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Prenatal cfDNA Sequencing and Incidental Detection of Maternal Cancer. BACKGROUND: Cell-free DNA (cfDNA) sequence analysis to screen for fetal aneuploidy can incidentally detect maternal cancer. Additional data are needed to identify DNA-sequencing patterns and other biomarkers that can identify pregnant persons who are most likely to have cancer and to determine the best approach for follow-up. METHODS: In this ongoing study we performed cancer screening in pregnant or postpartum persons who did not perceive signs or symptoms of cancer but received unusual clinical cfDNA-sequencing results or results that were nonreportable (i.e., the fetal aneuploidy status could not be assessed) from one of 12 different commercial laboratories in North America. We used a uniform cancer-screening protocol including rapid whole-body magnetic resonance imaging (MRI), laboratory tests, and standardized cfDNA sequencing for research purposes with the use of a genomewide platform. The primary outcome was the presence of cancer in participants after the initial cancer-screening evaluation. Secondary analyses included test performance. RESULTS: Cancer was present in 52 of the 107 participants in the initial cohort (48.6%). The sensitivity and specificity of whole-body MRI in detecting occult cancer were 98.0% and 88.5%, respectively. Physical examination and laboratory tests were of limited use in identifying participants with cancer. Research sequencing showed that 49 participants had a combination of copy-number gains and losses across multiple (≥3) chromosomes; cancer was present in 47 of the participants (95.9%) with this sequencing pattern. Sequencing patterns of cfDNA in which there were only chromosomal gains (multiple trisomies) or only chromosomal losses (one or more monosomies) were found in participants with nonmalignant conditions, such as fibroids. CONCLUSIONS: In this study, 48.6% of participants who received unusual or nonreportable clinical cfDNA-sequencing results had an occult cancer. Further study of DNA-sequencing patterns that are suggestive of occult cancer during prenatal screening is warranted. (Funded by the NIH Intramural Research Programs; ClinicalTrials.gov number, NCT04049604.).

Sequencing of circulating cell-free DNA (cfDNA) in maternal plasma has had a substantial impact on prenatal screening for fetal aneuploidy. Owing to its superior accuracy compared to serum biochemical and nuchal translucency screening,1,2 as of October 2020, cfDNA sequencing is now routinely offered to all pregnant women.3 It has resulted in a 50-70% global reduction of invasive diagnostic procedures, such as amniocentesis, demonstrating its clinical utility.4 As with many new genomic technologies, the potential for unexpected results has been realized. Retrospective studies from large commercial or national laboratories have reported an association between unusual sequencing results, such as those with multiple aneuploidies or an autosomal monosomy, and maternal malignancies.5–10 During a typical pregnancy, circulating cfDNA derives from the placenta (~10%) and the maternal hematopoietic system (~90%). The sequenced cfDNA in the test sample is compared to a reference sample, and gains or losses across the genome are used in bioinformatics algorithms to establish ratios. If a maternal tumor is present, it can also shed cfDNA into the circulation, distorting the expected ratios for a euploid or aneuploid fetus. In the United States (US), this most often triggers a nonreportable sequencing result since the fetal aneuploidy status cannot be assessed.

matics algorithms to establish ratios. If a maternal tumor is present, it can also shed cfDNA into the circulation, distorting the expected ratios for a euploid or aneuploid fetus. In the United States (US), this most often triggers a nonreportable sequencing result since the fetal aneuploidy status cannot be assessed. Although various approaches have been suggested,9,11–13 there is currently insufficient evidence to inform the subsequent management of pregnant women who receive nonreportable or unusual sequencing results. Additional data are needed to better understand the ability of cfDNA sequencing to detect maternal malignancies and to determine the optimal evaluation.3,14 To fill this knowledge gap, we launched the Incidental Detection of matErnal Neoplasia Through non-Invasive cell-Free DNA analYsis (IDENTIFY) study. The goals of the study were to identify DNA sequencing patterns and other biomarkers that could distinguish the subset of asymptomatic women who are likely to have malignancy and determine the best approach for diagnostic work-up of pregnant women who receive these results.

