Combined Merkel cell carcinoma and cutaneous squamous cell carcinoma with lymph node metastases: Report of two cases.

Merkel cell carcinoma (MCC) is an infrequent, aggressive cutaneous neoplasm, that typically affects the photodamaged skin of elderly individuals, and immunosuppressed patients. Because a subset of MCC is closely related to UV radiation, MCC can develop concurrently with other tumors, most commonly, as a combined tumor with squamous cell carcinoma (SCC). These combined tumors appear to represent a distinct disease process from pure MCC, as they are mostly Merkel cell polyomavirus (MCPyV) negative, and show a more aggressive behavior. We present two additional cases of combined MCC and SCC with nodal metastases, one of which was MCPyV positive. Two different subtypes of MCC have been proposed based on their origin: a true neuroendocrine carcinoma, that is MCPyV positive and has a dermal origin, and a UV-related SCC with neuroendocrine differentiation. This theory could explain why MCC can develop concurrently with SCC, and why these combined cases are generally MCPyV negative. However, it fails to explain the minority of combined MCC and SCC tumors that are MCPyV positive. Because both our patients had a history of chronic UV exposure, we hypothesize that UV radiation probably played a major role in the pathogenesis of these tumors, while MCPyV integration probably acted as an additional trigger.

Superficial GLI1-amplified mesenchymal neoplasms: Expanding the spectrum of an emerging entity which reaches the realm of dermatopathology.

Mesenchymal neoplasms with GLI1 alterations (rearrangements and/or amplification) have been reported recently in several anatomic locations, which include head and neck, soft tissue, and gastrointestinal tract. Herein, to the best of our knowledge, we describe the first three cases of superficial/subcutaneous mesenchymal neoplasm with GLI1 amplification. The neoplasms exhibited low-grade cytologic features with predominant round cell morphology, glomangioma-like areas and a rich background capillary network. There were two to three mitotic figures per 10 HPF and focal necrosis in one case. The tumors exhibited variable expression of CDK4, MDM2, STAT6, D2-40, CD56 and cyclin D1. p16 had strong and diffuse nuclear and cytoplasmic expression in two cases. Numerous other stains were negative. Fluorescence in situ hybridization detected GLI1, DDIT3, and CDK4 coamplification in all cases, while next generation sequencing did not detect a GLI1 gene fusion. The overall features were compatible with a GLI1-amplified mesenchymal neoplasm. In Case 1 a new distant skin lesion appeared 1 month after the surgery exhibiting similar morphology albeit with a higher mitotic index. In Cases 2 and 3, there is no evidence of local recurrence or systemic disease after 8 years and 1 month of follow-up, respectively. These new cases of superficial GLI1-amplified neoplasm expand its clinical spectrum and enter the realm of dermatopathology. The combination of CDK4, cyclin D1, D2-40, and p16 expression with variable MDM2, STAT6, CD56, and S100 immunoreactivity in a low-grade neoplasm with round/ovoid cytomorphology resembling a vascular or adnexal neoplasm may suggest the possibility of GLI1-amplified neoplasm.

Trichoepithelioma on the Finger: A Rare Location and Dermoscopic-Histopathologic Correlation.

ABSTRACT: Trichoepithelioma is a benign adnexal neoplasm of follicular germinative cells, with bulbs, papillae, and sheaths of perifollicular connective tissue as signs of follicular differentiation. Accordingly, trichoepithelioma may arise in any hair-bearing location, mostly on the face. That is why trichoepithelioma cannot appear in glabrous skin, and, although the dorsum of the hands and feet are a hair-bearing area, acral location is exceptional. We report the first case of trichoepithelioma localized in the finger of a 79-year-old man. It was a brown-pink, firm, 7-mm diameter, solid papule on the dorsal aspect of his left index finger. The lesion was completely asymptomatic, and he remembered to have it for many years. We describe this case highlighting its rare anatomical location and correlate its dermoscopic features with the histopathological appearance.

Differences by Anatomical Site of Non-Acral Lentiginous Melanomas of the Lower Limb.

