Detection of Non-Hodgkin Lymphoma Within Ovarian Tissue

Introduction

Adolescents and young adults include the largest age group affected by Hodgkin (HL) or non-Hodgkin lymphomas (NHL). Fertility preservation options depend on various chemotherapy regimens, particularly those with alkylating agents, which are known to be gonadotoxic since they damage all types of ovarian follicles from primordial to antral stages. ¹ The degree of ovarian toxicity depends on the patient's age and type/dose of treatment, and can become permanent, leading to infertility or premature ovarian failure (POF).

Ovarian tissue cryopreservation (OTC) is a valid strategy for preserving endocrine and reproductive function in patients at high risk of POF. Upon remission, cryopreserved ovarian tissue transplantation (COTT) allowed the recovery of endocrine ovarian function (95% of cases), induction of puberty, and resumption of spontaneous and/or induced fertility, as demonstrated by the birth of numerous (>200) healthy babies. ²

In this study, we describe the detection of NHL within ovarian tissue after cryopreservation.

Case Report

In June 2021, a 27-year-old woman went to the emergency room of the city where she lives for severe dyspepsia, nausea, lumbar and chest pain, and she was hospitalized for diagnostic investigations. The patient had no gynecological symptoms such as pelvic pain or abnormal uterine bleeding. After performing X-ray and computed tomography scan of the chest and abdomen, an oval mediastinal mass measuring 75 × 44 × 63 mm was found. A further positron emission tomography (PET) scan confirmed the diagnosis. At the level of both ovaries, the examination showed hyperfixation of the contrast, which was attributed to normal ovarian activity.

At the reference oncological center histological diagnosis of stage IV mediastinal large B cell lymphoma involving various thoracoabdominal organs was made using video-assisted thoracoscopic surgery and specific mediastinal mass biopsies. Immunohistochemical (IHC) investigation on mediastinal mass biopsies revealed a high proliferation index (ki67 > 80%), positivity for CD20, CD30, BCL2, BCL6, and negativity for cell cycles regulator (MYC). Laboratory studies showed elevated levels of tumoral markers CA125 (58.9 U/mL) and CA19-9 (99.3 U/mL).

The established treatment program included six cycles of dose-adjusted etoposide phosphate, prednisone, vincristine sulfate, cyclophosphamide, doxorubicin hydrochloride and rituximab (EPOCH-R) (etoposide 50 mg/m² per day for 5 days; prednisone 60 mg/m² per bid for 5 days; vincristine 0.4 mg/m² per day for 5 days; cyclophosphamide 750 mg/m² at day 5; doxorubicin 10 mg/m² per day for 5 days; and rituximab) dose-adjusted EPOCH-R (DA-EPOCH-R) chemotherapy.

Owing to the potential risks of chemotherapy-induced gonadotoxicity with potential loss of fertility, the patient was referred to our Gynecology and Physiopathology of Human Reproduction Unit for fertility-sparing advice. Given the urgency of anticancer treatments, it was decided to opt for OTC.

Serum Anti-Mullerian Hormone level was 5.35 ng/mL. Transvaginal ultrasound showed slightly enlarged ovaries (right 13.6 cc and left 13.00 cc) and antral follicle count >20. Both ovaries presented multiple well-defined homogeneously hypoechoic cysts. Doppler ultrasound showed no blood flow in the lesions.

A written informed consent for OTC was obtained from the patient.

About 40% of both ovaries was removed according to our surgical techniques ³ (Fig. 1A, B) and cryopreserved according to our laboratory procedures. ⁴ During surgery the cysts were removed. Macroscopically, the ovaries appeared markedly modified and some solid content cysts were highlighted (Fig. 1C, D). Microscopically the cysts revealed the presence of lymphoid infiltrates similar to those of primary mediastinal large B cell lymphoma. Typing of the infiltrate showed proliferation of elements with round nucleus and clear cytoplasm, growth in nests and cords with subsequent phenotype: CD45+, CD20+, CD10−, BCL6+, IRF4+−, CD23+, BCL2−+ (about 50%), cell cycles regulator MYC 15%, CD5−, CD3−, D1−, CD117−, variable proliferative index up to areas with KI67 70%, CD200+, PD-L1 focally +, IRTA1− (Fig. 2). Fluorescence in situ hybridization was performed using a probe specific for MYC gene rearrangements (8q24 translocation). One hundred nuclei were analyzed, and no presence of the translocation was observed.

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FIG. 1.

(A) Left and (B) right ovary observed during laparoscopy. Macroscopic appearance of (C) left and (D) right ovary.

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FIG. 2.

Typing of the ovarian infiltrate. CD45 (A) and CD20 (B) immunohistochemical positivity. Scale bar 50 μm.

Subsequent histological evaluation of some fragments of the cortical ovarian tissue revealed no evidence of pathological lymphoid infiltration. In particular, a good preservation of the follicular and stromal component was shown in all the ovarian fragments; the follicular density (primordial follicle number per mm² of the overall section area) was 12 in right ovarian fragments and 15 in left ones (Fig. 3).

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FIG. 3.

Histological analysis of follicular and stromal component. Scale bar 50 μm.

