The present study was approved by the Ethical Committee of Fudan University Shanghai Cancer Center (FUSCC; Shanghai, China). Written informed consent was obtained from the patients. A total of 11 male patients with LN-metastatic EMPD on the scrotum, who were treated at FUSCC between January 2009 and January 2016, were retrospectively reviewed. Patients who did not undergo surgery were excluded. A PET/CT scan was conducted on each of them prior to surgery, during which the primary lesion and lymph nodes were excised.

Clinicopathological characteristics, including age, metastatic sites, PFS and PET/CT scan results, were obtained from electronic records. Patients were regularly followed up by telephone or in the clinic every 3 months. Physical and radiographic examinations were retrieved from electronic medical records. Patients who had follow-up in their local hospital sent the information via the internet. With regard to patients who were followed up by phone, their vital status was followed using scheduled phone calls, independent of their physician. Events such as tumor recurrence, progression and metastasis were recorded.

Circulating tumor DNA was also conducted in one of the cases (case No. 11). A blood sample was taken from the patient and sent to BGI company (The Beijing Genomic institute, Beijing, China). BGISEQ-500 platform (http://www.genomics.cn/navigation/show_navigation?nid=4201) was used to get the results.

IHC and FISH were used to evaluate HER2 gene amplification in samples from LNs according to the American Society of Clinical Oncology/College of American Pathologists guidelines (15). Dual-probe ISH assays were conducted and HER2 amplification was defined by examining the HER2/chromosome enumeration probe 17 (CEP17) ratio followed by the mean HER2 copy number (15). The probe used in the FISH test was from the PathVysion HER2 DNA Probe kit (Abbott Laboratories, Abbott Park, Chicago, IL, USA).

Samples of dissected LNs were stored at −80° in the tissue bank at FUSCC. Genomic DNA was extracted using a TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China). Polymerase chain reaction (PCR) was performed on all DNA samples. The intron-based primers for 7 exons of the tyrosine kinase domain and exon 8 of the extracellular domain of HER2 were used according to a previous study (16). Distilled water was used as a negative control for the PCR. Primer sequences are shown in Table I. The denaturation, annealling and extension temperature was 95, 58 and 72°C (32 cycles), respectively. All PCR products were subjected to direct sequencing using an Applied Biosystems 3730×L DNA sequencer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

PFS time was calculated from the date of surgery to the progression of the disease. Patients without events or mortality were recorded as censored at the time of last follow-up. SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was used to perform the statistical analysis. PFS was analyzed using the Kaplan-Meier method, with log-rank tests used to assess the differences between the groups. A two-sided P-value of <0.05 was considered to indicate a statistically significant difference.

A total of 11 patients were included in the present study. The median age of these patients was 63 years (range, 48–76 years). Of these patients, 6 were diagnosed with EMPD and 5 were diagnosed with underlying apocrine carcinoma with epidermal manifestation of EMPD (Table II).

All patients underwent surgical treatment of the primary lesion and the affected LNs. PET/CT was performed on each patient in the month prior to dissection of the LNs. The mean size of the metastatic LNs on PET/CT was 2.3 cm. The mean SUVmax of the metastatic LNs was 7.2. Generally, the PET/CT results were highly associated with the pathology. A false-negative case (no. 11) of inguinal LNs and a false-negative case (no. 6) of pelvic LNs were identified using PET/CT (Tables III and IV). Although LN metastases were not identified in these 2 cases, the patients had invasive EMPD, which was strongly associated with LN metastasis in our previous study (3); therefore, a prophylactic LN dissection was discussed with the patients and they each accepted the surgical intervention. The sensitivity and specificity of PET/CT was 90.9 and 100.0% for inguinal LNs, and 85.7 and 80.0% for pelvic LNs, respectively.

The median time from LN dissection to disease progression (LN progression or novel LN metastasis or distant metastasis) was 15.9±1.5 months (primary site without vs. with underlying carcinoma, 16.2 vs. 14.6 months, respectively). The median time from discovery of the primary disease to LN metastasis was 12.0±2.3 months (primary site without vs. with underlying carcinoma; 12.0 vs. 6.0 months, respectively). Primary sites with underlying carcinoma were indicated to be more aggressive.

Following discovery or treatment of the primary lesion, all patients developed inguinal LN metastasis; 6 (54.5%) patients also developed pelvic LN metastasis (Table II). LN progression was identified in case no. 10 and 11, in 1 patient with lung metastasis and in 1 patient with liver metastasis. During the postoperative follow-up, 2 patients exhibited no recurrence of the disease. The disease-free survival times of these 2 patients were 16.2 and 54.8 months, respectively.

Adjuvant chemotherapy was administered to 3 patients. Following disease progression, different chemotherapy regimens were used as shown in Table V. The PFS of chemotherapy ranged from 3–16 months.