BACKGROUND: Acral location of melanomas is associated with poor survival. It can be due, at least in part, to the fact that acral lentiginous melanoma, a distinct melanoma subtype, has a particular biological profile and a bad clinical behavior. However, since almost 50% of acral melanomas are not of acral lentiginous melanoma subtype, the worse clinical behavior could also be attributable to the intrinsic characteristics of the location. OBJECTIVE: This study aimed to investigate if melanomas of the lower limb excluding acral lentiginous melanoma differ by location. METHODS: This retrospective, observational study recruited patients from an oncology referral center in Spain. We included 285 patients with superficial spreading and nodular melanomas of the lower limb. We compare melanomas by site, clinical and pathological characteristics, and the differences by location of disease-free and melanoma-specific survival by the Kaplan-Meier method and Cox proportional hazard method. RESULTS: Patients with melanomas on the foot, compared to those on the rest of the limb, were older and reported having suffered less sunburns; the melanoma more frequently appeared in areas that had been rarely sun exposed, were more frequently of nodular type, presented thicker tumors, with more ulceration, less regression, and more advanced stage of the disease. Foot location increased the risk of relapse and decreased melanoma-specific survival. CONCLUSION: Melanoma development in foot is less related to sun exposure and is associated with pathological features that can account for the worse prognosis and poorer survival.

Cutaneous Ossifying Amyloidoma.

ABSTRACT: Amyloidoma, otherwise known as tumoral amyloidosis, is a localized deposition of amyloid (AL-type or AA type) without systemic amyloidosis. It is the rarest form of tissue amyloid deposition, and up to 7% of amyloidomas develop systemic amyloidosis.Cutaneous AL-type amyloidoma is considered by many authors as an unusual variant of primary cutaneous marginal zone lymphoma. Although cutaneous amyloidoma can form calcifications, ossification is extremely unusual, with only 1 case previously published to date.We report the case of a 75-year-old woman with voluminous and strikingly ossifying AL-type amyloidoma in the left pretibial skin. Her medical history included excision of hepatic hydatidic cysts 25 years prior and diffuse large B-cell lymphoma of the left parotid gland 8 years prior treated with chemotherapy and radiotherapy with complete response. After the diagnosis of amyloidoma, an extension study with cervical, chest, abdominal, and pelvic TC was performed, with no additional lesions found. Serum and protein electrophoresis revealed elevations in kappa light chain and IgA immunoglobulin levels but did not reveal monoclonal bands. In situ hybridization for immunoglobulin light chains showed monotypic kappa expression in plasma cells infiltrating the amyloidoma.Extensive ossification in amyloidomas can make diagnosis difficult; therefore, we describe an interesting case of this histopathologically peculiar amyloidoma.

Adherence to Primary Prevention and Skin Self-Examination Practices by Spanish Melanoma Patients.

BACKGROUND: The incidence of cutaneous melanoma, an important global public health problem, has been increasing over the last several decades. OBJECTIVES: In order to decrease melanoma-related mortality, ways to communicate and implement the correct methods for conducting primary and secondary prevention measures (such as early detection via self-examination) should be investigated. MATERIALS AND METHODS: An observational, cross-sectional, retrospective study consisting of 409 patients diagnosed with cutaneous melanoma was conducted. An online questionnaire was created to evaluate knowledge levels, attitudes, and adherence to primary preventive measures and to skin self-examination practices. RESULTS: The results revealed that even when 43% of the patients perform cutaneous self-examinations, only half of them fully followed the recommendations. Patients aged <45 years, female, with a I-II phototype, with an intermediate/high level of education, and with a history of NMSC were more likely to have an adequate degree of knowledge. Moreover, patients aged <45 years and with an adequate degree of knowledge more frequently showed an adequate adherence to the primary prevention measures. Finally, patients aged 45-60 years and with an adequate degree of knowledge presented a good adherence to the self-skin examination measures. LIMITATIONS: Possible limitations of this study were memory bias through the influence of age within the study population, and bias due to a greater proportion of subjects with a high education level. CONCLUSION: Within the population of patients with melanoma, a high percentage of patients do not rigorously follow the recommended prevention measures. Our study highlights the need to implement awareness in this population to improve the prevention of cutaneous cancer.

Usefulness of ultrasound in dermatofibrosarcoma protuberans and correlation with histopathological findings: A series of 30 cases.

