibody negative during the first trimester of pregnancy, and did not have subsequent serologic testing, yet gave birth to a congenitally infected newborn, were also considered to have a proven primary maternal CMV infection. Both proven primary and presumptive primary maternal CMV infections were analyzed together as primary maternal CMV infections.Recurrent, or non-primary, maternal CMV infection was defined as the presence of CMV IgG antibody before pregnancy, or the presence of CMV IgG antibody and the absence of CMV IgM antibody during the first trimester of pregnancy. Recurrent infections during pregnancy may be from reactivation of the mother’s endogenous strain or strains/genotypes of CMV (in latently infected mothers) or reinfection with a new strain or strains/genotypes of CMV, but it is not possible with present serologic techniques to determine which type of recurrent maternal CMV infection resulted in the birth of a congenitally infected newborn. Hence these infections were categorized as just non-primary.Uninfected maternal status was defined as mothers who were CMV IgG seronegative in the first trimester and remained CMV IgG seronegative throughout pregnancy, and who delivered an uninfected newborn without congenital CMV infection. These mothers were categorized as having never been infected with CMV.Unknown type of maternal CMV infection during pregnancy was defined as the presence of CMV IgG antibody and the absence of CMV IgM antibody in the mother at delivery. This classification, which was assigned for some symptomatic, asymptomatic and control subjects, could have been applied to mothers who would have had either an unverified primary CMV infection or an unverified non-primary infection during pregnancy, or (for mothers of control subjects only), unverified non-infection. Included in this category were mothers in whom no or incomplete serologic data were available.CMV IgG antibody was determined by enzyme-linked immunosorbent assay and anticomplement immunofluorescence . CMV IgMUrine from newborns was collected by externally applied urine collection bag, usually obtained in the first 3 days of life, and always confirmed again with a second urine sample obtained before the first 21 days of life, and cultured for CMV using traditional cell culture techniques of human foreskin fibroblast (HFF) monolayers, and examined daily for 21 days for evidence of cytopathic effect characteristic for CMV . Isolati3). The presence of hearing loss alone at birth was not part of the defining condition of symptomatic cCMV disease for this study [Infants were examined at birth by their private pediatricians at the birthing hospital, and by one of the authors (GJDH) subsequently, and determined to be symptomatic at birth or asymptomatic at birth. Confirmed symptomatic cCMV disease was defined as a newborn with CMV detected by culture of urine samples collected within 3 weeks of life, who presented with at least one of the following classic CMV-related signs at birth: purpura/petechiae, jaundice, hepatosplenomegaly, microcephaly, unexplained neurological abnormality, elevated liver enzymes , hyperbilirubinemia , hemolytic anemia, or thrombocytopenia  and the multidisciplinary members of the Houston Congenital CMV Longitudinal Study Team, for growth, hearing, vision, neurodevelopmental milestones and cognitive assessments , 33. ThiMaternal demographic information for age, race/ethnicity, parity, and gestational age at delivery were collected and described. Maternal CMV infection type  and category of newborn by birth symptoms  were tabulated and described. The proportions and confidence intervals of children with SNHL up to 18 years of age were analyzed according to maternal CMV infection category. The primary and presumptive primary mothers were analyzed as one category.Sensorineural hearing loss in relation to the timing of CMV infection in pregnancy was also examined. SNHL rates by trimester of infection (when this could be determined) was tabulated for symptomatic and asymptomatic cases. The presence of SNHL by first or second half of pregnancy was also tabulated and examined for significance with a chi-square test.Socioeconomic status of the mothers was determined using a formula that took into account various economic indicators based on the zip code of residence at birth from the US Census Bureau and the presence or absence of private insurance assessed from patient records. This methodology allows the assessment of various components of SES at the neighborhood level such as income ranges, housing characteristics, employment and poverty, and has been used to associate SES with health outcomes, disease risk and health disparities –51.Survival analysis methods employed Life Table analysis –54. ThisSeparate Life Tables were computed for maternal primary infection, non-primary infection, uninfected, and unknown maternal infection categories of patients. For primary vs. non-primary maternal infection groups, relative risks were computed for proportions with hearing loss, probability densities, and hazard rates in the first year of life. Life Table analysis excluding Control (uninfected) subjects was also done.For a more granular look at hearing loss survival, the Kaplan-Meier Product Limit Method was used to take into account the exact times of documented hearing loss and censorship in symptomatic cCMV and asymptomatic cCMV cases in the four maternal infection categories rather than aggregating these into time intervals . Only syMean age at censoring was compared across the three maternal infection categories with Analysis of Variance; equality of mean censorship times was further assessed with Welch and Brown-Forsythe statistics, and pairwise comparisons were made with t-tests. Survival curves for symptomatic, asymptomatic and control subjects were also generated and compared as above.Since the type of maternal infection was not completely ascertained on all mothers, but symptom classification at birth  and hearing loss status were known for all children, simulations were undertaken to further estimate the influence of maternal CMV infection type on hearing outcomes. These were done separately in symptomatic and asymptomatic groups because of suspicions that most cCMV infections among symptomatic patients result from primary maternal infection, but among asymptomatic patients this assumption is less clear.Because the maternal CMV infections in the unknown category were likely to be a mix of both primary and non-primary maternal CMV infections, simulations were conducted to reassign symptomatic and asymptomatic newborns with unknown type of maternal infection into existing primary or non-primary maternal infection categories in various ratios. The hearing loss rates in these simulated, pooled maternal infection categories was then reassessed after augmentation with the reassigned children. Controls were not used in the simulations. The rate of hearing loss in the simulated, pooled primary maternal infection category then resulted from the existing rate of hearing loss in children known to have primary maternal infection combined with the hearing loss rate in those who were reassigned from unknown to primary maternal infection. Similarly, the hearing loss rate in the simulated, pooled non-primary infection category was determined from the hearing loss rate in those already known to have