IDENTIFY is an ongoing natural history study being conducted at the National Institutes of Health (NIH) in Bethesda, Maryland. We are enrolling women who underwent cfDNA sequencing during their routine obstetrical care and received unexpected results for which maternal malignancy was included in the differential diagnosis. Here we present results from the initial cohort of IDENTIFY participants. Participants were informed of the option to participate in the IDENTIFY study by the sequencing laboratory or their health care provider. The NIH institutional review board approved the study protocol. All participants provided written informed consent. The study was designed by the second and last authors. All authors were involved in the collection, analysis, and/or interpretation of the data. The first author wrote the first draft of the manuscript, which was then revised by the last author. All authors have reviewed the manuscript, vouch for the completeness and accuracy of the data, and made the decision to submit the manuscript for publication, according to the protocol (available at NEJM.org).

the data. The first author wrote the first draft of the manuscript, which was then revised by the last author. All authors have reviewed the manuscript, vouch for the completeness and accuracy of the data, and made the decision to submit the manuscript for publication, according to the protocol (available at NEJM.org). Eligible participants were at least 18 years of age, pregnant or up to two years postpartum, and had received sequencing results from one of 12 commercial laboratories (Table S1) that were either (1) abnormal and inconsistent with a viable fetus on sonogram, (2) abnormal and discordant with the fetal karyotype or chromosome microarray analysis, or (3) nonreportable. Our knowledge of the initial cfDNA-sequencing result was limited to what was documented on the written laboratory report (Figure S1) and/or communicated by the referring provider. These eligibility criteria reflect the current clinical landscape in the US, where multiple commercial laboratories offer either targeted or genome-wide prenatal cfDNA sequencing and have variable reporting practices.12 Patients were ineligible if their sequencing results were nonreportable due to insufficient fetal fraction or other technical or sample-related issues. The final protocol specified enrollment of 120 women without known cancer, based on operational rather than scientific considerations.

e variable reporting practices.12 Patients were ineligible if their sequencing results were nonreportable due to insufficient fetal fraction or other technical or sample-related issues. The final protocol specified enrollment of 120 women without known cancer, based on operational rather than scientific considerations. The cancer screening protocol included rapid whole-body magnetic resonance imaging (WB-MRI), blood tests, serum tumor markers, fecal occult blood test, family and medical history intake, and physical examination with oncologic symptom review. Participants who were not up to date with their cervical cancer screening underwent a pap smear with human papillomavirus testing. Blood tests included complete blood count with differential, complete metabolic panel with liver and renal function tests, and serum vitamin B12 levels. The serum tumor markers analyzed7 were CA-125 (cancer antigen-125), CA 15-3, CA 27.29, CA 19-9, and CEA (carcinoembryonic antigen). Various approaches to imaging have been suggested when cfDNA-sequencing results raise concern for maternal malignancy.9,15,16 We selected WB-MRI because of its safety during pregnancy and its proven effectiveness at detecting occult malignancies in other high-risk patient populations.17 Non-pregnant participants were studied with and without gadolinium contrast, whereas in pregnant participants no contrast was used (Table S2).

ignancy.9,15,16 We selected WB-MRI because of its safety during pregnancy and its proven effectiveness at detecting occult malignancies in other high-risk patient populations.17 Non-pregnant participants were studied with and without gadolinium contrast, whereas in pregnant participants no contrast was used (Table S2). All participants met with a medical oncologist. If cancer was detected, the oncologist provided referral for biopsy and subsequent management in the participant’s locale. If cancer was not detected, placental biopsies were collected at the time of delivery to evaluate for confined placental mosaicism (Supplementary Methods). Research cfDNA sequencing was performed on all participants as a fee-for-service by a CAP/CLIA certified contract laboratory. Peripheral blood (10 mL) was collected in Streck® tubes (Omaha, NE), subjected to plasma separation and DNA extraction, library preparation, and genome-wide massively parallel sequencing using previously described methods.18 The primary outcome was the presence of cancer in each participant following the initial clinical evaluation.

