Germ Cell Neoplasia in Situ Recognized Incidentally with Complaining of Discomfort in the Right Testis: A Case Report

A 27-year-old man experienced discomfort in his right testis in early September, 2021, and visited the hospital five days later. Physical examination did not detect any abnormalities in the scrotum. However, an ultrasound revealed a tumor in the central part of the right testis, and a Magnetic Resonance Imaging (MRI) showed a tumor 2.7cm in diameter with clear boundaries and a marginally smooth surface. The level of alpha-fetoprotein, human chorionic gonadotropin, human chorionic gonadotropin-β subunit, and lactate dehydrogenase were within normal limits. A Computed Tomography (CT) scan showed no abnormalities. We can't rule out the possibility of malignancy, right radical orchiectomy was performed with a diagnosis of right testicular tumor in mid-September 2021. The macroscopic lesion was 1.5×1.3 cm in size, and no viable tumorous cells were found pathologically. Atypical cells were observed in the seminiferous tubules from the spermatic cord, which were positively stained with immune-histochemical staining CD117 (c-kit), D2-40, and MIB-1 but negatively with alpha-fetoprotein, human chorionic gonadotropin, and human chorionic gonadotropin-β subunit. The pathological diagnosis was germ cell neoplasia in situ, and no continuity was observed between these cells and bleeding necrosis. The patient has been followed up for 1 year and 4 months after surgery, with no recurrence or metastasis observed.


Introduction
Recently, germ cell neoplasia in situ (GCNIS) has been well recognized as a precursor lesion to testicular germ cell tumor.Although GCNIS is frequently found in association with germ cell tumors such as seminoma, isolated reports of GCNIS are relatively rare in the literature.In this report, we present a case of GCNIS incidentally discovered in a patient who presented with discomfort in the right testis.We also provide a review of previously reported cases of GCNIS.sound revealed heterogeneous neoplastic lesion in the central part of the right testis (Figure 1).MRI revealed 2.7cm clear boundary and marginally smooth tumor in the center of the testis (Figure 2).Alpha-fetoprotein level was 1.38 ng/ml, human chorionic gonadotropin level was less than 1.0 mIU/l, human chorionic gonadotropin-β subunit level was less than 0.1ng/ml, and lactate dehydrogenase level was 178 U/l.CT revealed no abnormalities.We can't rule out the possibility of malignancy, right radical orchiectomy was performed with a diagnosis of right testicular tumor in mid-September 2021.The cut surface showed a solid mass 1.5cm in diameter in the right testis (Figure 3).
Bleeding necrosis and granulation tissue were observed in the tumorous lesion, but no viable tumorous cell component was observed pathologically.The granulation tissue intervened on the boundary line between necrosis and existing testicular tissue, but no findings suggestive of a tumor were obtained (Figure 4).The lesion contained atypical cells that were observed in the seminiferous tubules from the spermatic cord.The atypical cells were like spermatogonia, large, and had clear reticulum.The nucleus had large irregularity and included a clear nucleolus.These cells were  arranged like lining the basement membrane.The collapse of the basement membrane or extravasation was not recognized.No extension to epididymis, vas deferens, and spermatic cord of atypical cells was seen (Figure 5).The surgical stump was negative for a tumor.The atypical cells were stained positively with immune-histochemical staining MIB-1 (Figure 6), CD117 (c-kit) (Figure 7) and D2-40 (Figure 8), but negatively with alpha-fetoprotein, human chorionic gonadotropin, and human chorionic gonadotropin-β subunit.No obvious continuity was observed between these cells and bleeding necrosis.From these results, the pathological diagnosis was germ cell neoplasia in situ (GCNIS).The patient's postoperative course was uneventful.After surgery, CT scans should be performed every 3 months according to the testicular tumor guidelines for the first year.Subsequently, CT scans should be conducted annually.He has no recurrence or metastasis 1 year and 4 months after surgery.

