人原发性小肠恶性黑色素瘤裸鼠原位移植高转移模型的建立

目的 建立人原发性小肠恶性黑色素瘤裸小鼠原位移植高转移模型.方法 将手术切除的人原发性小肠恶性黑色素瘤原发灶和肝转移灶新鲜瘤组织块分别植入裸鼠小肠黏膜层内,观察原位移植的成瘤率、移植瘤的侵袭性和转移率,并进行形态学、流式细胞分析和染色体核型分析.结果 人小肠恶性黑色素瘤原发灶和肝转移灶新鲜组织均移植成功,建成人原发性小肠(原发灶)恶性黑色素瘤裸鼠原位移植高转移模型(ttSIM-0602)和人原发性小肠(肝转移灶)恶性黑色素瘤裸鼠原位移植肝转移模型(HSIM-0603).HSIM4)602和HSIM-0603模型分别传至21代和23代,共移植裸鼠227只,其肿瘤移植生长率和液氮冻存复苏成活率均为100%.HSIM-0602模型肝转移率为65.7%,肺转移率为84.8%,淋巴结转移率为63.8%.HSIM-0603模型肝转移率为100%,肺转移率为46.7%,淋巴结转移率为71.3%.移植瘤组织病理学为小肠高度恶性黑色素瘤.免疫组织化学显示,S-100蛋白和HMB-45均为阳性表达.电镜下,瘤细胞浆内可见大量的黑色素小体,也可见黑色素复合体.HSIM-0602模型移植瘤细胞DNA指数为1.59±0.07,HSIM-0603模型移植瘤细胞DNA指数为1.71±0.12,均为异倍体.染色体核型分析显示,HSIM-0602模型移植瘤细胞染色体数为55～57条,HSIM-0603模型移植瘤细胞染色体数为57～59条.结论 HSIM-0602和HSIM-0603模型是成功的人原发性小肠恶性黑色素瘤裸鼠原位移植自发性高转移模型,完整地模拟了人原发性小肠恶性黑色素瘤患者的自然临床病理过程,为研究原发性小肠恶性黑色素瘤转移生物学和抗转移治疗提供了理想的动物模型.     

Rapid growth and spontaneous metastasis of human germinal tumors ectopically transplanted into scid (severe combined immunodeficiency) and scid-nudestreaker mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F2 hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c-nu/nu and CD1-nu/nu). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J-nustr/nustr) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nustr (scid/scid; nustr/nustr) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid-nustr mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.     

Metastatic dissemination of B16 melanoma: pattern and sequence of metastasis

The progressive metastatic spread from subcutaneous transplants of two subpopulations of the mouse B16 melanoma, slow-growing clone G3.5 and fast-growing clone G3.12, was examined during tumor growth in C57BL/6 mice and after surgical excision of tumors of various sizes. In addition to enumeration of visible and lethal or potentially lethal ("clinically relevant") metastases, the occurrence of visibly undetectable proliferating (occult) or nonproliferating (dormant) micrometastases was assessed by implanting lymph nodes and organs subcutaneously into normal mice and monitoring for resulting tumor growth. Occult or dormant metastases were disseminated initially to the lungs from G3.5 tumors of 3-4 mm in mean geometric diameter (MGD) and G3.12 tumors of 6-7 mm in MGD. The ipsilateral axillary lymph node (IALN), the regional draining lymph node for these tumors, received metastases after the lungs, initially from 10 to 12-mm tumors. Subsequently, occult or dormant and visible metastases first appeared in systemic organs and lymph nodes (kidneys, adrenal glands, ovaries, and contralateral axillary lymph node) at tumor sizes of about 26 mm in MGD. Systemic metastases occurred only in mice with large and numerous lung metastases and did not depend on the continuing presence of the subcutaneous tumor or on the presence of IALN metastases, which indicated that established lung metastases were a generalizing site from which systemic metastatic spread initiated. After tumor excision, death generally resulted from extensive lung metastasis. Occasional lethal or clinically relevant metastases were also observed in the IALN, kidneys, adrenal glands, ovaries, brain, eyes, and urinary bladder; liver involvement was evident exclusively as occult or dormant micrometastases. Terminal metastatic patterns of these B16 melanoma transplants were as widespread and indiscriminate as those of malignant melanoma in humans.     