BACKGROUND: Small series of ultrasound findings in dermatofibrosarcoma protuberans (DFSP) have been published, but the usefulness of this technique as a preoperative planning tool for tumor resection has not been studied. MATERIALS AND METHODS: We retrospectively reviewed patients with DFSP at our hospital that underwent ultrasound examination. Depth of invasion was evaluated by ultrasound and histopathology. Accuracy of ultrasound for assessing depth of tumor invasion was estimated. RESULTS: Thirty histopathologically confirmed DFSPs were studied. Classic finger-like projections were observed in 73.3% of cases. A posterior hyperechoic area extending deep into the subcutaneous tissue correlated with the honeycomb DFSP pattern and was observed in 53.3% of patients. Concordance between ultrasound and histopathologic depth measurements was excellent. Lateral tumor extension and Doppler activity were not evaluated in our series. CONCLUSION: Ultrasound showed excellent prediction of depth of invasion. Further studies are required to define the usefulness of ultrasound for determining lateral tumor extension.

Locoregional Lymph Node Recurrence of Trunk Melanoma in Non-sentinel Lymph Node Basins: An Observational Retrospective Study.

Locoregional lymph node recurrences of primary trunk melanoma can occur in basins not identified during sentinel lymph node biopsy. However, the factors associated with recurrences in non-sentinel lymph node basins are unknown. To evaluate these factors, this observational retrospective study examined the patterns of first lymph node recurrence and the factors associated with recurrence in non-sentinel lymph node basins. A total of 305 patients with primary trunk melanoma who had undergone sentinel lymph node biopsy from 2000 to 2015 were evaluated. Twenty-three patients presented locoregional lymph node recurrence; 8 of which (34.8%) were in non-sentinel lymph node basins. Non-sentinel lymph node recurrences were more frequent in patients with positive sentinel lymph nodes and in those patients whose number of tumour-involved nodes was > 3. These results suggest that clinical examination and ultrasound surveillance should be performed on all potential lymph node drainage basins of trunk melanomas.

Solid Microcystic Adnexal Carcinoma on the Thigh: An Unusual Location.

Solid carcinoma, probably the solid variant of microcystic adnexal carcinoma, is an apocrine adnexal tumor first described in 1998. The authors report an additional new case of the tumor at an unusual localization. A 78-year-old man presented with an asymptomatic firm plaque on his right thigh that had been present for 15 years. A biopsy was taken, and then, the lesion was removed. A pathological study showed a huge number of islands made up of aggregations of neoplastic epithelial cells. The epithelial islands showed variable sizes and shapes at scanning magnification, arranged columns, cords, and strands at the basis of the tumor. The neoplastic cells were embedded within a fibrotic stroma. Ductal differentiation, cystic structures, and neurotropism were also observed. Immunohistochemically, the neoplastic cells expressed high-molecular-weight keratin (cytokeratin 5/6), broad-spectrum keratin (AE1/AE3), p40, and p63. No immunoreactivity was found for BerEP4, cytokeratin 7, cytokeratin 19, cytokeratin 20, chromogranin A, carcinoembryonic antigen, and S-100. The lesion was completely removed with slow-Mohs micrographic surgery. Two stages and previous debulking were necessary to obtain free margins. The second stage included the muscular fascia. The patient remains free of tumor after a year of follow-up. Solid microcystic adnexal carcinoma is a rare skin tumor that seems to occur more frequently in the scalp than in the face, but no area of the body can be excluded, as reported in our case. Differential diagnosis should include sclerosing and clear-cell basal cell carcinoma, clear-cell dermal duct tumor, or desmoplastic trichoepithelioma. Mohs micrographic surgery is the treatment of choice.

Multiple facial plaque variant of trichoblastoma.

BACKGROUND: The plaque variant of trichoblastoma has been described as a solitary tumor with diffuse infiltration of the lower dermis and hypodermis, with poorly defined borders. Herein, we report a new variant of multiple centrofacial trichoblastoma. OBJECT: To describe clinical and pathological features of a new multiple kind of plaque variant of centrofacial trichoblastoma. METHODS: Case series of patients with a multiple-plaque variant of centrofacial trichoblastoma treated in our department between 2005 and 2017. We identified eight patients with the centrofacial plaque variant of trichoblastoma treated in our department from 2005 to 2017. RESULTS: The final study sample comprised 13 trichoblastomas from four patients. All patients also developed at least one basal cell carcinoma. Mohs surgery was the method of treatment in the majority of the cases of trichoblastoma and in all the cases of basal cell carcinoma. We needed between 2 and 6 stages to obtain free margins in our cases of facial plaque trichoblastomas treated by Mohs surgery. CONCLUSION: To the best of our knowledge, a multiple-plaque variant of trichoblastoma has not been described in the literature. We suggest a genetic origin of this variant of trichoblastoma and describe its remarkable infiltrative nature, with poorly defined surgical margins.