Discussion
World Health Organization (WHO) revised its classification regarding urinary tract and male genital tumors in 2016.One significant change was observed with testicular germ cell tumors.Until now, the histopathological classification of testicular germ cell tumors was based only on morphological similarities.In contrast, the new classifica-    tion prioritizes similarities in histogenesis over morphological similarities.Germ cell neoplasia in situ (GCNIS), previously known as intratubular germ cell neoplasia of unclassified type, is now considered to be a precursor of most testicular germ cell tumors except for spermatocytic tumors, yolk sac tumors, and mature teratomas.
According to the general rule for clinical and pathological studies on testicular tumors, the 2018 4th edition, GCNIS is regarded as a large tumor cell similar to germ cells that appear or in a row on basement membrane.GCNIS cells stain positively with immune-histochemical staining such as Placental alkaline phosphatase (PLAP), CD117(c kit), OCT3/4, SALL4, and D2-40.In the current case, large tumor cells were recognized as standing in a row, and those cells stained positively with immune-histochemical staining MIB-1, CD117 and D2-40, but negatively with alpha-fetoprotein, human chorionic gonadotropin, and human chorionic gonadotropin-β subunit.
As far as searched in Japan, there were only three cases where testicular germ cell tumors were not recognized other than biopsy, and the tumor was GCNIS alone 1) .Testicular cancer occurs in 1% of men worldwide 2) .On the other hand, CNIS is a precursor lesion to testicular germ cell tumors, and there is a report that 50% of them in progress to testicular cancer in 5 years and 70% of them in 7 years 3) .There is a 1.9～5.2%chance that the testicular germ cell tumors occur contralaterally in heterochronous, and contralateral testicular biopsy during radical orchiectomy has been considered for the detection of GCNIS [4][5][6] . Te frequency of identification in contralateral testicular biopsy has been reported to be 3～5% 7) .
It has been pointed out that the risk of contralateral onset of GCNIS is high in cases with low semen concentration, small testis volume, irregular internal echo image of the testis, and young patients 8) .On the other hand, even if the result of contralateral testicular biopsy is negative, the development of tumors was about 1%, which can be considered a cause that the tumor was not detected due to low tumor burden 7,9) .Two-part biopsy is considered to enhance the sensitivity of discovery.GCNIS was detected in 5.1% of prospective 2,318 case studies.The pathology of the biopsy specimens from the paired side was reported to be different in 31.1% of GCNIS-positive patients, and it was shown that the detection frequency increased with a two-part biopsy 10) .
The complication rate of contralateral testicular biopsy includes hematoma and infection.The complication rate by two-part biopsy is reported to be less than 3%, and most complications resolved with conservative management, and the case requiring additional treatment was 0.6% 11) .The treatment of GCNIS is radiation therapy, which can result in a complete cure.However, due to differences in the radiosensitivity of testicular cells, only Sertoli cells remain in the seminiferous tubules, causing azoospermia.As Leydig cells have low radiosensitivity, hormone replacement therapy is often not needed 12) .It is reported that the an irradiation dose of 14 Gy causes 8% heterochronous incidence rate of germ cell tumor.
GCNIS will be 98% curable by irradiation dose of 18-20 Gy.But if the irradiation dose increases more than this, the possibility of hypogonadism will be increase.
Therefore, irradiation dose of 16-20 Gy is recommended for treatment 13) .Although contralateral testicular biopsy is not recommended in Japan, it may be considered in high-risk patients groups.
Because testicular cancer primarily affects young people, cryopreservation of sperm is an important consideration if radiation therapy is chosen.In such case, providing enough information to the patient and obtaining informed consent is necessary.
As this case involves a young patient and there is some possibility of contralateral onset, careful longer-term follow-up is required 14) .

Conclusion
We described a case of GCNIS and reviewed the relevant literature.Since the patient is still young, longer-term follow-up is required.

Figure 1 Figure 2
Figure 1 Ultrasound reveals heterogeneous neoplastic lesion in the right testis

Figure 3 AFigure 4
Figure 3 A solid mass 1.5cm in diameter in the right testis

Figure 5 H&E
Figure 5 H&E staining × 200 Atypical cells do not extend beyond the basement membrane.Tumor cells are large, composed of a pale cytoplasm and large, irregular nuclei with well-defined nucleoli.