Rapid Growth and Spontaneous Metastasis of Human Germinal Tumors Ectopically Transplanted into Scid (Severe Combined Immunodeficiency) and Scid-nudestreaker Mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F₂ hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c- nu/nu and CDl- nu/nu ). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J- nu^str/nu^str ) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nu ( scid/scid; nu^str/nu^str ) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid- nu^str mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.     

Isolation and properties of cell lines from the metastasising rat mammary tumour SMT-2A

A new cell line Rat mammary (Rama) 900 was isolated from the ascitic version of the SMT-2A metastasising rat mammary tumour by stepwise adaptation of the tumour cells to tissue culture. The cells grew mainly as loosely-adherent aggregates, and were dependent during the first 18 passages in vitro on a feeder layer of mesothelial-like cells (Rama 950) obtained from the same tumour. Subcutaneous injection of Rama 900 cells in fat pads of syngeneic Wistar Furth rats yielded anaplastic primary tumours and extensive, gross metastases including those in lungs, lymph nodes, liver and bones, similar to the parental transplantable tumour. The extent of metastatic spread from subcutaneous fat pads was increased by passage 17 in vitro for the Rama 900 cells. A similar extent of metastatic spread was achieved at earlier times by injecting the original cells with the non-tumorigenic Rama 950 cells in vivo. Subcutaneous injection of Rama 900 into thymectomised rats or MF1 nu/nu mice yielded fewer tumours, most of which regressed. No metastases occurred in the thymectomised rats and fewer metastases, mainly in lungs but not in lymph nodes, livers or bones, were seen in the nude mice. The ascitic tumours formed by intraperitoneal injection of nude mice contained both anaplastic rat cells similar to Rama 900 and mouse mesothelial-like cells similar to Rama 950. Although these anaplastic ascites cells failed to yield any tumours in syngeneic or thymectomised rats, they still produced tumours and metastases, including those in lymph nodes, in nude mice.     

Metastases of human tumor xenografts in nude mice

In the present study, using systematic microscopic examination, we tried to determine the true incidence of metastases in nude mice bearing a wide variety of human tumors. A total of 63 malignant tumors were successfully transplanted subcutaneously and 831 nude mice bearing tumors were examined. It appeared that 17 of the 63 tumors (26.9%) retained their metastatic ability in nude mice. Most of these tumors were adenocarcinomas (11/17 cases). Generally the metastatic deposits in the lungs and, to a lesser extent, in the lymph nodes were small and thus only detectable on microscopic examination. We also found a positive correlation between the presence of metastases and neoplastic infiltration of the lymphatic and/or blood vessels around the subcutaneous tumors. Metastatic human tumors, including neoplastic cells from effusion, exhibited higher metastatic ability than primary tumors (p less than 0.005). However, the expression of this metastatic potential depends on several factors including tumor volume, survival time after inoculation and murine hepatitis infection. Thus, animals with metastases bore larger tumors (9.56 cm3) than those without metastasis (6.35 cm3; p less than 0.0001). Moreover, survival time after inoculation was longer in mice with metastases (104 days) than in mice without metastases (81 days; p less than 0.0001). A negative influence of viral hepatitis on the incidence of metastases was observed. This may simply be related to the shortened life span of the animals. Death due to this infection may precede the expression of the metastatic potential.     

A technetium-labeled monoclonal antibody for imaging metastatic melanoma

Twenty patients with histologically proven metastatic melanoma were scanned with a 99mtechnetium (99mTc)-labeled melanoma antibody to determine the detection rate of known malignant lesions and to evaluate the antibody's ability to discover occult metastases. Isotope localization in different organs was as follows: liver 100%, bone 100%, subcutaneous lesions 80%, lymph nodes 54%, and lung 33%. Four unsuspected bone lesions and 16 occult subcutaneous lesions were found. False positive lesions were noted in two instances--one benign thyroid adenoma, and one arthritic bone lesion. One patient developed an atypical serum sickness reaction with a rash and arthralgias that responded rapidly to treatment. The 99mTc antimelanoma antibody is a safe and effective method to detect metastatic melanoma. It has potential use for screening newly diagnosed melanomas that carry an increased risk of recurrence.     

Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction-based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastases = 54.5, lymph node metastases = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.     

