DAI Geyang,CHEN Gaowen,LI Xiaoxuan,ZHENG Youhong,WANG Yuan,Li Xingsong,LI Jing,ZHOU Jing,XIE Yu,WANG Yifeng

1Department of Obstetrics and Gynecology,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China;2Novogene Bioinformatics Institute,Beijing 100020,China;3Obstetrics and Gynecology Hospital of Fudan University,Shanghai 200111,China;4Department of Information,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China

Abstract:Objective To explore the strategy of pregnancy-preserving and maternal-fetal management in patients with primary gynecologic neuroendocrine tumors(gNETs)during pregnancy.Methods We performed whole genome sequencing(WGS)for analyzing maternal and fetal somatic and germline single nucleotide variations(SNVs)and small insertions and deletions(InDels)for a 29-year-old pregnant woman diagnosed with stage IB2 large cell neuroendocrine carcinoma(LCNEC)and adenocarcinoma in the cervix.A systematic literature review was performed to explore the strategies for treatment of such rare histological type while maintaining pregnancy.Results Global case analysis of cervical NETs during pregnancy suggested that negative lymph node metastasis and an early FIGO stage were potentially associated with a good prognosis of the patients.In the case presented herein,a pregnancy-preserving strategy was adopted and favorable maternal-fetal outcomes were achieved after neoadjuvant chemotherapy,radical surgery and postoperative systemic chemotherapy.At 35+5weeks,the fetus was delivered by caesarian section,and the patient has by now had a disease-free survival of 19 months postoperatively.WGS analysis revealed 6 missense somatic pathogenic mutations in two cancer tissues of the patient,and among them KARS and VEGFA were related with targeted therapy.Five pathogenic germline variants were detected in the patient and her son,suggesting the necessity of a long-term follow-up schedule including precise genetic counselling for both the mother and the child.Conclusion Although gNETs in pregnancy are rare and highly risky,pregnancy-preserving managements of gNETs can still be considered and favorable maternalfetal outcomes are possible with proper assessment of the clinical indications and implementation of multimodal treatments.Precise treatment and follow-up strategies based on the results of WGS for risk-reducing intervention of cancer recurrence or occurrence can potentially benefit the patient and the neonate.

Keywords:primary gynecologic neuroendocrine tumors;large cell neuroendocrine carcinoma;pregnancy-preserving management;maternal-fetal management;cancer predisposing genes

INTRODUCTION

Primary gynecologic neuroendocrine tumors(gNETs)are rare primary tumors in the gynecologic tract,accounting for approximately 2% among gynecological malignancies［1,2］,but its incidence tends to increase rapidly by 4 folds from 0.3 per million in 1987 to 1.30 per million in 2012［3］.Histologically,gNETs is classified into large cell neuroendocrine carcinoma(LCNEC),small cell neuroendocrine carcinoma(SCNEC),and typical and atypical carcinoids.LCNEC and SCNEC are poorly differentiated and have a more aggressive clinical course,while typical and atypical carcinoid tumors generally show benign behaviors.The uterine cervix is the most common site of involvement by gNETs,followed by the ovary,endometrium,vagina and vulvar.Compared with those with more common gynecological malignancies,the patients with neuroendocrine carcinoma of the cervix(NECC)have a higher risk of cancer-related death［4］with a mean recurrence-free survival of 16 months and a mean overall survival of 40 months［5］.Among all ovarian cancers,NECC is associated with the worst survival outcome and the mean overall survival of the patients is only 27.4 months［6］;distal metastasis is present in around 70% of the patients at the time of diagnosis,whose prognosis can be dismal［3］.

Pregnancy-associated cancer(PAC)is defined as a cancer diagnosed during pregnancy and up to 1 year postpartum.Cervical and ovarian cancers account for 8.7% and 5.2% among PACs,respectively.The clinical management of PAC is much complicated by the pathophysiological changes and the relationship between the reproductive organs and the developing fetus［7,8］.gNETs during pregnancy are even rarer and because of its aggressive nature,a tendency for distal metastasis and the unclear origin,most clinicians are more inclined to recommend the termination of pregnancy to deliver radical treatments,although current treatment strategies have enabled pregnancy maintenance during treatment of some common histological types of gynecologic cancer［9,10］.But still,the management for rare subtypes of PAC remains clinically challenging and awaits further investigation.