non-primary infection, combined with the hearing loss rate in those reassigned from unknown to non-primary infection, assuming those with unknown type of maternal infection took their hearing loss rate with them when they were reassigned. The new hearing loss rate in each simulated maternal infection category was determined by the number with hearing loss divided by the new denominator population.Kaplan-Meier curves were generated for hearing loss and censorship events in these simulated, pooled maternal infection categories. Survival distributions, including those of symptomatic cases before and after the simulated reassignment, were compared similarly to the above.Age distributions in subjects from primary and unknown maternal CMV infection categories were compared with the Kolmogorov-Smirnov test..Demographic data showed most of the mothers were white, of non-Hispanic ethnicity, married, with a medium to high socioeconomic status (SES), who delivered term or near-term newborns. Their mean age overall was 27 years. The mean age of the mothers who delivered newborns with symptomatic cCMV infection were younger than those mothers who delivered newborns with asymptomatic cCMV . Symptomatic newborns were more likely to be from the referral group rather than the screened group, and more likely delivered to younger mothers of lower SES status .Both primary and non-primary maternal infections resulted in symptomatic and asymptomatic cCMV in the newborns. Of the 77 newborns with symptomatic cCMV infection, 12 (16%) of mothers had proven or presumed primary CMV infection during pregnancy, 4 (5%) had known non-primary CMV infection during pregnancy, and 61 (79%) were categorized as having unknown type of maternal CMV infection. Of the 109 newborns with asymptomatic cCMV infection, 51 (47%) of mothers had proven or presumed primary CMV infection during pregnancy, 18 (17%) had known non-primary CMV infection during pregnancy, and 40 (37%) were categorized as unknown type of maternal CMV infection. Of the 51 uninfected newborn controls, 10 (20%) of mothers had proven or presumptive primary CMV infection during pregnancy, five (10%) had non-primary CMV infection during pregnancy, 10 (20%) were never infected with CMV during pregnancy, and 26 (51%) were categorized as unknown type of maternal CMV infection .Of the 77 symptomatic newborns, 57 (74%) had hearing loss by 18 years of age. Of the symptomatic newborns whose maternal infection type could be determined, 9 out of 12 (75%) born to mothers with primary maternal CMV infection had hearing loss versus zero out of 4 with non-primary maternal CMV infection (p = .009). Of the 61 symptomatic patients with unknown type of maternal infection, 48 (79%) had hearing loss .Among the 109 asymptomatic patients, 22 (20%) developed hearing loss. Of the 51 asymptomatic newborns born to mothers with known primary maternal CMV infection, 14 (28%) developed hearing loss, versus 2 out of18 (11%) of asymptomatics with non-primary infection (p = .157). There were 40 asymptomatic newborns born to mothers with unknown type of maternal infection, six (15%) of whom had hearing loss .Among the 51 controls, three (6%) developed hearing loss: one born from the 10 mothers with primary CMV infection versus 0/5 of those were born to mothers with non-primary CMV infection (p = .462)); one was born from the 10 mothers confirmed to have no maternal CMV infection; and one born from the 26 mothers with unknown maternal CMV infection type .There was no significant relationship identified for the timing of primary maternal infection in relation to sensorineural hearing loss in the child. There were no significant differences in the proportions of subjects with sensorineural hearing loss in those born to mothers who acquired primary infections in the first half vs. the second half of pregnancy. This held true for symptomatic, asymptomatic and control subjects born to mothers with primary infections Tables Fig 2).Life tables for hearing loss were computed for each maternal CMV infection type. These tables show that most hearing loss events occurred in the first year of life. Of the ten uninfected control subjects who were born to mothers known to have no evidence of CMV infection during pregnancy (the “no infection” category), and who all had periodic assessments for hearing loss, one developed significant hearing loss at age 15 years. This subject was a known hunter with environmental exposure to gunshots, and did not consistently use ear protection. This is not characteristic of a typical pattern of congenital CMV-associated hearing loss. This one subject provides strong evidence that very late-onset hearing loss may not always be associated with, or caused by, congenital CMV infection, but rather more likely by environmental exposures such as firearms or loud music . However, the mean age at first hearing loss onset was significantly different between primary and unknown maternal CMV infection categories (p = .001 by student t-test) .Graphs generated from Kaplan-Meier survival analysis show that subjects with cCMV who were born to a mother with a non-primary maternal CMV infection had a relatively lower cumulative proportion of hearing loss over the age span, and the onset of hearing loss in this group was identified either at birth or within the first year of life. Subjects born with cCMV as a result of primary maternal CMV infection had a relatively higher cumulative proportion of hearing loss, with the hearing loss in this group either identified at birth or continuing to accrue up to 17 years of age. Subjects with cCMV born to mothers with an unknown-type maternal CMV infection had an even higher proportion of hearing loss at birth and in the first year of life, and continued to accrue hearing loss up to 11 years of age. The survival distributions were significantly different between primary and non-primary maternal CMV infection (p = .026), and non-primary and unknown maternal CMV infection (p = .001) by log-rank tests. There was no significant difference in survival distributions between primary and unknown maternal CMV infection (p = .068) by log-rank tests (.Subjects who did not develop known hearing loss were censored on average 14.2 years after the average age of first hearing loss onset among those who did develop hearing loss (p< .001). It is therefore highly unlikely there was significant undetected or hidden hearing loss among the censored study subjects. Many subjects who withdrew (censored cases) did so after age 15 years, but most hearing loss occurred well before that age Subjects with mothers who were in the unknown maternal infection category were reassigned to either primary or non-primary infection categories in simulations.In the first simulation, involving only the symptomatic at birth category of subjects, 90 percent of those with unknown type of maternal infection were reassigned into primary maternal infection and 10 percent reassigned into the non-primary maternal infection category. This 90/10 reassignment into the known maternal infection categories was chosen based on current estimates from epidemiologic studies, which suggest that 90% of symptomatic cCMV cases result from primary maternal infection. This simulation was also reformulated to reassign 80/20, 70/30, and 60/40 of unknowns to primary and non-primary maternal infection, respectively, for