Comparative study of the expression of DNA mismatch repair genes, the adenomatous polyposis coli gene and growth arrest DNA damage genes in melanoma recurrences and metastases

The main goal of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2), the adenomatous polyposis coli (APC) gene and growth arrest DNA damage inducible (GADD) genes (GADD34, GADD45 and GADD153) in the different stages of melanoma recurrences and metastases, and to identify any mutual consistencies in their expression pattern. All the cases of primary melanoma examined showed a reduced expression of DNA repair genes. These results demonstrate that disturbances of DNA repair begin in the early stages of melanoma. No significant differences were found in the expression of these markers between cutaneous melanomas and their recurrences and metastases (P> 0.05). Eighteen significant correlations between markers were found in the primary melanomas, and 10 significant correlations were observed in the first recurrences of melanoma. In contrast, 27 statistically significant relationships were demonstrated in metastatic lymph nodes. The different correlations found in primary and metastatic tumours confirmed the hypothetical difference in marker interaction in the diagnostic groups investigated. Our results suggest that DNA repair genes may play an important role in the recurrence and metastasis of melanomas.     

Methylation state of cellular genes and oncogenes as a marker of malignancy in human carcinomas

The methylation pattern of the human HLA-DR alpha gene was analyzed in primary tumors and lymph node metastases isolated from patients with a variety of tumors, including thyroid, pancreas, breast and gastric carcinomas and melanomas. In normal tissues (including breast, muscle, brain, sperm, T-and B-lymphocytes) the HLA-DR alpha gene is hypermethylated at CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region was located in the 5' portion of the gene. Our results indicate that the HLA-DR alpha gene is hypomethylated in metastatic lymph nodes, as well as in the carcinomas and melanomas studied. These findings lend support to the hypothesis that DNA hypomethylation of the human HLA-DR alpha gene may represent a molecular marker of malignant tumors.     

The utility of ultrasound in patients with melanoma

The highest quality gray-scale ultrasound images are obtained with high-frequency transducers; however, such high frequencies do not penetrate more than a few centimeters into body tissue. Fortunately, in patients with melanoma, the structures of interest are close to the skin surface, making them ideal targets for examination with high-resolution ultrasound. These include primary cutaneous melanomas, uveal melanomas and the regional lymph nodes draining the skin that lie in the axilla, groin, neck and other locations. Although ultrasound study of primary melanomas arising in the skin and eye has provided some insights, a major role for ultrasound has evolved recently, to provide early detection of metastatic melanoma in regional lymph nodes. Ultrasound is clearly superior to clinical palpation of the nodes during follow-up and, when combined with guided fine-needle biopsy, allows the earliest possible surgical intervention for regional nodal metastases. In the future the use of ultrasound contrast agents may improve the sensitivity of ultrasound in the detection of very small metastatic deposits.     

Effect of an anti-CD54 (ICAM-1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice

We have shown that administration of a novel anti-CD54 monoclonal antibody (UV3) results in long-term survival of SCID mice bearing human myeloma xenografts. Previous studies have demonstrated a link between the expression of CD54 and the progression of uveal melanoma. Our study assessed the expression of CD54 on 7 human uveal melanoma cell lines and 3 cell lines established from uveal melanoma metastases. In vivo studies examined the efficacy of systemic and local administration of UV3 antibody on the progression of uveal melanoma cells transplanted either heterotopically or orthotopically into SCID mice. Five of the 7 primary uveal melanoma cell lines and all 3 of the metastases cell lines expressed CD54. Intraperitoneal injection of either IgG or F(ab')2 fragments of UV3 significantly inhibited the growth of subcutaneous and intraocular melanomas. Subconjunctival injection of either IgG or F(ab')2 fragments of UV3 produced a significant reduction in the growth of intraocular melanomas, even if the antibody was administered after the appearance of intraocular tumors. The results indicate that both primary and metastatic human uveal melanoma cells express CD54. The marked inhibition of intraocular and subcutaneous uveal melanoma progression suggests that UV3 antibody is a promising therapeutic agent for further evaluation in patients with uveal melanoma. This is especially noteworthy, as no existing therapeutic modality prevents metastasis of uveal melanoma or prolongs the survival of patients with uveal melanoma.     