Research cfDNA sequencing was performed on all participants as a fee-for-service by a CAP/CLIA certified contract laboratory. Peripheral blood (10 mL) was collected in Streck® tubes (Omaha, NE), subjected to plasma separation and DNA extraction, library preparation, and genome-wide massively parallel sequencing using previously described methods.18 The primary outcome was the presence of cancer in each participant following the initial clinical evaluation. To explore whether the sequencing data could distinguish women with malignancies from those with non-malignant findings, two investigators (AET and DWB) performed an exploratory analysis of the 50-kb sequencing traces. All 50-kb traces were blindly inspected and visually stratified to establish different groups of sequencing patterns. Any differences in stratification were resolved through discussion and further review of the chromosome ideogram data. Once the groups were established, the clinical outcome information was reviewed (Figure S2). To evaluate the test performance of the standardized cancer screening protocol, we calculated the sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals and estimated the area under the nonparametric receiver-operator-characteristic (ROC) curve with 95% confidence intervals using the R package caret.19

Eligible participants were at least 18 years of age, pregnant or up to two years postpartum, and had received sequencing results from one of 12 commercial laboratories (Table S1) that were either (1) abnormal and inconsistent with a viable fetus on sonogram, (2) abnormal and discordant with the fetal karyotype or chromosome microarray analysis, or (3) nonreportable. Our knowledge of the initial cfDNA-sequencing result was limited to what was documented on the written laboratory report (Figure S1) and/or communicated by the referring provider. These eligibility criteria reflect the current clinical landscape in the US, where multiple commercial laboratories offer either targeted or genome-wide prenatal cfDNA sequencing and have variable reporting practices.12 Patients were ineligible if their sequencing results were nonreportable due to insufficient fetal fraction or other technical or sample-related issues. The final protocol specified enrollment of 120 women without known cancer, based on operational rather than scientific considerations.

The cancer screening protocol included rapid whole-body magnetic resonance imaging (WB-MRI), blood tests, serum tumor markers, fecal occult blood test, family and medical history intake, and physical examination with oncologic symptom review. Participants who were not up to date with their cervical cancer screening underwent a pap smear with human papillomavirus testing. Blood tests included complete blood count with differential, complete metabolic panel with liver and renal function tests, and serum vitamin B12 levels. The serum tumor markers analyzed7 were CA-125 (cancer antigen-125), CA 15-3, CA 27.29, CA 19-9, and CEA (carcinoembryonic antigen). Various approaches to imaging have been suggested when cfDNA-sequencing results raise concern for maternal malignancy.9,15,16 We selected WB-MRI because of its safety during pregnancy and its proven effectiveness at detecting occult malignancies in other high-risk patient populations.17 Non-pregnant participants were studied with and without gadolinium contrast, whereas in pregnant participants no contrast was used (Table S2). All participants met with a medical oncologist. If cancer was detected, the oncologist provided referral for biopsy and subsequent management in the participant’s locale. If cancer was not detected, placental biopsies were collected at the time of delivery to evaluate for confined placental mosaicism (Supplementary Methods).