symptomatic subjects..In simulations with the asymptomatic at birth category of subjects, reassignments 60/40, 50/50, 40/60, and 30/70 were made to maternal primary and non-primary infection categories, respectively For symptomatic subjects, after random reassignment of children with unknown type of maternal infection to primary/non-primary categories in ratios of 90/10, 80/20, 70/30 and 60/40, the point estimates for the proportion of children with hearing loss ranged from 77.6% to 78.7% in the simulated primary maternal infection group. Among these symptomatic children, the hearing loss rates in the simulated primary infection groups was similar to the rate for those with known primary infection before simulation with the augmented cases, regardless of reassignment ratio. This is due to the similar rates of hearing loss in the primary (75%) and unknown (79%) groups for symptomatic children before the reassignments..For symptomatic subjects in the simulated non-primary infection category in the above ratios, 50.0% to 67.9% had hearing loss. The estimated rate of hearing loss was higher than in those with known non-primary maternal infection before the simulation, owing to the fact that so few symptomatic patients were ascertained to have a known non-primary infection, none of whom had hearing loss, and to the high rate of hearing loss among those with unknown type of maternal infection who were reassigned into this non-primary category. For these symptomatic subjects, as the proportion of those reassigned from unknown to non-primary maternal CMV infection got higher in the simulations, the rate of hearing loss in the simulated non-primary category increased .For asymptomatic subjects, the estimated point estimates for hearing loss in the simulated groups after augmentation with those of unknown maternal infection type ranged from 23.9% to 25.4% in the simulated primary maternal infection group, and from 11.8% to 14.3% in the simulated non-primary maternal infection group. These estimated rates of hearing loss did not differ much across the different reassignment ratios and did not greatly differ from the hearing loss rates in the known primary or known non-primary maternal CMV infection categories for asymptomatic subjects before the reassignments Results of these simulations show that if the maternal CMV infection status of subjects was fully ascertained, hearing loss rates among symptomatic subjects born to mothers with primary maternal infection would likely not meaningfully change from our actual observed rate of 75%, regardless of the ratio of primary/non-primary maternal infection. However, higher hearing loss rates would be expected among symptomatic subjects born to mothers with non-primary maternal CMV infection, compared to the 0/4 or 0% rate of hearing loss that was documented in symptomatic subjects who were known to have been born to mothers with non-primary maternal CMV infection in this study. Again this is due to the low number of symptomatic subjects who were known to have maternal non-primary infections, and the high (79%) rate of hearing loss in the symptomatic subjects with unknown maternal infection type.These results also suggest that among asymptomatic subjects, full ascertainment of maternal infection category would not likely meaningfully change the known hearing loss rates that were actually observed in asymptomatic subjects with known primary or known non-primary maternal CMV infection.To further elucidate the results of simulated reassignments of those with unknown maternal serology type to either primary or non-primary maternal infection, Kaplan-Meier graphs were generated. For the symptomatic subjects, we graphed a random reassignment of 90% of subjects born to mothers with unknown maternal serology type to primary maternal infection category, and 10% to the non-primary maternal infection category, where they were combined with those already known to be in those categories as described above. The actual hearing loss status and dates of onset for the known and randomly-reassigned patients were used. The graphs show the hearing loss survival distributions of subjects born to mothers with known primary and non-primary maternal infection categories, before any reassignments were made, and after the addition of reassigned unknowns to these categories .Fig 2). The similarity of hearing loss survival distributions, before and after reassignments, supports the assumption that many or most of the symptomatic subjects born to mothers with unknown maternal CMV infection type, may result from primary maternal CMV infections.Results show that, in the symptomatic group, there is no statistical difference between hearing loss survival distributions of the known primary maternal infection group before reassignments were made and after reassignments of unknowns to this category (p = .576 by Log-Rank test). The distributions also show no difference between the known non-primary maternal infection group before reassignments and after reassignments of unknowns to that group. All three variants of the log-rank test show no significant differences between the before and after groups . In addition, separate analysis of only those 92 asymptomatic at birth subjects identified through the hospital-based newborn screening program showed no statistical differences in hearing loss survival distributions before and after simulated reassignments, for either primary or non-primary maternal CMV infections.The survival distributions for the 109 asymptomatic subjects also show no statistical differences between hearing loss survival distributions before and after simulated reassignments, for either primary or non-primary type of maternal CMV infection  was controlled for.Hearing loss occurred in subjects born to mothers with primary or non-primary CMV infections, and in subjects with cCMV infection who were asymptomatic at birth or symptomatic at birth. Hearing loss was more common in subjects born with symptomatic cCMV compared to asymptomatic cCMV infection. In addition, most hearing loss occurred in children born with cCMV infection as a result of maternal primary CMV infection than non-primary maternal CMV infections (in both symptomatic and asymptomatic subjects), although the timing of maternal primary infection in pregnancy appeared to have no effect on the rates of sensorineural hearing loss. In all subjects with cCMV infection, hearing loss was common and usually occurred at birth or within the first year of life. The latest age of onset of hearing loss observed was 17 years of age. Kaplan-Meier survival curves also showed a higher proportion of onset of hearing loss in subjects over time born to mothers with primary CMV infection, as well as those with an unknown type of maternal CMV infection during pregnancy, compared to those born to mothers with a known non-primary CMV infection, although the number of mothers ascertained to have known non-primary maternal CMV infection was small.In the uninfected controls, hearing loss also occurred, but at a later time in childhood, at a much lower rate, and exhibited a different pattern than in the subjects with cCMV infection. This comparative baseline of hearing loss in uninfected children, compared to congenitally infected children, may be useful in planning a vaccine trial to prevent maternal CMV infection with a trial endpoint of reducing