Differences of ploidy status in progression of head and neck melanomas

Thirty-three common naevi, 26 dysplastic naevi, 58 primary melanomas of facial skin, 24 oral melanomas, 32 lymph nodes and 12 distant melanoma metastases were stained using Feulgen method to evaluate the ploidy status by image analysis GAS-200 system. Eight percent of common naevi, 22% of dysplastic naevi, 43% of facial melanomas, 65% of oral melanomas, 40% of lymph nodes with melanoma metastases and 66% of distant melanoma metastases were classified as aneuploid. In facial melanomas the percentage of aneuploid cases increased with Clark level. Survival time of patients was significantly shorter for aneuploid cases as compared to euploid ones (p <0.01).     

Evaluation of premalignant and malignant lesions during the induction of mouse melanomas

The objective of the present study was to evaluate the malignant and premalignant lesions that arise in C57BL/6 mice after treatment with 7,12-dimethylbenz(a)anthracene and croton oil. Tissues from 70 treated mice were evaluated by histological and transplantation techniques, and 17 (24%) were found to have malignant tumors. Eleven of the tumors were diagnosed as malignant melanomas, three as spindle cell sarcomas, and three as squamous cell carcinomas. The incidence of malignant melanomas (15.7%) in this group of mice was similar to that in our initial study on the induction of melanomas with 7,12-dimethylbenz(a)-anthracene and croton oil, in which two of 20 mice developed malignant melanomas. Mice that developed melanomas had been treated with croton oil for an average of 7 mo, and the mean latent period for tumor development was 11 mo. Seven of eight melanomas grew rapidly after transplantation to syngeneic C57BL/6 mice. Pigmented nevi and/or draining lymph nodes from nine of 11 mice with melanomas grew progressively after transplantation to athymic nude mice. Pooled nevi from one mouse with no apparent tumors grew into a histologically malignant melanoma after transplantation to a nude mouse. Nevi from three mice with sarcomas, one mouse with a carcinoma, and 42 tumor-free treated mice failed to grow in nude transplant recipients. Thus, only nevi from mice with either apparent or occult malignant melanomas exhibited progressive growth in nude mice. These results confirm that malignant melanomas can be induced in C57BL/6 mice at a regular, predictable rate and further indicate that this is an excellent system in which to study melanoma induction, progression, and therapy.     

Long-term prognostic significance of HSP-70, c-myc and HLA-DR expression in patients with malignant melanoma.

AIM: Use of molecular markers indicative of the tumour oncogenic potential and host response may enhance our prognostic information for more effective treatment of melanoma patients. The roles of HSP-70 protein, c-myc oncogene and HLA-DR antigen expression were examined in melanoma patients and related to prognostic factors, recurrence rate and long-term survival. METHODS: Forty patients with tumours thicker than 1 mm were included in this study. All had elective node dissection and were followed for at least 7 years. Twenty-two had microscopic nodal metastases. Both primary melanoma tumour and lymph nodes were examined for the immunohistochemical expression of HSP-70 protein, c-myc oncogene and HLA-DR antigen. RESULTS: Eighteen patients had a recurrence (45%) and 23 patients survived overall (57.50%). Positive HSP-70 expression was observed in 52.50% of the primary melanomas and was associated with improved overall survival, especially in the patient group with tumours > or = 1.5 mm (70%vs 26.70%, P=0.0159). C-myc oncogene was overexpressed in 47.50% and HLA-DR antigen in 42.50% of the primary melanomas, but no correlation with survival was observed. The expression profile of these molecular markers in the primary tumour did not predict the status of regional nodes. HLA-DR expression in lymph nodes was observed exclusively in the nodal tissue surrounding the metastatic melanoma tumour in five patients. CONCLUSIONS: The immunohistochemical expression profile of HSP-70 but not of c-myc oncogene or HLA-DR antigen in the primary melanoma tumour could be of certain value in the identification of patients with graver prognosis who may benefit from more aggressive therapeutic strategies. Copyright Harcourt Publishers Limited.     