Research cfDNA sequencing was performed on all participants as a fee-for-service by a CAP/CLIA certified contract laboratory. Peripheral blood (10 mL) was collected in Streck® tubes (Omaha, NE), subjected to plasma separation and DNA extraction, library preparation, and genome-wide massively parallel sequencing using previously described methods.18

To explore whether the sequencing data could distinguish women with malignancies from those with non-malignant findings, two investigators (AET and DWB) performed an exploratory analysis of the 50-kb sequencing traces. All 50-kb traces were blindly inspected and visually stratified to establish different groups of sequencing patterns. Any differences in stratification were resolved through discussion and further review of the chromosome ideogram data. Once the groups were established, the clinical outcome information was reviewed (Figure S2). To evaluate the test performance of the standardized cancer screening protocol, we calculated the sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals and estimated the area under the nonparametric receiver-operator-characteristic (ROC) curve with 95% confidence intervals using the R package caret.19

From December 23, 2019, to December 4, 2023, 117 participants without a known cancer diagnosis were enrolled, and 107/117 had complete data available for analysis. Ten women were excluded because they did not undergo cancer screening or had incomplete clinical data (Figure 1). Participant characteristics are presented in Table 1. The mean maternal age was 32.7 years (range, 19-53 years). Eighty-nine participants were pregnant at the time of their cancer screening, and the mean gestational age was 22.2 weeks (range, 13.3-36 weeks). Most participants (90/107) were referred with nonreportable or abnormal results from genome-wide cfDNA sequencing (Figure S1).

ternal age was 32.7 years (range, 19-53 years). Eighty-nine participants were pregnant at the time of their cancer screening, and the mean gestational age was 22.2 weeks (range, 13.3-36 weeks). Most participants (90/107) were referred with nonreportable or abnormal results from genome-wide cfDNA sequencing (Figure S1). Cancer was present in 52/107 participants (48.6%) (Figure 1). Fifty-one participants underwent biopsies to confirm their diagnoses (Table S5). Lymphoma was the most common diagnosis (31/52, 59.6%), followed by colorectal cancer (9/52, 17.3%) and breast cancer (4/52, 7.7%). Additional malignancies included two cases of cholangiocarcinoma and one case each of adrenocortical carcinoma, non-small cell lung cancer, pancreatic cancer, Ewing sarcoma, and renal carcinoma. Of the 31 confirmed lymphoma cases, 20 were Hodgkin lymphoma and 11 were non-Hodgkin lymphoma. Of the 20 solid tumor cases, two participants had stage 1 disease, five participants had stage 2 or 3 disease, and 13 participants had stage 4 disease. Thirteen of 20 participants were eligible for potentially curative treatment, including five participants with colon cancer and one with Ewing sarcoma who had metastases to a single area (Figure S3). Of the participants with cancer, 29/52 (55.8%) were asymptomatic and 13/52 (25%) had symptoms that were ascribed to pregnancy or other etiologies, such as epigastric pain attributed to reflux in a patient with pancreatic cancer. In 10/52 participants (19.2%), clinical symptoms were either not recognized by the patient, or when worked up, revealed unconcerning results (Table S3).

tic and 13/52 (25%) had symptoms that were ascribed to pregnancy or other etiologies, such as epigastric pain attributed to reflux in a patient with pancreatic cancer. In 10/52 participants (19.2%), clinical symptoms were either not recognized by the patient, or when worked up, revealed unconcerning results (Table S3). The remaining 55/107 (51.4%) participants were not diagnosed with cancer. In 30/107 (28.0%) cases, there was an explanation that did not involve cancer for the initial clinical sequencing result, including fibroids (17/107, 15.9%), confined placental mosaicism (8/107, 7.5%), a fetal finding (3/107, 2.8%), or clonal hematopoiesis (2/107, 1.9%). In 25/107 (23.4%) cases, the initial clinical sequencing result was unexplained, and in 10 of these participants, there is ongoing concern for malignancy (Figure 1). Participants will be followed for up to five years.