hearing loss in children and adolescents. Our findings suggest that a CMV vaccine trial would not require an extended followup time in order to capture most cCMV-related hearing loss. It also shows that study dropouts after the early followup period would not obscure significant undocumented hearing loss in controls.Among symptomatic children, the hearing loss rate of those born to mothers with an unknown type of maternal CMV infection was similar to the rate in in those born to mothers with a known primary CMV infection. And in simulations where children born to mothers of unknown type of CMV infection were reassigned to the symptomatic group with known maternal primary infections, the rate of hearing loss in the augmented symptomatic group was not significantly different from before the simulated reassignments. This similarity lends support to the belief that symptomatic congenital CMV infection is usually the result of primary maternal CMV infection, which confers a higher risk of hearing loss than non-primary maternal CMV infection.Only a small number of mothers were ascertained to have known non-primary CMV infection and gave birth to children categorized in the symptomatic cCMV category at birth (n = 4), none of whom developed hearing loss. However, the simulated reassignments of symptomatic children with unknown type of maternal infection into the non-primary maternal infection category show that non-primary maternal infections would likely confer a considerable hearing loss rate as well. This finding suggests children born with symptomatic cCMV infection, as a result of non-primary maternal CMV infection during pregnancy, are at a relatively higher risk for hearing loss than was previously appreciated for those with non-primary maternal infection.Subjects categorized in the asymptomatic group at birth and born to mothers with known non-primary type of maternal CMV infection (n = 18), also had a considerable 11% (95% CI = 0–25%) known hearing loss rate, and after augmentation with the simulated reassignments of subjects with unknown maternal serology category to this category, the hearing loss rates for this category were the same or slightly increased.It is likely that most of the unknown types of maternal CMV infections in this study were actually primary CMV infections, especially for the children born with symptomatic cCMV infection.Our results suggest that prevention of both primary and non-primary maternal CMV infection during pregnancy is likely to significantly reduce the incidence of hearing loss in children.Life table analysis may be helpful in planning trials to evaluate the effect of an intervention to prevent cCMV-associated hearing loss in children. CMV is a dynamic infectious agent with multiple transmission vectors whose timing and impact on cCMV incidence rates are not fully understood. It is therefore not clear which vectors of CMV transmission are the most important for maintaining incident cCMV in a population and could be disrupted by vaccination, e.g., toddler-to-toddler, toddler-to-mother, adult-to-mother-to-fetus, etc. Life table analysis is especially useful in this regard to determine the incidence and prevalence of cCMV-related hearing loss in various age groups and would allow the computation of age-adjusted hearing loss rates. It allows the determination of the probability density  for different age groups or subpopulations, and to compare hearing loss hazard ratios between these groups. These comparisons could be made within a cohort or between entire cohorts that do and do not receive a CMV vaccine. Life table analysis would be especially informative in community trials of a CMV vaccine, for example where community cohorts of subjects may be enrolled all at once to establish the ongoing incidence and prevalence of cCMV and its related hearing loss before and after a vaccine is introduced. In a community setting, incident cases of CMV-related hearing loss in one-time interval or age group would diminish the number of persons at-risk for CMV-related hearing loss in subsequent time intervals; and conferred immunity to CMV in one age group might decrease incident CMV infection rates in younger or older age groups going forward. Even for an imperfect CMV vaccine, life table analysis can aid in determining the optimal targets, age groups and timing for vaccination that would be needed to disrupt transmission or significantly mitigate hearing loss outcomes. It also allows the delayed effects of immunity, waning immunity, and herd immunity to be observed in a cohort, depending on the study design. It is informative to determine baseline cCMV-related hearing loss rates in different age strata, as our study has done, before CMV vaccine trials are initiated. Although our life table methodology might have been more amenable to a larger community cohort study of cCMV, it did show from a natural history perspective that most cCMV-related hearing loss occurred in the first year of life or by three years of age at a time when few if any prenatal or public health messages about cCMV were extant and no CMV vaccine yet exists.This study is the first to document the relative role of maternal primary vs maternal non-primary CMV infection in hearing loss in children born with asymptomatic cCMV infection, and to relate the long-term outcome of hearing loss through adolescence to maternal serologic status. Previous studies have focused on symptomatic newborns, but asymptomatic newborns, because they represent the more common manifestation of cCMV, likely constitute the greater public health burden from cCMV. Prevention of both primary and non-primary types of maternal CMV infection, and transmission to the fetus, may therefore reduce the public health impact of cCMV infection.This study has limitations. Although diversity is represented in our study, this population provides information on primarily white, non-Hispanic, middle to upper SES mothers, in Texas, and may not be generalizable to all maternal populations or geographic areas.Another limitation is a lack of definitive ascertainment of maternal CMV serologic status for many of the subjects enrolled. This limitation was minimized through simulated reassignments of subjects born to mothers with unknown type of maternal CMV infection into primary or non-primary categories in biologically plausible ratios, and the determination of the resulting hearing loss rates. Furthermore, since the women were identified and tested serologically for CMV infection over 30 years ago, contemporary methods, including CMV IgG avidity, interferon gamma release tests, and molecular nucleic acid detection methods to determine CMV reactivations versus reinfections in the non-primary CMV infection category, were not available. However, since most practicing obstetricians will use only the standard CMV IgG and CMV IgM antibody tests to screen or diagnose pregnant women for CMV infection, the results from this study still have great clinical significance.The results from this study have relevance for CMV vaccine development . ProgresWhile development of a CMV vaccine continues, prevention of both primary and non-primary CMV infections in pregnancy remains a challenge. Studies have shown prenatal behavioral interventions, consisting of CMV awareness education about common behaviors associated with CMV transmission to pregnant women, are effective in changing behaviors and may reduce transmission of CMV to pregnant women . BecauseSince cCMV infection occurs after both maternal primary and non-primary CMV infections, identification of newborns with cCMV infection through universal newborn screening is likely to be the best way to detect cCMV infection in all newborns, both symptomatic and asymptomatic at birth, born to mothers with both primary and non-primary CMV infections , 58. Ear