Telomere length, telomerase reverse transcriptase promoter mutations, and melanoma risk.

Telomere repeats at chromosomal ends, critical for genomic integrity, undergo age-dependent attrition and telomere length has been associated with different disorders including cancers. In this study, based on 1469 patients and 1158 healthy controls, we show a statistically significant (P = 6 × 10-10 ) association between increased telomere length and melanoma risk. Mendelian randomization, using 5 telomere length-associated polymorphisms, ruled out confounding factors or reverse causality and showed association between increased telomere length and melanoma risk with odds ratio of 2.66 (95% confidence interval: 2.07-3.25). Age-dependent telomere attrition was faster in melanoma cases than controls (P = .01). The carriers of a highly penetrant germline -57A>C TERT promoter mutation, in a previously reported melanoma family, had longer telomeres than the noncarriers. The mutation causes increased TERT and telomerase levels through creation of a binding motif for E-twenty six (ETS) transcription factors and the carriers develop melanoma with an early age of onset and rapid progression to metastasis. In analogy, we hypothesize that increased telomere length in melanoma patients reflects stochastic increased telomerase levels due to common genetic variation. Paradoxically, we observed shorter telomeres (P = 1 × 10-5 ) in primary tumors from unrelated melanoma patients with (121) than without (170) somatic TERT promoter mutations that similar to the germline mutation, also create binding motifs for ETS transcription factors. However, the age-dependent telomere attrition was faster in tumors with the TERT promoter mutations than in those without such mutations. Besides a robust association between increased telomere length and risk, our data show a perturbed telomere homeostasis in melanoma.

Sentinel lymph node biopsy versus observation in thick melanoma: A multicenter propensity score matching study.

The clinical value of sentinel lymph node (SLN) biopsy in thick melanoma patients (Breslow >4 mm) has not been sufficiently studied. The aim of the study is to evaluate whether SLN biopsy increases survival in patients with thick cutaneous melanoma, and, as a secondary objective, to investigate correlations between survival and lymph node status. We included 1,211 consecutive patients with thick melanomas (>4 mm) registered in the participating hospitals' melanoma databases between 1997 and 2015. Median follow-up was 40 months. Of these patients, 752 were matched into pairs by propensity scores based on sex, age, tumor location, histologic features of melanoma, year of diagnosis, hospital and adjuvant interferon therapy. The SLN biopsy vs. observation was associated with better DFS [adjusted hazard ratio (AHR), 0.74; 95% confidence interval (CI) 0.61-0.90); p = 0.002] and OS (AHR, 0.75; 95% CI, 0.60-0.94; p = 0.013) but not MSS (AHR, 0.84; 95% CI, 0.65-1.08; p = 0.165). SLN-negative patients had better 5- and 10-year MSS compared with SLN-positive patients (65.4 vs. 51.9% and 48.3 vs. 38.8%; p = 0.01, respectively). As a conclusion, SLN biopsy was associated with better DFS but not MSS in thick melanoma patients after adjustment for classic prognostic factors. SLN biopsy is useful for stratifying these patients into different prognostic groups.

Dysplastic vs. Common Naevus-associated vs. De novo Melanomas: An Observational Retrospective Study of 1,021 Patients.

The aim of this case-case study was to determine the differences between dysplastic and common naevus-associated melanomas (NAM) and de novo melanomas. A total of 1,021 prospectively collected patients with invasive cutaneous melanoma from an oncology referral centre were included in the study. Of these, 75.51% had de novo melanomas, 12.93% dysplastic NAM, and 11.56% common NAM. Dysplastic NAM, compared with de novo melanomas, were associated with intermittently photo-exposed sites, atypical melanocytic naevi, decreased tumour thickness, and presence of MC1R non-synonymous variants. Common NAM were more frequent on the trunk and of superficial spreading type. Comparison of dysplastic with common NAM showed significant difference only with regard to mitoses. Both subtypes of NAM shared less aggressive traits than de novo melanomas, albeit with no significant differences in survival after multivariate adjustment. In conclusion, NAM present with less aggressive traits, mostly due to a greater awareness among patients of changing moles than due to their intrinsic biological characteristics.