acental mosaicism (8/107, 7.5%), a fetal finding (3/107, 2.8%), or clonal hematopoiesis (2/107, 1.9%). In 25/107 (23.4%) cases, the initial clinical sequencing result was unexplained, and in 10 of these participants, there is ongoing concern for malignancy (Figure 1). Participants will be followed for up to five years. Research cfDNA sequencing was performed in all participants; results were unavailable in two participants (one with cancer, one with fibroids) because their samples failed to amplify. Our blinded inspection of the sequencing data revealed there were six patterns represented: normal (no copy number gain/loss present) (n=28); chromosomal gains and losses (sub-chromosomal and/or whole chromosome copy number gains and losses across multiple (≥3) chromosomes) (n=49); chromosomal gains only (one or more duplications or whole chromosome trisomies) (n=11); chromosomal losses only (one or more deletions or whole chromosome monosomies) (n=4); maternal copy number variants (n=2); or borderline abnormal (no clear copy number abnormalities but borderline z-scores across multiple chromosomes) (n=11) (Figure 2).

y (one or more duplications or whole chromosome trisomies) (n=11); chromosomal losses only (one or more deletions or whole chromosome monosomies) (n=4); maternal copy number variants (n=2); or borderline abnormal (no clear copy number abnormalities but borderline z-scores across multiple chromosomes) (n=11) (Figure 2). In 49 samples, the research cfDNA-sequencing results showed chromosomal gains and losses (Figure 2B). In most cases, the abnormalities were genome-wide and profound, but in some, the abnormalities were subtle or affected only a few autosomes (Figure S4). Of the 49 samples, 47 (95.9%) were in women with cancer. There is ongoing concern for malignancy in the other two participants. There were four participants who had cancer but did not have the suspicious pattern of chromosomal gains and losses: two participants (one with stage 1 breast cancer and one with recurrent Hodgkin lymphoma) had borderline abnormal results; two participants (one with stage 1 primary mediastinal large B-cell lymphoma and one with stage 1 renal carcinoma) had normal results (Figure S2).

uspicious pattern of chromosomal gains and losses: two participants (one with stage 1 breast cancer and one with recurrent Hodgkin lymphoma) had borderline abnormal results; two participants (one with stage 1 primary mediastinal large B-cell lymphoma and one with stage 1 renal carcinoma) had normal results (Figure S2). The other research cfDNA-sequencing patterns (Figure 2, panels C–F) had a biologic source other than cancer. In 11 samples there were only chromosomal gains: 10 showed whole chromosome trisomies of two to four autosomes, and one sample showed trisomy 13. These cases were explained by the fetus (n=3) and/or placenta (n=8) (Figure S2, Table S4). In four samples, there was one or more sub-chromosomal and/or whole-chromosome monosomy present; these occurred in women with fibroids detected by WB-MRI (Figure S2). Eleven participants with fibroids were referred with multiple monosomies (n=5) or nonreportable results (n=6) on clinical sequencing, but we detected no abnormalities in these women on research sequencing. Three participants with fibroids also had cancer; all three had a combination of chromosomal gains and losses on research sequencing (Table S5).

ids were referred with multiple monosomies (n=5) or nonreportable results (n=6) on clinical sequencing, but we detected no abnormalities in these women on research sequencing. Three participants with fibroids also had cancer; all three had a combination of chromosomal gains and losses on research sequencing (Table S5). Two samples showed a maternal copy number variant that was determined to reflect clonal hematopoiesis (Supplementary Methods). In 11 samples, the sequencing results were borderline abnormal; two of these were in women with cancer, and one was in a participant with fibroids. The other eight cases were unexplained, and these participants are being followed. Of the 15 participants with normal research cfDNA and cancer screening results, seven were postpartum; the abnormalities detected on the original clinical sequencing may have been confined to the fetus or placenta, and therefore were not present in the postpartum blood sample.

and these participants are being followed. Of the 15 participants with normal research cfDNA and cancer screening results, seven were postpartum; the abnormalities detected on the original clinical sequencing may have been confined to the fetus or placenta, and therefore were not present in the postpartum blood sample. Results of the test performance calculations are shown in Table 2. Rapid WB-MRI showed suspicion for malignancy in 48/101 participants, all of whom were confirmed to have cancer (Figure 3, Table S6). The single false negative result was in a participant who was diagnosed with stage 1 breast cancer three months after her evaluation at NIH. There were six indeterminate WB-MRI findings that required follow-up: two liver lesions that could not be fully characterized without contrast, one breast lesion, one thyroid lesion, one axillary nodule, and one lung lesion. Through subsequent imaging, all were determined to be benign. The AUC for WB-MRI was 93.2% (95% CI 88.4 to 98.0) (Table 2).