A number of studies found that serotonin plays a vital role in the development of depression and irritable bowel syndrome. Recent studies showed that vitamin D was associated with regulating the synthesis of serotonin. This review focuses on the recent progress in the relationship between vitamin D and serotonin synthesis. Serotonin is a monoamine neurotransmitter derived from tryptophan , synthesVitamin D (VD) is a fat-soluble steroid that is a key regulator of calcium and phosphorus metabolism , which mFirmicutes , IBS with diarrhea (IBS-D), and IBS with constipation (IBS-C), it was found that colonic mucosal serotonin concentrations in UC, IBS-C, and IBS-D specimens were significantly lower than those in healthy control specimens . HoweverSerotonin regulates a wide variety of brain functions and behaviors. Previous findings have demonstrated that serotonin regulates executive function, sensory gating, and social behavior and that attention deficit hyperactivity disorder, bipolar disorder, schizophrenia, and impulsive behavior all share in common defects in these functions. Serotonin synthesis was controlled by VD . There iNowadays, distinct vitamin D response elements (VDREs) have been identified in the regulatory regions of both TPH2 and TPH1 , which sIn people with insufficient VD levels, VD supplementation may improve the mood of patients by increasing the availability of serotonin in the brain through the expression of TPH2 , while iThese findings provide important evidence that VD affects the synthesis of serotonin to relieve anxiety and inflammation symptoms in patients, thereby improving IBS.Marya SS et al. quantified the mRNA expression of serotonin synthesis isoenzymes TPH1 and TPH2 as well as the serotonin reuptake transporter (SERT) and the enzyme responsible for serotonin catabolism monoamine oxidase-A (MAO-A) expression to explore the response of neuronal cells to 1.25D. The results show that 1.25D not only induces serotonin synthesis but also acts at an indirect molecular genomic stage, mimicking SSRI and MAO inhibitors, which may elevate serotonin in the central nervous system, and this also explains the effect of VD on neuronal trajectories and causes of development of mental disorders . MeanwhiAlthough VD has TPH2 expression in cultured rat serotonergic neurons, a randomized clinical trial of VD supplementation in children with autism spectrum disorder (ASD) showed that VD supplementation had no significant effect on serum serotonin levels of ASD children . AdditioNumerous studies have shown that serotonin is related to mental and intestinal functions, and VD may affect serotonin levels by regulating TPH, thereby affecting human mental and intestinal functions. This has been confirmed in animal experiments, but there is a lack of sufficient evidence in clinical trials, which provides directions for future research.


Recent larger-scale studies of patients with cancer and longitudinal population cohorts have revealed how age-related expansions of mutant hematopoietic cells  have differential associations with incident and prevalent cancers and their outcomes. Increasing recognition and deeper understanding of genetic subtypes of CH are yielding insights into the tumor-immune interface that may help to explain the heterogeneous impact of CH on tumorigenesis and treatment. Herein, we update the expanding influence of CH in precision oncology and propose important research and clinical questions to address to effectively manage and harness CH in oncology patients. This article explores the potential consequences of clonal hematopoiesis for patients with solid tumor. There are no specific bounds to define CH, while the currently accepted VAF threshold for CHIP is at least 2%, indicating that 4% of total peripheral blood cells are affected, assuming a heterozygous mutation. Most studies have adopted this 2% VAF threshold for identifying CHIP and its corresponding clinical associations; however, this threshold is somewhat arbitrary and was initially determined by the standard limit of detection of next-generation sequencing (NGS) technologies. Newer technologies such as error-corrected (EC)-NGS can detect small CH clones with VAFs as low as 0.01% that normally escape detection, although the clinical implications of these clones should decrease with diminishing clone size.11 Owing to the lack of sufficient differentiation between CH and CHIP in most studies, we refer to both CH and CHIP as CH in this review for its broader definition.Clonal hematopoiesis (CH) is the clonal expansion of hematopoietic stem cells (HSCs) and their progeny as driven by somatic mutations acquired during aging. CH describes any clonal expansions found in the blood and HSCs driven by somatic mutations and is thus inclusive of hematologic malignancies such as acute myeloid leukemia (AML) but typically refers to patients with premalignancy and no established disease.enia Fig . BetweenKey ObjectiveClonal hematopoiesis (CH), the common age-associated expansion of somatically mutated blood cells, is associated with immune dysregulation, increased inflammatory disease, and hematologic malignancy risk. This review explores the implications of CH for solid cancers to provide recommendations for future research and practice.Knowledge GeneratedCH is common in patients with solid tumors, attributable to age, cancer treatments, and the possibility that CH is a risk factor for some cancers. Current applications in oncology include the optimization of tumor molecular profiling and management of therapy-related neoplasms. The effects of CH in the tumor microenvironment are diverse, varying between CH driver and cancer type.RelevanceThe frequent copresentation of CH with solid cancer presents a tremendous opportunity to improve clinical outcomes for patients with cancer. Implications of CH for some clinical applications are already clear, although much more research is needed to leverage CH as a tool in precision oncology.Recent studies have revealed differential associations of CH with incident and prevalent cancers and their outcomes. Increasing recognition and deeper understanding of genetic subtypes of CH are yielding insights at the tumor-immune interface that may help to explain the heterogeneous impact of CH on tumorigenesis and treatment. Herein, we update the expanding influence of CH in precision oncology and propose important research and clinical questions to address in order to effectively manage and harness CH in oncology patients.6 As noted previously, these estimates rely on older sequencing methods and higher VAF clones. With EC-NGS and the ability to detect lower VAF clones, <2% VAF CH appears almost ubiquitously in younger populations age 50-70 years, reaching rates of 95%, compared with 5% in the aforementioned studies.11CH is notable as a model of somatic mutations and aging in a variety of tissues, but its high prevalence and relationship with various