Herein we present our experience with fertilitypreserving treatment of LCNEC in pregnancy in a 29-year-old Cantonese woman with a history of thyroid carcinoma.We discuss the management strategy focusing on maternal-fetal outcomes with integrated plans for diagnosis,treatment and follow-up,reviewed the literature documenting such rare histological type during pregnancy,and explored the value of high-throughput whole genome sequencing(WGS)in such cases.

CASE PRESENTA TION

A 29-year-old Chinese women was referred to Zhujiang Hospital (Guangzhou, China) with complaint of intermittent vaginal bleeding from the 16th week of gestation in March,2019.The patient reported a history of thyroid papillary carcinoma with neck lymph node metastasis and received radical surgery in December 2015.Regular postoperative examinations showed no signs of recurrence until the current admission.Gynecological examination revealed a 3 cm×1 cm×2 cm exophytic lesion on the posterior lip of the uteri cervix without invasion of the parametrium or the vaginal fornix.Human papillomavirus(HPV)testing showed a persistent HPV-18 infection, and serum alpha fetoprotein(AFP)level was 235.55 ng/mL.Pathological examination of the lesion revealed LCNEC with adenocarcinoma,which showed positive immunoreactivity for CK,CK-7,synaptophysin,CgA,P16,CD-56 and Ki67(＞90%)(Fig.1).Magnetic resonance imaging (MRI) and diffusion-weighted imaging(DWI)detected no evidence of lymph node involvement or pelvic metastasis.The tumor was classified as clinical stage IB2 based on FIGO staging of cancer of the cervix uteri(2018).

As the patient strongly wished to continue pregnancy,neoadjuvant chemotherapy(NACT) with cisplatin and etoposide(CDDP 20 mg/m2,d1-d5 and VP-16 100 mg/m2,d1-d5)was administered at 27 and 31 weeks,respectively.The treatment did not produce obvious toxicity,and no signs of fetal malformation were detected.At35+5week,the fetus was delivered by caesarian section, and the patient subsequently underwent a radical hysterectomy combined with bilateral salpingectomy, bilateral ovarian biopsy,and sentinel lymph node biopsy(SLNB)(Fig.2).The first-minute Apgar score of the neonate was 9,and the 5th-minute Apgar score was 10.As examination of frozen sections of the surgical specimens showed a negative result,both of the ovaries were preserved and systematic lymph node dissection was not performed.Histological examination also showed negative margins on all the resected tissues,including the uterus,parametrium,bilateral fallopian tube,and the placenta.No signs for maternal-fetal tumor transmission were found.After the operation,the patient received 4cycles(21days per cycle)of cisplatin and etoposide treatment(CDDP 20 mg/m2,d1-d5 and VP-16 100 mg/m2,d1-d5).Regular follow-up examinations for both the patient and her son were scheduled(every 3 months in the first year and 3 to 6 months in the second year).At 19 months after the operation,physical and cytological examinations,tumor marker level monitoring,and ultrasound scans of the patient revealed no evidence of tumor recurrence or metastasis,and the baby remained healthy without signs of neurodevelopmental disorders.

Fig.1 Histology of large cell neuroendocrine carcinoma and adenocarcinoma of the cervix.A:HE staining shows a trabecular solid growth pattern of the tumor cells(Original magnification:×40).B:HE staining shows pleomorphic tumor cells with prominent nucleoli,frequent mitoses and apoptosis(× 80).C:Ki-67 staining shows positivity in over 90% of the tumor cells(× 80).D:Immunohistochemistry shows positivity for neuroendocrine marker synaptophysin in the tumor cells(×80).

Fig.2 Imaging diagnosis with DWI/MRI and cesarean section.A:DWI/MRI demonstrates abnormal high signal in the posterior lip of the uterine cervix with a size of 28 mm×15 mm×24 mm(red arrow).The vaginal fornix and parametrium remain intact,and no pelvic lymph node swelling is observed.B:The patient underwent cesarean section on the lower middle part of the corpus uteri.