WB-MRI findings that required follow-up: two liver lesions that could not be fully characterized without contrast, one breast lesion, one thyroid lesion, one axillary nodule, and one lung lesion. Through subsequent imaging, all were determined to be benign. The AUC for WB-MRI was 93.2% (95% CI 88.4 to 98.0) (Table 2). Pertinent medical history and clinical details are presented in Table S7 and Figure S5. Physical examination was performed in all participants and was abnormal in 9/52 participants with cancer, a sensitivity of 17.3% (95% CI 8.2 to 30.3). Screening blood tests did not show concern for malignancy in any participant. Serum tumor marker results were available in 103 participants. Of the 52 participants with cancer, 34 had at least one abnormal serum tumor marker (true positives), a sensitivity of 68.0% (95% CI 53.3 to 80.5). Fourteen participants without cancer had abnormal serum tumor marker results, a false positive rate of 26.4% (95% CI 15.3 to 46.7). Assessing only CA 19-9, CA 15-3, and CEA, which are not typically affected by pregnancy,20 did not improve accuracy. Of the nine participants with colorectal cancer, eight underwent fecal occult blood testing, and only four had abnormal results (Table 2).

Cancer was present in 52/107 participants (48.6%) (Figure 1). Fifty-one participants underwent biopsies to confirm their diagnoses (Table S5). Lymphoma was the most common diagnosis (31/52, 59.6%), followed by colorectal cancer (9/52, 17.3%) and breast cancer (4/52, 7.7%). Additional malignancies included two cases of cholangiocarcinoma and one case each of adrenocortical carcinoma, non-small cell lung cancer, pancreatic cancer, Ewing sarcoma, and renal carcinoma. Of the 31 confirmed lymphoma cases, 20 were Hodgkin lymphoma and 11 were non-Hodgkin lymphoma. Of the 20 solid tumor cases, two participants had stage 1 disease, five participants had stage 2 or 3 disease, and 13 participants had stage 4 disease. Thirteen of 20 participants were eligible for potentially curative treatment, including five participants with colon cancer and one with Ewing sarcoma who had metastases to a single area (Figure S3). Of the participants with cancer, 29/52 (55.8%) were asymptomatic and 13/52 (25%) had symptoms that were ascribed to pregnancy or other etiologies, such as epigastric pain attributed to reflux in a patient with pancreatic cancer. In 10/52 participants (19.2%), clinical symptoms were either not recognized by the patient, or when worked up, revealed unconcerning results (Table S3).

Research cfDNA sequencing was performed in all participants; results were unavailable in two participants (one with cancer, one with fibroids) because their samples failed to amplify. Our blinded inspection of the sequencing data revealed there were six patterns represented: normal (no copy number gain/loss present) (n=28); chromosomal gains and losses (sub-chromosomal and/or whole chromosome copy number gains and losses across multiple (≥3) chromosomes) (n=49); chromosomal gains only (one or more duplications or whole chromosome trisomies) (n=11); chromosomal losses only (one or more deletions or whole chromosome monosomies) (n=4); maternal copy number variants (n=2); or borderline abnormal (no clear copy number abnormalities but borderline z-scores across multiple chromosomes) (n=11) (Figure 2). In 49 samples, the research cfDNA-sequencing results showed chromosomal gains and losses (Figure 2B). In most cases, the abnormalities were genome-wide and profound, but in some, the abnormalities were subtle or affected only a few autosomes (Figure S4). Of the 49 samples, 47 (95.9%) were in women with cancer. There is ongoing concern for malignancy in the other two participants. There were four participants who had cancer but did not have the suspicious pattern of chromosomal gains and losses: two participants (one with stage 1 breast cancer and one with recurrent Hodgkin lymphoma) had borderline abnormal results; two participants (one with stage 1 primary mediastinal large B-cell lymphoma and one with stage 1 renal carcinoma) had normal results (Figure S2).