inflammatory processes and age-related diseases makes it of great interest to clinicians and researchers. Initial population screenings for CH surveyed blood-derived genomic sequencing data from more than 30,000 individuals for evidence of clonal expansion. Together, these studies found that CH prevalence increases dramatically with age, detected in 10%-20% of individuals older than 70 years while being almost undetectable in those younger than 40 years. Of great concern, carriers of CH were found to have a 30%-40% increase in all-cause mortality.12). Initial studies revealed an approximately 10-fold increase in relative risk and 1% annual risk of malignant transformation, but it is again important to note the bias toward larger VAF clones because of study methodologies, and increasing CH clone size and the number of mutations were associated with increased risk of hematologic malignancy.3 Recent estimates using more sensitive sequencing methods predict a 3 to 5-fold increase in AML risk and when distinguishing between myeloid- and lymphoid-associated CH drivers, a 7-fold and 4.2-fold increase in relative risk of myeloid and lymphoid cancers, respectively.14 In addition to clone size, the risk of developing a hematologic malignancy is also modified by the affected gene(s). The two most commonly affected genes in CH, DNMT3A and TET2 exhibit some of the lowest rates of AML progression while the less frequently mutated but still prevalent TP53 and U2AF1 demonstrate a much higher risk of malignant transformation and are associated with poorer AML prognosis.15 Certain mutations within genes also confer varying risks of progression, such as the DNMT3A R882H mutation—the most prevalent CH mutation overall—which is underrepresented in CH versus AML and myelodysplastic syndrome (MDS), indicating a potential for increased progression risk.16Although CH is a precursor state to hematologic malignancy, not all CH-mutant clones develop into cancer, and the risk of malignant transformation varies between subtypes of CH -α, interleukin (IL)-6, and IL-1β through the activation of various inflammatory pathways.20 The effects of DNMT3A and TET2 mutations have been best characterized in relation to inflammation, although knowledge is still incomplete. These epigenetic regulators have broad roles in restricting inflammation in the immune system, such as in monocyte/macrophages, where loss of function mutations in these genes promote the excessive release of proinflammatory cytokines.23 Importantly, the hyperinflammatory environment created by CH also acts alongside inflammatory stressors such as infection to promote the further development and expansion of CH clones, acting cyclically to exacerbate inflammation . For example, the elevated levels of TNF-α commonly seen in CH have been found to provide a fitness advantage for TET2-mutant HSCs, allowing them to continue to expand and proliferate to occupy a larger proportion of the active HSCs.25Moving beyond the risk of overt hematologic malignancy, the altered inflammatory milieu imposed by CH promotes systemic inflammation and increased morbidity and mortality.JAK2 specifically conferring a 12-fold risk increase for coronary heart disease.26 Subsequent cohort analyses and mechanistic studies have verified these initial findings while also expanding to show that CH is associated with increased risk of early-onset myocardial infarction, chronic heart failure, and thrombosis.32 CVD is not the only major clinical association of CH aside from hematologic malignancy. CH-associated inflammation has recently been linked to chronic kidney disease, with patients found to have elevated levels of CH versus the general population and had a more than doubled risk of kidney failure during the study follow-up period.33 Other adverse clinical outcomes associated with CH include autoimmune diseases such as antibody-associated vasculitis, chronic obstructive pulmonary disease, osteoporosis, and, albeit controversially, severe COVID-19.38 Curiously, the hyperinflammatory milieu conferred by CH may provide a protective effect in some diseases, such as in Alzheimer disease, where CH was found to reduce the risk for dementia and neuropathological features.39A primary consequence of CH-associated inflammation and one of the main drivers of increased morbidity and mortality in CH carriers is an elevated risk of developing cardiovascular disease (CVD). Initially, the associations between CH and CVD were limited to coronary heart disease and ischemic stroke, where relative risk of incident events were, respectively, 2 and 2.5 in CH carriers, with mutations in 40 Subsequent analyses of an overlapping but expanded cohort from MSK similarly found the prevalence of CH in patients with cancer to be 30%. Interestingly, the incident risk of CH was not uniform across cancer types—patients with thyroid and ovarian cancer demonstrated an elevated risk of CH, whereas melanoma, prostate cancer, colorectal cancer, and renal cell carcinomas were associated with a lower risk of CH.41 An additional analysis of cancer patient samples identified an increased risk of CH in thymoma patients and a reduced risk in bladder and breast cancers.42 The risk of confounding because of relationships with cancer treatment  and shared CH and cancer risk factors such as age and smoking limit the ability of these studies to make causal conclusions about the risk associations between CH and cancer.41Several population cohort studies have already begun exploring another significant clinical association of CH—solid cancers. The first project specifically focused on CH in solid cancers was an analysis of 8,810 patients treated at Memorial Sloan Kettering (MSK). CH was common in this cohort, appearing in just over 25% of analyzed patients with solid cancer and associating with age, smoking, and previous exposure to therapy.DNMT3A-mutant CH was additionally associated with incident stomach and bladder cancer while mutations in splicing factors SF3B1 and SRSF2 were uniquely linked with higher rates of colorectal and head/neck cancers.43 A similar analysis using 628,388 individuals from the UK Biobank found relationships between CH status and incident risk of lymphoid cancer, lung cancer in both smokers and nonsmokers, breast cancer, and prostate cancer.44 Strengthening the link between CH and lung cancer, CH conferred a 36% risk increase for disease across several cohorts, even when controlling for other confounders.45 Conversely, a recent evaluation of CH and prostate cancer risk found no association with the risk of prostate cancer.46Larger, non–cancer-specific longitudinal studies have the added benefit of monitoring healthy people with CH over an extended period, better modeling incident disease risk. An analysis of 200,453 individuals enrolled in the UK Biobank study found various associations between CH status and the risk of developing a solid tumor. CH, especially with mutation VAF >10%, was linked with incident lung cancer, kidney cancer, lymphoma, and sarcoma. Notably, certain driver genes also carried their own incident risk associations—47 It was previously thought that cancer therapies such as cytotoxic chemotherapy and radiotherapy were directly causing tMNs via mutagenic effects on HSCs; however, recent evidence has demonstrated that the presence of CH in patients with cancer significantly increases the