Discordant noninvasive prenatal testing results in a patient subsequently diagnosed with metastatic disease
Funding sources: None.
Conflicts of interest: Patricia Devers is an employee of Verinata Health, Inc.
What's already known about this topic?
- Noninvasive prenatal testing for detection of trisomies 21, 18, and 13 is clinically available and is reported to have a false positive rate of 1% or less
- This technology utilizes massively parallel shotgun sequencing of cell-free DNA, of maternal and placental origin, present in maternal plasma
What does this study add?
- Unexplained abnormal noninvasive prenatal testing results should prompt consideration of a maternal source of the abnormal cell-free DNA, such as malignancy
Noninvasive prenatal testing (NIPT) for fetal aneuploidy is clinically available for women at an increased risk of fetal aneuploidy. At the time of the present case report, detection of aneuploidy through NIPT was only available for Down syndrome, trisomy 18, and trisomy 13. NIPT has been quoted to have a detection rate for trisomies 21 and 18 of approximately 99%, and a detection rate for trisomy 13 of between 79 to 92%. The false positive rate for all autosomal aneuploidies is reported to be 1% or less.[1, 2]
Noninvasive prenatal testing utilizes massively parallel shotgun sequencing of cell-free DNA, of maternal and placental origin, present in maternal plasma. With massively parallel shotgun sequencing, 36 base pair fragments of the cell-free DNA obtained from maternal plasma are sequenced, aligned, and uniquely mapped to sites from a reference human genome.[3-5] Although multiple statistical measures are utilized by competing laboratories, the laboratory utilized in this case report (Verinata Health, Inc., Redwood City, CA) then established a normalized chromosome value (NCV) for each chromosome of interest. An NCV > 4.0 was required to classify a chromosome as affected (i.e. trisomic for that chromosome). An NCV < 2.5 was required to classify a chromosome as unaffected. Samples with an NCV between 2.5 and 4.0 were called ‘unclassified’.[1, 5]
Noninvasive prenatal testing relies on the presence of cell-free fetal DNA in maternal plasma. Over 99% of the cell-free fetal DNA in maternal plasma is thought to be placentally derived; as such, it is theorized that an NIPT result that is discordant with prenatal or postnatal fetal karyotype could be indicative of confined placental mosaicism. Alternatively, as circulating cell-free DNA is a combination of maternal and placental DNA, an abnormal maternal chromosome complement may also lead to a discordant NIPT result.
In the current report, we present a case of maternal metastatic disease as causative of a very abnormal NIPT result.
A 37-year old gravida 2 para 1 presented to High Risk Clinic regarding advanced maternal age at approximately 13 weeks' gestational age. Her prior medical, obstetrical, and gynecologic history was negative. She had a normal Pap smear noted 1 month prior. After appropriate genetic counseling, the patient elected to proceed with NIPT. NIPT results were suggestive of fetal aneuploidy for chromosome 13 and chromosome 18. Of note, an NCV of +10.9 was detected for chromosome 13, consistent with trisomy 13. An NCV of −10.7 was detected for chromosome 18, consistent with monosomy 18. The test was repeated by the laboratory on a second aliquot of maternal plasma from the same blood draw, with similar results (NCV 13: +8.8; NCV 18: −10.2) obtained. At that time, the patient was counseled on the high probability of an abnormal fetal karyotype. The patient elected to proceed with amniocentesis for determination of the fetal karyotype.
Final results from the patient's amniocentesis revealed a karyotype and direct interphase aneuploidy fluorescence in situ hybridization (FISH) results consistent with those of a normal male fetus (46,XY). A whole genome oligonucleotide microarray for detection of submicroscopic genomic imbalances was also reported as normal. We discussed the possibility of lab error, faulty tube, or sample switch on the initial serum specimen with the patient. A second maternal serum specimen assayed at approximately 17 weeks' gestational age to rule out these possible etiologies for the patient's abnormal NIPT result also demonstrated fetal aneuploidy for both chromosomes 13 and 18 (NCV 13: +17.6; NCV 18: −20.8).
A fetal anatomic ultrasound survey demonstrated normal fetal anatomy and growth with normal appearing placenta at 19 weeks gestational age. As confined placental mosaicism was a suspected etiology for the abnormal NIPT, serial ultrasound assessment of fetal growth was performed, with normal interval growth at 31 weeks gestational age. In the third trimester, the patient was admitted for an episode of vaginal bleeding, and a friable mass was noted on the cervix. The clinical diagnosis was that of a prolapsing leiomyoma. After consultation with Gynecologic Oncology, biopsy and removal were deferred until after delivery.
Prior to delivery, we received consent from the patient to obtain and biopsy the placenta after delivery to screen for confined placental mosaicism. Of note, at 36 weeks' gestational age, the patient also elected to proceed with collection of a third maternal serum specimen. This specimen was sent to a separate laboratory (Sequenom CMM) for repeat NIPT. Results of the third NIPT were ‘non-reportable,’ significant for reduced chromosome material for both chromosomes 13 and 18. Although an official report was not issued for this pro bono sample, Sequenom representatives stated that the chromosome levels measured in maternal serum were well below the established range for a diploid fetus, possibly consistent with monosomy for both chromosomes 13 and 18.
