Tuberous sclerosis complex (TSC) is an autosomal dominant genetic syndrome that was first described >150 years ago.1,2 The clinical manifestations are quite distinct, which include seizures and hamartomatous lesions involving many organs including the central nervous system (CNS), skin, heart, lung and kidneys.3 The diagnostic criteria of TSC includes a genetic diagnosis based on a pathogenic alteration in TSC1/TSC2 or a clinical diagnosis based upon major and minor features (Table 1).4

Tumors develop as a result of the inactivation of both alleles of either the TSC1 or TSC2 genes, which act as tumor suppressors, usually by a loss of function alteration, most of which are truncating nonsense, insertion or deletion mutations.1 Germline loss-of-function mutations will occur in TSC1/2 along with somatic loss-of-function of the residual wild-type allele in the tumors.5 TSC1 and TSC2 along with TBC1D7, form a complex which controls the activity of the MTOR complex 1 (MTORC1) by regulating GTPase activity and maintaining RHEB in its inactive form.1,6 Inactivation of TSC1/TSC2 eradicates the downregulation of MTORC1, resulting in cell growth, elevated protein translation and oncogenesis.1,7

The tumors in TSC affect multiple organ systems including:4,5,8

CNS: subependymal giant cell astrocytomas (SEGA), subependymal nodules (SEN), cortical dysplasias (tubers and white matter heterotopias)

Heart: cardiac rhabdomyomas

Kidney: renal cysts, angiomyolipomas (AML), renal cell carcinoma (RCC)

Lung: lymphangioleiomyomatosis (LAM), pulmonary cysts, micronodular pulmonary pneumocyte hyperplasia

Eye: retinal hamartomas, retinal achromic patch

Skin: angiofibromas or fibrous cephalic plaque, shagreen patches, ungual fibromas, “confetti” skin lesions, hypomelanotic macules

Bone and Dental: dental enamel pits, intraoral fibromas, bone cysts

Other: AML of other organs such as liver and adrenal gland, hamartomatous rectal polyps, thyroid papillary adenomas

TSC1 located on chromosome 9q34, results in the formation of hamartin and is mutated in approximately 20% of patients.1TSC2 located on chromosome 16q13, results in the formation of tuberin and is mutated in approximately 70% of patients.1 Patients with TSC2 mutations develop a more severe phenotype than those with TSC1 mutations. Approximately two-thirds of TSC cases are somatic or de novo and occur in the absence of a family history, while the remaining one-third is familial.1 Most of the remaining 10% of patients with clinical features of TSC likely have genetic mosaicism and can sometimes be detected at very low levels.1 Therefore, use of clinical criteria is recommended for the primary diagnosis of TSC.8

Though occurring in a minority of patients, SEGA is the most common CNS neoplasm identified in TSC patients and is considered a major criterion in the clinical diagnosis of TSC.9 SEGAs present within the first two decades of life and are often identified on routine radiologic screening but may be the initial presentation of TSC with symptoms of increased intracranial pressure or worsening seizure activity 10. They are well-circumscribed, multinodular tumors which typically arise within or adjacent to the lateral ventricles.11 SEN, defined by size of less than 5 mm, may be precursor lesions to SEGA in some cases.12 The presence of 2 or more SENs is also a major criterion for TSC diagnosis and warrants more frequent imaging to assess progression.9

Microscopically, SENs and SEGAs are composed of a mixture of glial and neuronal cells confirmed by immunohistochemical stains, leading some to suggest subependymal giant cell tumor (SGCT) as a more appropriate name.10 The glial cells predominate and have a variety of appearances, most commonly gemistocytic-like, epithelioid to polygonal cells with abundant eosinophilic and glassy cytoplasm and eccentric nuclei with prominent nucleoli. In other cases, the glial cells have a ganglionic appearance with abundant pink, granular cytoplasm and round, vesicular nuclei and occasional nuclear pseudoinclusions or a spindled appearance with long cytoplasmic processes gathering in fascicles (Figure 1A-C). The background blood vessels are dilated, hyalinized, and occasionally calcified. Scattered large pleomorphic, ganglion-like cells and an inflammatory infiltrate composed of lymphocytes and mast cells are common.11 SEGAs are benign (CNS WHO Grade 1), and—unlike other glial neoplasms—this benign designation does not change with the finding of mitotic activity, microvascular proliferation, or necrosis in the tumor.13 These tumors are positive for TTF-1 which has been found to be a helpful diagnostic marker.14,15 SEGA is treated surgically and more recently with mTOR inhibitors.16, 17, 18

Skin manifestations of TSC include angiofibromas on the face and ungual and subungual fibromas. These lesions are well-circumscribed, skin-colored papules, which often occur in a malar distribution.19 Three or more angiofibromas on the face and two or more ungual fibromas are major criteria for the clinical diagnosis of TSC.9

Microscopically, the lesions consist of a proliferation of dermal stromal cells with increased numbers of ectatic blood vessels and an overlying normal to atrophic squamous epithelium (Figure 1D).19 The stromal cells can have variable histologic features including multinucleation and a plump, spindled, or stellate morphology. Rare variants have been described including granular20 or clear cell,21,22 hypercellular, pigmented, pleomorphic, inflammatory, and epithelioid fibrous.19,23 Solar elastosis is notably absent within the lesion in these sun-exposed locations. Occasionally, a perivascular lymphocytic infiltrate is seen.19 TSC-associated fibromas are benign but can be treated with laser therapy or topical mTOR inhibitors for cosmetic reasons.24,25