Results of the test performance calculations are shown in Table 2. Rapid WB-MRI showed suspicion for malignancy in 48/101 participants, all of whom were confirmed to have cancer (Figure 3, Table S6). The single false negative result was in a participant who was diagnosed with stage 1 breast cancer three months after her evaluation at NIH. There were six indeterminate WB-MRI findings that required follow-up: two liver lesions that could not be fully characterized without contrast, one breast lesion, one thyroid lesion, one axillary nodule, and one lung lesion. Through subsequent imaging, all were determined to be benign. The AUC for WB-MRI was 93.2% (95% CI 88.4 to 98.0) (Table 2). Pertinent medical history and clinical details are presented in Table S7 and Figure S5. Physical examination was performed in all participants and was abnormal in 9/52 participants with cancer, a sensitivity of 17.3% (95% CI 8.2 to 30.3). Screening blood tests did not show concern for malignancy in any participant. Serum tumor marker results were available in 103 participants. Of the 52 participants with cancer, 34 had at least one abnormal serum tumor marker (true positives), a sensitivity of 68.0% (95% CI 53.3 to 80.5). Fourteen participants without cancer had abnormal serum tumor marker results, a false positive rate of 26.4% (95% CI 15.3 to 46.7). Assessing only CA 19-9, CA 15-3, and CEA, which are not typically affected by pregnancy,20 did not improve accuracy. Of the nine participants with colorectal cancer, eight underwent fecal occult blood testing, and only four had abnormal results (Table 2).

Using a standardized sequencing and cancer screening protocol, we detected cancer in 48.6% of women who initially received nonreportable or unusual cfDNA-sequencing results from 12 commercial laboratories in North America. All participants were referred with concern for malignancy. Our cohort is not representative of the general pregnant population (Table S8). The US differs from other countries in that clinical laboratories use different sequencing technologies, proprietary bioinformatics algorithms, and test-reporting practices. This is confusing for obstetric providers and poses challenges to the identification of women at highest risk for cancer.21 Our results support WB-MRI and the further investigation of certain cfDNA-sequencing patterns in the evaluation of cancer in persons who receive nonreportable or unusual cfDNA-sequencing results.

This is confusing for obstetric providers and poses challenges to the identification of women at highest risk for cancer.21 Our results support WB-MRI and the further investigation of certain cfDNA-sequencing patterns in the evaluation of cancer in persons who receive nonreportable or unusual cfDNA-sequencing results. In the research sequencing results, the combination of sub-chromosomal and/or whole chromosome copy number gains and losses across multiple chromosomes was observed in 47/51 (92.2%) participants with cancer and available sequencing data; patients with this sequencing pattern appear to have the highest risk of cancer and should be identified on the written laboratory report so that timely cancer screening can be pursued. Other cfDNA-sequencing patterns appeared to not be associated with malignancy. For example, cancer was not present in participants with multiple trisomies or one or more monosomies. Although prior studies have suggested that multiple aneuploidies or an autosomal monosomy is suggestive of cancer, we found that multiple whole-chromosome trisomies, commonly double aneuploidies, were more likely to be explained by anomalies in the fetus and/or placenta, and sub-chromosomal and/or whole chromosome monosomies to be explained by uterine fibroids.