Human IgM monoclonal anti-GD2 antibody: reactivity to a human melanoma xenograft

The cell membranes of human melanoma express a tumor-associated ganglioside, GD2. We previously established a human melanoma cell line, M14-A, that metastasizes to the lung, liver, skin, lymph nodes, and abdominal organs of nude mice in addition to forming ascites and pleural effusions. We also reported the successful in vitro production of human IgM monoclonal antibody to GD2. In the present study, we evaluated the GD2 expression of human melanoma cells at the primary and metastatic sites and their reactivity to human monoclonal anti-GD2 antibody in vivo. GD2 was expressed strongly on the melanoma cells from both primary and metastatic sites, except for cells from pleural effusions and ascites. When M14-A-bearing nude mice received systemic injections of the human monoclonal antibody, the anti-GD2 titer in the sera was reduced markedly at 2 hours, whereas the reduction was minimal in sera from tumor-free mice and mice bearing GD2-negative human M24 cells. The immune adherence test confirmed that antibody was fixed on cells of primary subcutaneous M14-A tumors and on their metastases to liver, lung, abdominal organs and skin. These results suggest that this large molecule protein can penetrate the blood-tumor barrier and bind immunologically to antigen-positive melanoma cells in vivo.     

The utility of ultrasound in patients with melanoma

The highest quality gray-scale ultrasound images are obtained with high-frequency transducers; however, such high frequencies do not penetrate more than a few centimeters into body tissue. Fortunately, in patients with melanoma, the structures of interest are close to the skin surface, making them ideal targets for examination with high-resolution ultrasound. These include primary cutaneous melanomas, uveal melanomas and the regional lymph nodes draining the skin that lie in the axilla, groin, neck and other locations. Although ultrasound study of primary melanomas arising in the skin and eye has provided some insights, a major role for ultrasound has evolved recently, to provide early detection of metastatic melanoma in regional lymph nodes. Ultrasound is clearly superior to clinical palpation of the nodes during follow-up and, when combined with guided fine-needle biopsy, allows the earliest possible surgical intervention for regional nodal metastases. In the future the use of ultrasound contrast agents may improve the sensitivity of ultrasound in the detection of very small metastatic deposits.     

A tumor-associated antigen expressed in melanoma cells with lower malignant potential

The antigen K-1-2, detectable by a MAb is found in nevi and melanomas. It is associated with melanoma cells of low invasive and metastatic potential as shown by immunoperoxidase studies with cell lines, biopsies and autopsies: K-1-2 occurs in melanoma cell line SK-Mel 25, but not in cell line A-375. A-375 has a higher malignant potential than SK-Mel 25 because, in contrast to SK-Mel 25, it produces plasminogen activator and grows in nude mice. K-1-2 was frequently strongly expressed (greater than or equal to 50% cells positive) in flat (less than 1.5 mm) and less frequently in medium and thick primary tumors. In thick primary melanomas K-1-2 positive cells were confined to the junctional zone or to marginal, flat areas of the tumor. Only rarely does K-1-2 occur in metastases. Strong expression of the K-1-2 antigen was found less often in primary melanomas, which develop early metastases, than in tumors that had not metastasized during an observation period of 18 months. In 5 patients with disseminated metastatic disease, metastases strongly expressing K-1-2 and those negative for this marker or containing only a minor percentage of K-1-2 positive cells were observed simultaneously or at different times. These findings suggest that a change from high malignancy to low malignancy--as observed in animal systems--may also occur in human melanoma.     

Spontaneous regional lymph node metastases of three variants of the B16 melanoma: relationship to primary tumor size and pulmonary metastases

We studied the patterns of spontaneous regional lymph node metastases of three variants (F1, F10, and BL6) of the B16 melanoma in C57BL/6 mice and related the incidence to primary tumor size and pulmonary metastases. The incidence of regional lymph node and pulmonary metastases correlated with increasing primary tumor size (p less than or equal to 0.0001). The incidence of pulmonary metastases in mice whose regional lymph nodes did not contain tumor also correlated with increasing primary tumor size (p less than or equal to 0.0001). This propensity for direct hematogenous spread was more apparent in BL6 tumors than in F1 and F10 tumors (p less than or equal to 0.0001). BL6 tumors also metastasized to regional nodes at smaller primary tumor sizes (p less than or equal to 0.04). Heterogeneous variants that metastasize earlier to regional lymphatic and hematogenous sites dictates the natural history of the primary tumor. Whether prophylactic lymphadenectomy for melanomas is therapeutic may depend on dissemination-related phenotypic characteristics.