Immunohistochemical and Fluorescence In Situ Hybridization Analysis of MYC in a Series of 17 Cutaneous Angiosarcomas: A Single-Center Study.

Cutaneous angiosarcoma (AS) is an uncommon, aggressive sarcoma whose incidence is rising because of the increasing use of radiation therapy, especially in breast cancer. The few studies on the relevance of prognostic factors, such as MYC status in cutaneous AS, have reported inconclusive findings, with some authors reporting MYC amplification only in postirradiation and lymphedema-associated AS and others reporting evidence of MYC amplification in idiopathic AS. We analyzed 17 cases of cutaneous AS (6 idiopathic AS, 10 postirradiation AS, and 1 lymphedema-associated AS) treated at our institute between 2000 and 2015. Follow-up data were available in all cases. We compared the presence/absence of MYC amplification by fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) MYC overexpression in the different AS subtypes. We also investigated potential associations between MYC amplification and prognosis. MYC amplification was observed by FISH in 6 of 14 informative cases. The positive cases were all secondary AS (5 postirradiation AS and 1 lymphedema-associated AS). IHC detected MYC overexpression in 8 of 15 informative cases (7 secondary AS and 1 idiopathic AS). In conclusion, MYC amplification and MYC overexpression were detected almost exclusively in secondary AS. No associations were found between MYC amplification/overexpression and prognosis. We found MYC amplification or overexpression in a similar proportion of the patients who died and who were still alive at the end of the study. In the group of 9 patients who died, MYC was detected by FISH in 4 cases and by IHC in 5. The corresponding figures in the group of 6 patients still alive were 2 by FISH and 3 by IHC.

Subcutaneous dermatofibrosarcoma protuberans, a rare subtype with predilection for the head: A retrospective series of 18 cases.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) typically affects the dermis and subcutaneous tissue. The subcutaneous variant is rare. OBJECTIVE: We sought to characterize the subcutaneous DFSP (SC-DFSP) variant and compare it with cutaneous DFSP (C-DFSP). METHODS: This work was a retrospective study of DFSP treated in our institution. RESULTS: Of 124 cases of DFSP, 18 were SC-DFSP (14.5%). Except for the deep location, the pathologic and genetic features were indistinguishable from the C-DFSP variant. Histologically, of 18 SC-DFSP cases, 13 were classic DFSP, 3 fibrosarcomatous DFSP (FS-DFSP), 1 Bednar tumor, and 1 giant-cell fibroblastoma. All tumors expressed CD34 and the COL1A1-PDGFB fusion transcripts. In our series, higher proportions of SC-DFSP tumors (61%) than C-DFSP tumors (8.5%) were located on the head (P < .001). Of the 20 DFSP tumors on the head (16.1%), 11 were SC-DFSP and 9 were C-DFSP. In addition, half the SC-DFSP tumors affected muscle or periosteum, compared with a quarter of the C-DFSP tumors (P = .009). SC-DFSP needed a higher number of Mohs stages than did C-DFSP (P = .009). Median follow-up time was 63 months, and 2 FS-DFSP tumors recurred (1 SC-DFSP, 1 C-DFSP). LIMITATIONS: Limitations include the retrospective aspect of the study. CONCLUSIONS: Most DFSP tumors involving the head were subcutaneous and required more complex surgery. Dermatologists should be aware of this atypical presentation, especially in lesions involving the head.

Hereditary progressive mucinous histiocytosis: 3 different phenotypes in 3 family members.

We describe 3 cases of multiple histiocytic cutaneous tumors that began in childhood and affected 3 members from 2 generations of the same family: a mother, a daughter and a nephew. The lesions were mostly skin-colored papules distributed symmetrically on the dorsum of the forearms and hands and on the face and thighs. There were no signs of spontaneous regression. The clinical and histopathological features were consistent with a diagnosis of hereditary progressive mucinous histiocytosis (HPMH), but phenotypic expression varied somewhat between the 3 patients. HPMH has only been described in 8 families to date, and just one of the reports included 3 well-documented cases. Our cases confirm that HPMH can affect males and expands the clinical spectrum of skin lesions in this disease.