multiple aneuploidies or an autosomal monosomy is suggestive of cancer, we found that multiple whole-chromosome trisomies, commonly double aneuploidies, were more likely to be explained by anomalies in the fetus and/or placenta, and sub-chromosomal and/or whole chromosome monosomies to be explained by uterine fibroids. Although fibroids are a well-documented etiology for nonreportable or discordant cfDNA-sequencing results,6,22 their presence does not preclude a co-existing malignancy. In this study, the three participants with fibroids and cancer had research cfDNA results showing multiple copy-number gains and losses, whereas the 16 women with only fibroids had a different pattern (Table S5). The DNA sample from one woman with multiple sub-serosal fibroids failed to amplify (Table S5). Further work on whether and to what extent sequencing patterns can prioritize patients with both fibroids and occult cancer for WB-MRI is warranted.

whereas the 16 women with only fibroids had a different pattern (Table S5). The DNA sample from one woman with multiple sub-serosal fibroids failed to amplify (Table S5). Further work on whether and to what extent sequencing patterns can prioritize patients with both fibroids and occult cancer for WB-MRI is warranted. In some countries other than the US, whole-body imaging is routinely performed when cfDNA-sequencing results suggest maternal malignancies.9,15 Current practice in the US is to consider imaging on a case-by-case basis depending on the patient’s medical and family histories, results of physical examination and laboratory tests, and, ultimately, insurance coverage.3,12 Suggested work-ups in the literature have prioritized targeted imaging, such as chest radiographs, over whole body approaches.11,12 We found that patient history, self-reported symptoms, physical examination, and laboratory tests had limited utility in identifying which patients had cancer or its location. A lack of overt symptoms and/or pregnancy are not reasons to delay imaging. WB-MRI was safe, efficient, and the most effective method for detecting cancer. The false positive rate of WB-MRI screening was 11.5% (95% CI, 4.4 to 23.4), considerably lower than that reported in other high-risk patient populations screened with WB-MRI.23 The effectiveness of cfDNA sequencing in identifying patients with an existing cancer likely explains this lower false positive rate.

cancer. The false positive rate of WB-MRI screening was 11.5% (95% CI, 4.4 to 23.4), considerably lower than that reported in other high-risk patient populations screened with WB-MRI.23 The effectiveness of cfDNA sequencing in identifying patients with an existing cancer likely explains this lower false positive rate. The median time between participants’ initial clinical sequencing and their cancer screening evaluation was 9.6 weeks (range, 2 to 114.6 weeks). Factors contributing to delays in referral to the IDENTIFY study included confusion about the significance of the initial sequencing results, providers ordering repeat sequencing and/or pursuing fetal diagnostic testing prior to maternal follow-up, and participants’ ambivalence about pursuing cancer screening.24 The importance of prompt cancer screening in patients who receive cfDNA-sequencing results suggestive of maternal malignancy is highlighted by the five women in this study who had stage 2 or 3 solid tumors identified, and the six women with stage 4 cancers with limited metastatic involvement that were eligible for potentially curative treatment.

ncer screening in patients who receive cfDNA-sequencing results suggestive of maternal malignancy is highlighted by the five women in this study who had stage 2 or 3 solid tumors identified, and the six women with stage 4 cancers with limited metastatic involvement that were eligible for potentially curative treatment. Strengths of our study include that participants had no known diagnosis at the time of study entry, a uniform sequencing and cancer screening protocol was performed, and eligibility criteria reflected current US clinical practice and the challenges clinicians face in identifying patients at risk for malignancy.21 Study limitations include subjective grouping of the cfDNA-sequencing patterns as an exploratory analysis, lack of pre-specification of hypotheses or analyses, lack of statistical justification of the sample size, our inability to directly compare the research and original clinical sequencing results, the need to exclude some participants due to incomplete clinical data, and variable follow-up periods for participants with ongoing concerns for malignancy. We may have underestimated the proportion of participants with cancer. And finally, the sequencing patterns described here are novel and require prospective validation in an independent population adequately sized to estimate diagnostic accuracy.