The patient presented in spontaneous labor at term and delivered a male infant with Apgars 8/9 and weight of 3040 g. The neonate was examined after birth and appeared normal with no dysmorphic features. The placenta was sent to pathology and to cytogenetics to rule out confined placental mosaicism as causative of the abnormal NIPT results. The aforementioned cervical mass was not visible at the time of delivery.
Following delivery, the patient complained of persistent and worsening pelvic pain. During the patient's evaluation, anteroposterior pelvic radiograph was significant for minimally displaced pathologic fractures involving the right superior and inferior pubic ramus. The inferior pubic ramus was noted to have an irregular mottled appearance, consistent with an infiltrative malignancy. MRI demonstrated increased signal in the right superior and inferior pubic rami with multiple lesions extending up to the proximity of the abdominal wall. A CT-guided biopsy of these lesions was performed. Fine needle aspiration cytopathology results of this biopsy were diagnostic for malignancy and classified as a metastatic neuroendocrine carcinoma (unknown site of origin).
At 2 weeks postpartum, the cervix appeared normal without mass or lesion. However, a separate mass was noted on the anterior vaginal wall. Vaginal biopsy performed as follow-up was diagnostic for a high-grade neuroendocrine carcinoma, consistent with a small cell carcinoma. The previous fine needle aspiration from the right hip was reviewed and was found to be identical to the cells retrieved during biopsy. Final diagnosis is consistent with metastatic disease of small cell carcinoma of vaginal origin.
Evaluation of 100 cells from a paraffin-embedded biopsy of the patient's tumor demonstrated evidence of aneuploidy in 80% of the examined cells; an increased number of chromosome 13 FISH signals (2–8 signals/cell) relative to chromosome 18 signals (1–3 signals/cell) were observed. These findings were consistent with the aneuploidy classification results from the NIPT testing. Postnatal karyotyping of 10 to 12 metaphase cells and interphase FISH analysis of 100 cells from each of the eight different placental biopsy specimens revealed no clear evidence of a chromosome 13 and/or chromosome 18 aneuploidy. These data suggest that the abnormal NIPT result obtained for this patient likely reflected the presence of her tumor rather than an aneuploid fetus or confined placental mosaicism.
The preceding case represents a discordant NIPT result likely arising because of the presence of maternal malignancy. We theorize that the abnormal result, consistent with aneuploidy for both chromosome 13 and chromosome 18, was driven by chromosome abnormalities present in circulating cell-free DNA arising from maternal cancer cells rather than being of fetal origin.
Maternal illness as the cause of an abnormal maternal serum screening result during pregnancy has been previously reported. Gonsulin et al. report a case of an abnormal maternal serum alpha-fetoprotein level in a woman at 16 weeks' gestation. Follow-up evaluations, including ultrasound examination and fetal chromosome analysis, revealed no abnormalities. In this case report, the patient was subsequently diagnosed with metastatic colon cancer. It was hypothesized that metastases to the liver had resulted in the elevated maternal serum alpha-fetoprotein level.
It is also well-documented that genetic and genomic aberrations are fundamental in the pathogenesis of malignancy. A recent study by Duijf et al. found that small chromosomes, such as chromosomes 21, 18, and 13, are lost more readily than large ones in analyzed cancer cells. It is interesting that our NIPT result was consistent with a significantly lower level of detected chromosome 18 material. The authors of this study note that aneuploidy, particularly the loss of chromosomes, also results in the loss of key tumor suppressor loci. It is also known that the gain of a chromosome may increase the levels of specific oncogenes.
Although the underlying etiology of maternal malignancy seen in this case is likely to be rare, discordant NIPT results are also possible in the context of confined placental mosaicism, undiagnosed maternal mosaicism, and possibly in cases of intrauterine demise of an affected co-twin. Therefore, pretest counseling for NIPT should include a discussion of the need for confirmation of abnormal results with chorionic villus sampling or amniocentesis.
The present report is a unique case of an abnormal NIPT result, likely the result of maternal metastatic disease. This case illustrates that unexplained abnormal NIPT results should prompt consideration of a maternal source of the abnormal cell-free DNA, including the remote possibility of malignancy.
WHAT'S ALREADY KNOWN ABOUT THIS TOPIC?
- Noninvasive prenatal testing for detection of trisomies 21, 18, and 13 is clinically available and is reported to have a false positive rate of 1% or less.
- This technology utilizes massively parallel shotgun sequencing of cell-free DNA, of maternal and placental origin, present in maternal plasma.
WHAT DOES THIS STUDY ADD?
- Unexplained abnormal noninvasive prenatal testing results should prompt consideration of a maternal source of the abnormal cell-free DNA, such as malignancy.