Desmoplastic melanoma may mimic a cutaneous peripheral nerve sheath tumor: Report of 3 challenging cases.

Desmoplastic melanoma (DM) and cutaneous malignant peripheral nerve sheath tumors (MPNST) reveal histological and immunohistochemical similarities, including S100 positivity and negative staining for conventional melanocytic markers. We present 3 cases of cutaneous S100-positive spindle cell tumors in elderly patients, in which first findings led to initial misdiagnoses as cutaneous MPNST and benign peripheral sheath nerve tumor (neurofibroma). The identification of adjacent atypical melanocytic hyperplasia in the overlying skin along with tumor cell proliferation, also in the superficial dermis, the neurotropic component and the absence of any relationship between the tumor and a major nerve, pre-existing neural benign tumor or the existence of stigmata suggestive of neurofibromatosis raised consideration of a DM. Careful attention should be paid to the presence of a firm dermal nodule and atypical scar lesions especially in sun-exposed areas (mainly head and neck region) in elderly patients associated with S100-positive spindle cell proliferation, solar elastosis and adjacent atypical melanocytic proliferation. In such cases, the possibility of a DM should be excluded with caution, especially if the tumor reveals a paucicellular morphology resembling various non-melanocytic neoplasms including malignant or benign peripheral sheath nerve tumors.

TERT promoter mutations in melanoma survival.

Despite advances in targeted therapies, the treatment of advanced melanoma remains an exercise in disease management, hence a need for biomarkers for identification of at-risk primary melanoma patients. In this study, we aimed to assess the prognostic value of TERT promoter mutations in primary melanomas. Tumors from 300 patients with stage I/II melanoma were sequenced for TERT promoter and BRAF/NRAS mutations. Cumulative curves were drawn for patients with and without mutations with progression-free and melanoma-specific survival as outcomes. Cox proportional hazard regression models were used to determine the effect of the mutations on survivals. Individually, presence of TERT promoter and BRAF/NRAS mutations associated with poor disease-free and melanoma-specific survival with modification of the effect by the rs2853669 polymorphism within the TERT promoter. Hazard ratio (HR) for simultaneous occurrence of TERT promoter and BRAF/NRAS mutations for disease-free survival was 2.3 (95% CI 1.2-4.4) and for melanoma-specific survival 5.8 (95% CI 1.9-18.3). The effect of the mutations on melanoma-specific survival in noncarriers of variant allele of the polymorphism was significant (HR 4.5, 95% CI 1.4-15.2) but could not be calculated for the carriers due to low number of events. The variant allele per se showed association with increased survival (HR 0.3, 95% CI 0.1-0.9). The data in this study provide preliminary evidence that TERT promoter mutations in combination with BRAF/NRAS mutations can be used to identify patients at risk of aggressive disease and the possibility of refinement of the classification with inclusion of the rs2853669 polymorphism within TERT promoter.

Skin involvement as the first manifestation of breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (ALCL) is a newly described clinical and pathologic entity that typically presents as seroma in the fibrous scar around the implant. Less frequently, it presents as a solid peri-implant mass, and there have been no reports to date of cutaneous lesions as the presenting manifestation. We report the case of a 56-year-old woman with a history of bilateral breast reconstruction following breast cancer of the right breast who consulted with several papules on the right breast suggestive of metastasis. Histopathology showed a proliferation of large epithelioid lymphocytes with highly pleomorphic cells and nuclei. The neoplastic cells were CD15 and CD30 positive and ALK-1 negative. The epithelial markers were all negative except for epithelial membrane antigen (EMA), which was weakly positive. Molecular analysis showed monoclonal T-cell receptor γ gene rearrangement, confirming a diagnosis of breast implant-associated ALCL. The non-specific morphology of the skin lesions, the epithelioid nature of the neoplastic cells and the expression of EMA can lead to an erroneous diagnosis of skin metastases from a poorly differentiated adenocarcinoma of the breast. We recommend immunohistochemical staining for CD30 and ALK-1 for patients with breast implants who develop anaplastic lesions.