GENOMIC ANALYSIS

WGS,whole exome sequencing(WES),and nextgeneration sequencing for exploring cancer driver genes and predisposing variants facilitate precise and individualized clinical management for patients with cancers including NECC［11,12］.The genetic background of LCNEC especially during pregnancy was rarely identified in previous studies.For establishing a more precise follow-up schedule,we performed WGS of blood samples from the patient and her son and microdissected tumor samples from paraffin-embedded tumor tissue sections(both LCNEC and adenocarcinoma)with further bioinformatic analyses under approval of Institutional Review Board at the Zhujiang Hospital.The patient had given written informed consent for publication of her case details.

The reference human genome(UCSC hg19)was utilized for validating sequencing data mapping by Burrows-Wheeler Aligner(BWA)software［13］,and the original mapping results were sorted by SAMtools［14］and duplications were marked by Picard(http://broadinstitute.github.io/picard/).Germline single nucleotide variations(SNVs)and small insertions and deletions(InDels)were called using SAMtools(version 1.10,mpileup with parameters,-q 1-C50-t DP,SP,DV-m2-F0.002-ugf)and bcftools(version:0.1.19-58-g3d123cd).Meanwhile,to call somatic SNVs and small InDels from paired tumor-normal samples,MuTect［15］and Strelka［16］were used respectively.Subsequently,the VCF(variant call format)file was annotated by ANNOVAR［17］.Both somatic and germline mutations were compared with 950 cancer related genes using Cancer Gene Census(CGC)［18］,the Familial Cancer Database(FaCD,http://www.facd.info),intOGen［19］and 152 cancer predisposing genes(CPGs)reported by Huang et al［20］.Tumor mutation burden(TMB)of tumor tissues samples were calculated as somatic non-synonymous mutations per megabase(MB)of the genomic coding region.Microsatellite instability(MSI)was assessed using the MSIsensor algorithm that yielded a quantitative MSIsensor score［21］.The corresponding target drug prediction was inferred using NovoDrug database integrated integrated PharmGKB［22］,Drugbank［23］,My Cancer Genome and KEGG［24］.

Microsatellite instability

WGS analysis of blood and tumor samples of the patient suggested a TMB in the adenocarcinoma of 1.17 per Mb and a LCNEC of 1.19 per Mb,which were lower than that of common cervical cancers but similar to SCNEC(1.7 per Mb)［12,25］.Neither of the cancer tissues of the patient showed positive finding in MSI,indicating a potentially poor response of the tumors to immunotherapy.

Somatic mutations

Six missense somatic pathogenic mutations were detected(Tab.1),and 4 of them(GNAS,IKBIP,INPP4B,and KRAS)were found in both tumor tissues from LCNEC and adenocarcinoma of the patient.Further analysis related with targeted therapy indicated that the KRAS(p.G12D)variant was associated with probable sensitivity to Cobimetinib,Trametinib or Binimetinib in all solid tumors and resistance to Panitumumab and Cetuximab in colorectal cancer,while the VEGFA(p.R333Q)variant may relate with potentially sensitive to Bevacizumab and Sunitinib in all massive tumors(Tab.2).In previous studies,the mutations of KRAS and GNAS has been detected from NECCs［11,12］.A patient of recurrent SCNEC of the cervix harboring the KRAS(p.G12D)variant was reported to have successful treatment using a MEK(Methyl ethyl ketone)inhibitor［26］.Moreover,recurrent mutations at a single codon(R201)in the GNAS were identified in cervical tumors,which may activate GNAS function and were associated with drugs targeting the guanine-binding protein-couple receptor(GPCR)［25］.These findings of somatic missense pathogenic variants KRAS,GNAS,and VEGFA by WGS suggest a potential precise targeted therapy for tumor recurrence.

Tab.1 Somatic mutations detected in tumor tissues of LCNEC and adenocarcinoma

Tab.2 Potential resistance or sensitivity of targeted drugs to different tumors

Germline mutations

Five pathogenic germline variants(PGVs)and 2 variants of uncertain significance(VUSs)were detected in the patient and her son(Fig.3).The child and the mother shared 1 PGV,indicating the presence of a genetic predisposition.In addition to GNAS(p.R186H)detected in both cancer tissues,another PGV of GNAS(p.H69N)was found in the patient.In a recent prospective,multicenter cohort study of 2984 patients with solid tumor cancer for assessing germline predisposing variants,PGVs were found in 394(13.3%)patients and VUSs in 1415(47.4%)patients［27］,and the findings resulted in modifications of the treatment in 42 patients;a younger age at cancer diagnosis was shown to be associated with the presence of PGVs.As hereditary factors are crucial risk factors for several cancers,cascade family variant testing can be essential for the disease-free relatives of patients positive for PGVs.In our case,based on the findings of PGVs and VUSs,we arranged a regular long-term follow-up schedule for the patient and her son and provided them with precise genetic counselling for risk-reducing intervention of cancer recurrence or occurrence,especially for tumors in the nervous system,endocrine glands,and genitourinary system.