Discussion

NHL is the most common type of lymphoma, accounting for 88% of lymphoma cases, typically diagnosed in adolescents and young adults.^5,6 In recent years, the survival rate for NHL has severely increased, mainly for young adults with good survival and fertility potential (5-year overall survival 84%). ⁷ The most common and aggressive form of NHL requires urgent start of chemotherapy and leaves no time to apply fertility preservation strategies. OTC and subsequent COTT are valid options for preserving endocrine and reproductive function in patients at high risk of POF, but these choice are risky due to possible contamination of ovarian tissue with lymphoma cells.

Possible presence of neoplastic cells in cryopreserved ovarian tissue remains a serious concern regarding of COTT in patients with hematological malignancies. ⁸ According to Donnez and Dolmans on the risk of reintroducing malignant hematological cells, COTT should be strongly contraindicated in all types of leukemia (high risk), although it may be performed in HL (low risk) and in NHL (moderate risk). ⁹ However, in a very recent study, Dolmans et al. ² report that in cases of pathologies at high risk of neoplastic contaminations the inducing of complete remission, with chemotherapy before OTC, reasonably reduces the risk of reintroducing neoplastic cells and does not modify the reproductive outcomes after transplantation. As a matter of fact, in patient in complete remission of their leukemia ∼10 live births have been reported after COTT.^2,10

In our case, no suspicion of ovarian involvement by NHL was hypothesized by gynecologists and hematologists. The PET scan showed hyperfixation in both ovaries attributed to normal ovarian activity, and the ultrasound features were different from those reported in case of ovarian metastasis. Testa et al. reported ultrasound features of three cases of metastases in the ovaries derived from lymphoma: all cases were monolateral solid masses with a mean diameter of 120 mm, irregular borders, and with rich vascularity at power Doppler. ¹¹ Considering the ultrasound appearance, the absence of previous transvaginal ultrasound examinations and the family history of endometriosis (patient's sister has endometriosis), the suspect was of bilateral endometriotic cysts. Therefore, given the urgency of anticancer treatments, after counseling with the patient and consultation of the team, it was decided to proceed with OTC and removal of the cysts.

Ovarian involvement from NHL can be primary or secondary. Primary ovarian lymphoma, a very unusual entity, is considered when full investigation should not have lymphoma anywhere else in the body: the lymphoma should be present only in the ovary with or without spread to adjacent lymph node. The adjacent structures, peripheral blood, and bone marrow should not also contain abnormal cells. ¹² Primary ovarian lymphoma arises from the lymphocytes surrounding the blood vessels of the hilum and are related to the corpus luteum. ¹³

Secondary involvement is either as premature metastasis in extra ovarian neoplasms or as generalized metastatic disorder. ¹³ Regardless the presence of the corpus luteum in only right ovary, it is possible to consider that the patient had a secondary ovarian lymphoma. Our hypothesis is supported by the presence of diffuse lymphadenopathy at the time of diagnosis and the absence of atypical cells in peripheral blood examination.

To date a number of studies failed to prove ovarian contamination with histology, molecular investigations, and long-term xenografting in NHL patients. ¹⁴ Although these reassuring outcomes, Dolmans et al. detected NHL cells by histological analysis and anti-CD20 IHC, in the medulla of one patient and in the cortex of another patient. ¹⁵

Similarly, in this study we described the presence of pathological lymphoid infiltration in ovarian cysts but not in the cortical tissue of a young woman with secondary NHL.

In our experience we performed an accurate analysis of cryopreserved ovarian cortex before transplantation in four women who received cancer treatment for NHL with diffuse lymphadenopathy. In none of the four cases abnormal ultrasound features or PET scan hyperfixation in the ovaries were detected at the time of cryopreservation. All women experienced POF after chemotherapy and subsequent reinfusion of autologous stem cells. In three cases histological analysis and IHC investigation of fixed and paraffinized ovarian tissues revealed no evidence of pathological lymphoid infiltration. Same results were achieved using molecular analysis on both cryopreserved and formalin-fixed paraffin embedded ovarian tissue (right and left) to determine the rearrangement of genes encoding for immunoglobulins heavy (FR1-FR2-FR3 regions) and light chains immunoglobulin kappa locus.

After COTT one healthy baby was born and ovarian function is still present in two of the three women (2 and 4 years after the transplant). ¹⁶ To date, no recurrences were observed 4 years after the first transplantation. On the contrary, in one case, no ovarian involvement was observed by histological analysis, whereas the molecular analysis revealed the presence of pathology in the cryopreserved ovarian tissue. For this reason, it was recommended to avoid the COTT to reduce the risk of reseeding the malignancy in the woman.

Based on literature and our outcomes, it was possible to emphasize that cryopreservation and autotransplantation should still be considered for restoring fertility in NHL patients after an accurate analysis of cryopreserved ovarian tissue.

According to this evidence, we hypothesize that a multidisciplinary approach that integrates different methods (including histological examination, in vitro culture, immunohistochemistry, polymerase chain reaction, multicolor flow cytometry, and long-term xenograft) should be proposed to analyze frozen ovarian tissue. However, all these tests are not mandatory and depending on the type of pathology of the patient. To date no standardized strategies are available to search neoplastic cells in the ovarian tissue of survivors. The use of advanced and patient-specific approaches may dramatically improve the detection of malignant cells in the ovarian tissue to increase the safety of ovarian tissue transplantation.