Out of 11 patients, 3 (27.3%) were HER2-positive. IHC results are listed in Table III and shown in Fig. 1. Patients with HER2 amplification showed a trend for shorter median times from disease discovery to LN metastasis (15.6 vs. 10.0 months) and from LN dissection to disease progression (16.2 vs. 13.6 months), although no statistically significant difference was observed due to the limited number of samples (Table II).

A previous study has indicated that the majority of HER2 mutational sites are clustered in two major regions of the gene. Overall, 20% of patients have extracellular domain mutations at residues 309 and 310, and 68% have kinase domain mutations between residues 755 and 781 (17). Therefore, in all 11 samples, the region of exon 8, including residues 309 and 310, and the 7 exons of the entire HER2 tyrosine kinase domains were sequenced. No HER2 mutations were identified. Trastuzumab-targeted therapy was administered to 2 of the patients with HER2 amplification with successful outcomes.

A 48-year-old man presented with scrotal erythema and was admitted to the FUSCC in February 2014. A CT scan showed metastatic LNs in the left inguinal. Pathomorphology of biopsy specimens were consistent with EMPD with underlying carcinoma. The patient underwent a wide local tumor resection with left inguinal and pelvic lymphadenectomy in February 2014. However, PET/CT revealed recurrence with left external iliac, retroperitoneal and right inguinal lymphadenopathy in August 2014. Examination of a biopsy specimen from the enlarged right inguinal LNs confirmed recurrence of the disease, and FISH analysis revealed HER2 gene amplification (Fig. 2).

Trastuzumab targeted therapy was administered at a maintenance dose of 2 mg/kg on days 1, 8 and 15, together with chemotherapy (80 mg/m² paclitaxel and 30 mg/m² cisplatin on days 1, 8 and 15) every 3 weeks. Following 4 cycles of treatment, a complete response was obtained with regression of the iliac lymph node (Fig. 3), and the disease remained stable until January 2016, when magnetic resonance imaging showed enlarged LNs in the retroperitoneum. The follow-up was performed once every 3 months. The PFS time following first-line trastuzumab monotherapy was almost 17 months.

A 54-year-old man was diagnosed with EMPD in June, 2013. Extensive resection of the primary lesion was performed. Disease recurrence was identified in August, 2014 and a second surgery together with adjuvant radiation therapy was conducted. The disease remained stable until December 2015, when PET/CT revealed left external iliac lymphadenopathy with an SUVmax of 5.2; swelling of a left inguinal LN was also observed. The patient underwent left inguinal and pelvic lymphadenectomy in January 2016. Pathological examination showed left external iliac and left inguinal lymphadenopathy. Subsequent to the surgery, the patient received 4 cycles of chemotherapy with docetaxel (75 mg/m²) and cisplatin (30 mg/m²) every 3 weeks. In February 2017, the patient developed headaches and chest discomfort. Another PET/CT showed multiple metastatic sites in the liver and bones. FISH analysis revealed HER2 genetic heterogeneity (Fig. 4). The HER2/CEP17 ratio was >2.0 for 10% of infiltrating tumor cells. The mean HER2/CEP17 ratio was 1.9, but >4 copies of HER2 per cell were observed. Therefore, this patient was considered to possess a positive result for HER2 amplification. Trastuzumab targeted therapy was administered at a dose of 360 mg every 3 weeks together with paclitaxel (80 mg/m²) every week. Following 2 cycles of trastuzumab and 9 cycles of paclitaxel, the disease remained stable for 5 months. In August 2017, the patient felt lumbar pain and another PET/CT revealed regression of the initial sites of liver metastases and those at certain bone sites. Also novel bone metastatic sites and progression were identified.

A blood sample was taken from the patient. Sequencing of circulating tumor DNA was conducted following the failure of the treatment, and human epidermal growth factor receptor 3 (HER3) mutation was identified at a rate of 0.6%. This may explain why the patient experienced resistance to trastuzumab. The patient was then treated with lapatinib and capecitabine. The follow-up was performed once every 3 months and the patient had stable disease at last follow-up on January 2018.

A literature review of studies that investigated the prevalence of HER2 amplification in patients with EMPD was conducted (Table VI). Altogether, 485 cases of EMPD were reported and 35 of them had metastases. Overall, 20.7% of patients with EMPD exhibited HER2 amplification. In addition, the probability of HER2 amplification was more notable in patients with metastatic EMPD. A slight increase in the percentage of HER2 positivity was observed when metastatic disease samples were assessed for HER2 gene amplification.

The published case reports regarding the efficacy of HER2-targeted therapy in EMPD patients with HER2 amplification are summarized in Table VII. These cases, including the present study, included a total of 10 patients. All patients showed a marked response to targeted therapy, including 4 with the complete remission of metastatic disease. The median PFS time was >12 months. None of the patients developed severe adverse effects during treatment.