LITERATURE REVIEW

Fig.3 Screening for germline pathogenic and uncertainly significant mutations from the patient and her child.A:Germline pathogenic mutations.B:Germline uncertainly significant mutations.AR:Androgen receptor;GNAS:Guanine nucleotide-binding protein G(s)subunit alpha;POLD1:Subunit of the DNA polymerase δ complex;RECQL:ATP-dependent DNA helicase Q1-like;CNTNAP2:Contactin associated protein-like 2;PMS2:Postmeiotic segregation increased 2.

We searched PubMed,Embase,and the China National Knowledge Infrastructure Databases(CNKI)for studies or case reports of gNETs in pregnancy in human subjects published in English or Chinese from 1981 to 2020.All the eligible publications described pathologically established diagnosis of gNETs and clinical staging of gNETs based on 2018 International Federation of Gynecology and Obstetrics(FIGO)staging system［28］.Multivariable Cox proportional hazards regression models and Kaplan-Meier analyses were used for inferring the association between the prognostic factors and the survival of the patients.

Clinical characteristics and outcomes of gNETs in pregnancy

We retrieved only 44 cases of pregnancy-related gNETs reported worldwide since 1976（Tab.S1,available from the corresponding author on request）.Uterine cervix was the most common site of involvement of gNET(cNET)in these cases(35/44)followed by the ovary(oNET)(8/44);only one case of gNET in the vagina(vNET)was reported.Among these 44 cases,a familial history of cervical cancer(in the cousin and maternal grandmother)was reported in only one case;in our case,we report the first case with a history of primary thyroid papillary carcinoma.

The clinical characteristics and outcomes of the patients with gNETs in pregnancy are listed in Tab.3.Apart from a younger age,the patients with gNETs in pregnancy had similar clinical characteristics including initial symptoms,tumor size,and histology type to those of the non-pregnant patients with gNETs［3］.Among the patients with gNETs in pregnancy,most of the patients were diagnosed in the second(23/44)and third(11/44)trimesters,and only 4 cases in the first trimester and 4 postpartum.In cases of cNETs,74%(26/35)were diagnosed at the localized stage(I-IIA),and 25.7%(9/35)had lymph node metastasis.The“benign”appearance of the lesions including polypoid,myoma,and cysts-like lesions and underestimated atypical symptoms often contribute to a delayed diagnosis during pregnancy.Paraendocrine syndromes were found in one cNET patient with Cushing syndrome and one oNETs patient with hypercalcemia during operation.For ma-nagement, 17 patients underwent pregnancypreserving managements,and antenatal neoadjuvant chemotherapy was administered in 12 women without any reports of fetal malformations.Twenty-four of the patients gave live births and only 3 babies were small for gestation age(SGA).In patients with cNETs,the tumors frequently present with multiple-organ metastasis,involving most commonly the liver(7/11).In total,48%(18/37)of the patients died,including 14 with cNET within 12 months,3 with oNET within 17 months,and 1 with vNET within 11months.

Maternal-fetal safety management for patients with gNETs inpregnancy

For cNETs in pregnancy,univariate analyses showed that negative lymph node(P=0.025),earlier FIGO stage(P=0.001)and NACT(P=0.040)were all associated with a better survival outcome.Multivariate analysis suggested that negative lymph nodes(P=0.012),earlier FIGO stage(P=0.008)and the combination of surgery and platinumbased chemotherapy(P=0.048)were associated with favorable outcomes of the patients(Tab.4).A tumor diameter＜40 mm was not found to be a protective factor in non-pregnant women(P=0.652 in univariate andP=0.062in multivariate analysis).