likelihood of tMN development.47 In fact, the tMN driver mutations in adult patients with cancer are repeatedly found circulating before the receipt of cancer therapy, indicating that cancer therapy is instead providing a selective pressure that favors the growth and ultimately malignant transformation of preexisting CH clones.41 The effects of cancer therapy on CH clones are not uniform, differing on the basis of the role of the mutated driver gene. Studies investigating this phenomenon have recurrently found enrichment of DDR gene-mutant CH in patients after cytotoxic chemotherapy and radiotherapy, with experimental and clinical evidence supporting a fitness advantage for these mutations in the context of therapy.51 This may explain the previously noted enrichment of CH in some groups of patients with solid cancer, with treatments such as radioactive iodine and peptide receptor radionuclides implicated with increased prevalence of CH in thyroid and neuroendocrine tumors, respectively, although the potential roles of CH as a causal driver in tumorigenesis should not be neglected, as seen with lung cancers.53 Beyond the elevated risk of tMN development, cancer therapy–related CH driven by DDR gene mutations has also been implicated in other treatment complications via excessive inflammation, namely chemotherapy-induced cardiomyopathies and nonischemic heart failure.56 Although studies have found that immunotherapies generally have no influence on CH clones in patients with cancer, a case report highlighted a patient with large B-cell lymphoma who experienced the fatal expansion of a TP53-mutant CH clone after anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, demonstrating a need for treatment-specific insights for immunotherapy and clonal dynamics in CH.57 In pediatric cancers, chemotherapy and radiation were found to predict CH presence in survivors, albeit these therapy-related clones remained stable in size during longitudinal follow-up.58CH can play an important role in predicting various clinical and treatment outcomes after a cancer diagnosis Fig . One of 40 The reason for this effect on cancer progression is yet to be determined, with different cancer types and CH-driving mutations each experiencing distinct relationships . With tumor progression being a potential driver of this relationship between CH and mortality in patients with cancer, the lack of literature examining CH in cancer progression and metastasis is surprising. A small study of patients with metastatic renal cell carcinoma found CH in 43% of patients, negatively affecting overall survival.59 Sequencing of metastatic breast cancer specimens found that CH may be dictating bone metastasis, with enrichment for DNMT3A mutations in the samples found in a pattern resembling CH.60 Although these findings are notable, they are nonetheless incidental, and the conclusions that can be drawn from them are limited. A recent analysis of the results of the FIRE-3 trial for metastatic colorectal cancer was able to focus primarily on CH and outcomes in metastatic cancer, finding that 36% of the patients in the trial had CH and that CH was associated with improved survival outcomes—driven specifically by mutations in DNMT3A.61 In an effort to expand on these findings, the influence of CH on survival was evaluated in metastatic esophagogastric and colorectal cancers. This study found that CH was associated with reduced overall survival in the esophagogastric cancers and had a null effect in colorectal cancers—although DNMT3A mutation status was not independently evaluated as in the FIRE-3 trial analysis.62Patients with cancer with >10% VAF CH have been found to experience poorer overall survival than CH-negative patients, even when controlling for factors such as age, sex, and smoking. Strikingly, however, most of this effect was not driven by tMN development, but rather the most common cause of death among patients in the cohort was progression of the primary tumor.63DNMT3A mutation status also identified a distinct subgroup of patients with metastatic non–small-cell lung cancer that saw improved responses to ICI, although it was unclear whether these mutations were found in tumor cells or CH-mutant tumor-infiltrating leukocytes.64 CH in CAR T-cell therapy has also demonstrated predictive value, although results vary between studies. An initial investigation showed that CH was associated with complete response and cytokine release syndrome but not survival in patients with non-Hodgkin lymphoma and multiple myeloma.65 Another study examining CAR T-cell therapy in non-Hodgkin lymphoma found contradicting results, with CH predicting improved overall survival but no change in response rates.66CH may also provide value as a predictive biomarker for cancer immunotherapy treatments. A study conducted on the same MSK cohort described above examined the relationship between CH and outcomes for patients undergoing immune checkpoint inhibitor  therapy, finding that CH was predictive of poorer survival for most cancer types, with the notable exception of colorectal cancer.The complete range of prognostic and predictive implications of CH are yet to be fully realized; however, CH has already made an unplanned entry into precision oncology by appearing as an incidental finding in tumor or liquid biopsy genetic testing for patients. As discussed previously, the common mutational drivers of CH fall on common cancer driver genes, and as such, mutations carried by CH clones may be misinterpreted as tumor mutations or even germline events.68 Although rare, appropriate measures must be taken to avoid the misdiagnosis of germline conditions such as Li-Fraumeni syndrome (LFS), a condition driven by mutations in the common CH gene TP53. A follow-up study for TP53 variants in germline testing was able to successfully distinguish LFS from CH and other somatic expansions using established LFS diagnostic criteria, tracking of variants through family history, and VAF evaluation.69Germline interference is generally rare among patients with cancer—CH has been recorded to interfere in just 0.3% and 0.05% of patients in two large studies of germline genetic testing.40 With tumor-only sequencing, CH mutations may lead to incorrect reporting of tumor variants, potentially leading to recommendations for inappropriate targeted therapies of little to no clinical benefit. In multiple cohorts, CH-associated mutations found in the blood are frequently detected in unpaired tumor sequencing, contributing to the erroneous calling of tumor variants in as many as 5% of patients.71 The contamination of liquid biopsies with CH variants is also problematic, with evaluations of cell-free DNA samples showing a significant proportion of somatic mutations that display features consistent with CH.75 As an incidental finding, CH with high-risk mutations can act as a point of referral for further hematologic consultation, helping to reveal occult hematological malignancies.76 The possibility of CH contaminating or interfering with diagnostic tests warrants consideration from precision oncologists, although knowledge of CH and the utilization of matched blood and/or tissue normal sequencing alongside tumor sequencing can drastically reduce the risk of any adverse consequences for patients.CH becomes slightly harder to differentiate when it is discovered alongside other somatic