Accumulating evidences for a "healthy mother effect" demonstrate that physiological changes during pregnancy do not cause a growth-promoting effect on female genital malignancies［29,30］.In the present study,we found that fewer patients in pregnancy were diagnosed at advanced stages(28.5%;12/42)as compared to the data for non-pregnant women in Surveillance,Epidemiology and End Results(SEER)cancer database analysis(67%,349/520)reported in 2019［3］.Moreover,neither univariate(P=0.485)nor multivariate(P=0.170)survival analysis showed a disease-deteriorating effect of delayed treatment.Antenatal NACT has been recommended for pregnancy-preserving treatment［10］,and in our case,considerable reduction in tumor size was achieved after antenatal platinum-based NACT.Cisplatin and etoposide therapy is the most commonly used regimen,but the exact dosage and duration varied among published studies,which described 2.4 cycles of chemotherapy on average.It is highlighted that a 3-week time window before delivery is significant for both maternal and fetal bone marrow to recover,and chemotherapy should not be implemented beyond 35weeks of gestation［10］.

Tab.3 Baseline demographic and clinical characteristics of 44 pregnant patients with primary gynecologic neuroendocrine tumors

Tab.4 Univariate and multivariate analysis of prognostic factors associated with overall survival of patients with neuroendocrine carcinoma of cervix in pregnancy

Ovary-sparing strategy currently remains experimental for cNET especially in pregnancy.The patient in our case and 3 patients with cNET in pregnancy in the 44 cases we retrieved had preserved their ovaries,but one of the reported patient experienced ovary metastasis after 8 months.No malignant histological findings were reported in the ovaries of patients who underwent bilateral salpingo-oophorectomy.Ovarian preservation surgery in non-squamous cell carcinoma can be applied in young patients at an early FIGO stage without deep stromal,endometrial,or perineural invasion,particularly in those without uterine corpus invasion,parametrial involvement,and infiltration of the vagina［31］.Additional data analysis is required to promote ovarian preservation,which is beneficial to the physiological and psychosexual well-being of premenopausal women, especially primipara.

For oNETs in pregnancy,the treatments are still based on the regimens for common ovarian tumors.Conservative treatment does not exacerbate typical carcinoid tumor,which is a low-grade malignancy.But there is no consensus on pregnancy maintenance in cases of small cell carcinoma of the ovary(SCCO).In a report by Timothy et al［32］,a patient with stage IIB SCCO and hypercalcemia,who showed elevated calcium level during the operation,experienced recurrence even after NACT and systemic treatment with a debulking cytoreductive surgery(R0)and EP regimen.But in another report,a stage IIIC patient in pregnancy achieved favorable outcome after NACT with cyclophosamide and carboplatin regimen and debulking cytoreductive surgery(R0)［33］.Zhu et al suggested that an early clinical stage and a low tumor grade were independently associated with better survival of the patients［34］.In patients with SCCO with normal blood calcium level,an age below 30 years,a tumor size＜10 cm,and the absence of large cells might be positive predictors for survival［35］,suggesting that more relevant clinical parameters need to be taken into account in clinical decision on the treatment especially for patients who strongly wish to continue pregnancy.We retrieved only one case of vNETs in pregnancy from the literature,in which favorable maternal and infant outcomes were not achieved due to the advanced stage IV.

CONCLUSION

In summary,although gNETs in pregnancy are highly malignant,pregnancy-preserving managements based on the patients'desire are still possible when the clinical indications are carefully evaluated and multimodal treatments are administered to control micro-metastasis and tumor dissemination,especially in cases in early FIGO stage with negative lymph nodes.In the case presented herein,the pregnancy-preserving strategy achieved favorable maternal-fetal outcomes in the patient with stage IB2 LCNEC and adenocarcinoma in the second trimester without lymph node metastasis.Furthermore,based on the results of WGS analysis,precise treatment and follow-up strategies were adopted,which might benefit the patient and her son for riskreducing intervention of cancer recurrence or occurrence.

ACKNOWLEDGMENTS

We thank Prof.DENG Yongjian (Department of Pathology,Nanfang Hospital and School of Basic Medical Sciences,Southern Medical University)for valuable comments on pathological identification,Prof.XIN Xuegang(School of Medicine of South China University of Technology)for critical suggestions on writing,and GUO Xiang and LI Li(School of Public Health,Southern Medical University)for assistance with statistical analyses.