variants sourced from a patient's tumor. If both tumor and matched blood have been sequenced, this distinction can be informed by variations in VAF of the clone between the two samples, with higher blood VAF indicating a clone of hematopoietic origin.77 Additionally, there is evidence that patients with solid tumors with CH, at least those with TET2 or DNMT3A variants, experience elevated levels of lymphocyte invasion in the TME.78 Given that these mutant immune cells are entering the TME, it is reasonable to predict that they might be disturbing the intricate balance of immunity and eliciting a direct effect on the growth and progression of the tumor.The clinical associations found between CH and cancer are becoming increasingly apparent with each upcoming study, so it is imperative that these are accompanied by basic research to support causal associations and identify potential confounding relationships. By using samples from primary breast tumors, researchers identified that immune cells carrying somatic mutations in CH driver genes were infiltrating the tumor microenvironment (TME).TET2 and PPM1D can drive elevated levels of IL-1β and IL-18 via increased activation of the NLRP3 inflammasome.56 The NLRP3 inflammasome has been shown to play a bidirectional role in the antitumor immune response, with activation of the pathway linked to the progression of several types of cancer such as breast cancer, lung cancer, and lymphoma, although of note, NLRP3 activation has been found to be protective in colorectal cancers.83 IL-6, another commonly elevated inflammatory marker in CH, is linked with not only numerous detrimental processes in tumorigenesis such as tumor cell proliferation and angiogenesis but also antitumorigenic processes such as T-cell trafficking to the tumor site.84 The disruption of common CH driver genes, namely DNMT3A and TET2, can also directly modulate the function of CAR T cells. With the loss of TET2, CAR T cells demonstrated a central memory phenotype that helped enhance the potency of the cells while DNMT3A regularly plays a role in inducing the epigenetic changes that underlie exhaustion, so deletion facilitated an enhanced antitumor response.86 These studies directly reference CH mutations in CAR T cells, although it is yet to be determined how these mutations in normal circulating human T cells can modulate their function.At the cellular level, changes to immune function induced by CH driver mutations have both protumorigenic and antitumorigenic effects. For example, macrophage-specific mutations in TET2 has been most extensively studied in this regard, with one study highlighting a protumorigenic effect of TET2 deletion that operates through increased populations of granulocytic myeloid-derived suppressor cells that deplete CD8+ T cells, driving immunosuppression and tumor growth in models of hepatocellular carcinoma and breast cancer.87 Other models of hematopoietic TET2 depletion have shown varying effects, promoting angiogenesis and tumor progression in lung cancer while reducing tumor burden in melanoma by facilitating a proinflammatory tumor-associated macrophage phenotype that augmented T-cell infiltration.89 Less data are available for other common CH drivers, but similar trends appear. DNMT3A loss of function mutations appeared to drive colitis-associated colon cancer growth and progression, although the exact mechanism has not yet been elucidated.90ASXL1 mutations were found to promote tumorigenesis in a variety of cancer models through disrupted T-cell development and functionality.91 Finally, wild-type p53 in myeloid cells was found to suppress M2 macrophage polarization and tumor invasiveness in an intestinal cancer model, indicating a potential cancer risk with TP53 loss-of-function mutations in CH.92Animal models have also provided evidence for distinct roles of the CH driver genes in tumorigenesis. The findings presented here depict an exciting future for research examining the role of CH in solid tumors. Building on this foundation, there are a number of additional steps that need to be taken to translate these findings into precision oncology practice. Primarily, current knowledge of CH in solid cancers depicts a relationship that is as heterogeneous as cancer itself, and more research will be required to better understand this relationship to determine the contexts where CH is helpful, harmful, both, or neutral for patients with solid tumors. Further analyses of longitudinal population and cancer-specific cohorts will provide a better estimate of the risk of specific cancers and clinical outcomes in relation to CH, hopefully while being able to separate confounding effects due to cancer therapy and shared risk factors between both conditions. Although connected by signs of systemic inflammation and immune dysfunction, the variety of CH driver mutations have demonstrated diverse effects on tumorigenesis at different sites, the most notable of which being the distinct protective effect of CH reported in colorectal cancers. These nuances must be studied further if we ever hope to use CH in cancer therapy—both to develop novel strategies and optimize existing ones, whether that be targeting CH directly, incorporating CH as a prognostic and/or predictive biomarker, or leveraging CH in the TME to our benefit.93 although some novel approaches have been proposed since then. For example, DNMT3A R882 mutations are noted as the most prevalent of all CH variants across many studies, and the herbal extract, oridonin, has been proposed as a promising candidate to suppress both CH and leukemias that are driven by such mutations.94 One novel approach for TET2-mutant CH is the utilization of eltrombopag, a thrombopoietin receptor agonist that can restrict the growth of malignant TET2-mutant clones while favoring the expansion of healthy cells.95 Targeting of TET2-mutant cells has also shown promise with the mutant IDH1/2 metabolite 2-hydroxyglutarate and similarly engineered small molecule inhibitors, as well as XPO1 inhibitors.97 Other advancements include the potential of PARP1 inhibition for antagonizing TET2-mutant CH; however, the risk of hematologic malignancy is elevated in patients undergoing this therapy and thus more research is warranted before this can be applied safely in a clinical setting.98 With the implication of inflammasome activity in TET2-mutant and other forms of CH, clinical trials such as IMPACT are now investigating the value of the anti–IL-1β antibody canakinumab in high-risk CH .Targeting CH directly remains elusive, largely driven by a lack of motivation for clinical trials because of the limited risk-benefit profile that has been proposed by existing research. Potential avenues of treatment have been reviewed previously with relevance to precision oncology by Miller and Steensma,Beyond cancer, developments in our understanding of the biology of CH will also prove beneficial in a variety of clinical settings. For example, the implications of more novel CH drivers such as lymphoid cancer–associated genes and mCAs are not yet fully elucidated in a more general sense, let alone in the context of cancer. Looking toward the future, the influence of CH in the era of precision medicine is expanding rapidly, and patients with solid cancer await the research and development of effective strategies for managing CH in a clinical oncology setting.