Cardiac rhabdomyomas are the most common pediatric heart neoplasm and TSC patients account for most of these cases, particularly when multiple tumors are present.26 Grossly, the tumors are well-circumscribed, white to yellow intramural masses usually found in the ventricles.27 The white to yellow gross appearance is microscopically due to enlarged myocytes with abundant clear, glycogen-rich cytoplasm. In the characteristic “spider cell,” strands of sarcoplasm extend from the centrally located nucleus through the glycogen rich cytoplasm and connect to the cell membrane, similar in appearance to a spider web. Rhabdomyomas may regress spontaneously, be surgically excised, or treated with mTOR inhibitors.28, 29, 30

Lymphangioleiomyomatosis (LAM) is one of the major criterion for clinical diagnosis of TSC and affects approximately one-third of TSC patients.9,31 Among TSC patients, LAM develops in women more frequently and with more severity as compared to men.31,32 Grossly, LAM forms thin-walled cysts throughout the lungs bilaterally, which may coalesce as the disease progresses.33

Microscopically, two neoplastic cell types characterize LAM: 1) plump smooth muscle-like cells with eosinophilic, granular cytoplasm and 2) cuboidal cells with clear cytoplasm resembling perivascular epithelioid cells (Figure 2).34 LAM cells form subtle thickening to discrete small nodules along the cyst walls. The nodules may also abut or invade vasculature and lymphatics in the lung, leading to hemoptysis and chylothorax, respectively. If invasive into blood vessels, hemorrhage into alveolar spaces and hemosiderin-laden macrophages may also be seen histologically. Specifically in LAM due to TSC, foci of small (<1 cm) proliferations of type II pneumocytes termed multifocal micronodular pneumocyte hyperplasia can develop and may mimic atypical adenomatous hyperplasia or adenocarcinoma in situ morphologically.35,36 However, the bland nuclear features and lack of mitoses help to differentiate LAM from malignant precursors.

LAM falls within the spectrum of perivascular epithelioid cell neoplasms (PEComas), and immunohistochemical stains can be used to confirm the characteristic combination of smooth muscle and melanocytic differentiation.37 Lung function tests are used to track disease progression and lung transplantation may ultimately be required.38 mTOR inhibitors, particularly sirolimus, can slow progression.39

The most common cause of significant morbidity and mortality in patients with TSC is due to chronic kidney disease as a consequence of surgical removal or embolization of TSC-associated tumors such as AMLs, RCCs and epithelial cysts, which can result in hemorrhage, flank pain and hypotension.40, 41, 42 Approximately 80-85% of renal tumors in TSC patients are AMLs and are the most common renal abnormality and abdominal imaging is part of the current recommendations at diagnosis.9,40,41

Unveiling the genomic landscape of renal epithelial tumors through molecular testing has aided in their classification and has led to the discovery that many of the tumors identified in TSC patients have a sporadic counterpart. Therefore, these tumors not only display similar morphologic phenotype, but also harbor similar genetic alterations.43 Tumors in TSC harbor alterations in TSC1/TSC2, while mutations in other mTOR pathway related genes have also been additionally identified in their sporadic counterparts. Subtle clinical differences such as earlier age at diagnosis (<45 years of age), bilateral and multifocal involvement can provide clues to the potential association with TSC in addition to the characteristic morphology.1,5 With that in mind, it is important to remember these tumors are uniformly more common in the sporadic rather than germline or TSC setting.

Multiple renal cysts develop in ∼35-50% of TSC patients or as part of a contiguous gene deletion syndrome involving the PDK1 and TSC2 genes which are direct neighbors on chromosome of 16p13.3. Therefore, deletions involving both genes occur in a small subset (∼2-5%) of patients with TSC and display an aggressive polycystic kidney disease (PKD) phenotype.8,44 Microscopically, these are simple cortical cysts which are often lined by cuboidal or hobnail cells with more cytoplasm compared to the thinner cysts with flattened cells in adult PKD.40

AMLs are characterized by a combination of 1) smooth muscle, 2) thick-walled vessels and 3) mature adipose tissue, giving rise to the classic triphasic morphology of these tumors. More recently, it has been characterized as a proliferation of perivascular epithelioid cells and is now considered as a type of PEComa. These tumors can have varying amounts of each component, and biopsies are often performed when they are “fat-poor” in which the differential includes a carcinoma by imaging modalities. Occasionally, solid or muscle-predominant AMLs can have a cystic component lined by cuboidal to hobnail cells with a mullerian-like subepithelial stroma which has been termed AML with epithelial cysts (AMLEC) (Figure 3).45,46

Immunohistochemically, the tumor cells are positive for lysosomal markers like cathepsin K, melanocytic markers including HMB45 and Melan-A in varying amounts and muscle markers including SMA and desmin.45 The mullerian-like subepithelial stroma of the cysts/AMLEC, are often positive for ER, PR and CD10. The epithelial lining of the cysts/AMLEC are negative for melanocytic, muscular and hormonal markers but positive for epithelial markers (e.g. pancytokeratin and PAX8), which are uniformly negative in AML.45,46

Although AMLs are most commonly identified in the kidney, they have been observed in other organs and are a part of the major clinical criteria when 2 or more are present.8 Renal AMLs can cause significant morbidity with risk of bleeding due to the prominent vasculature resulting in need for hemodialysis and renal transplantation.8 By 2 years of age, ∼20% of children with TSC will develop AML and the incidence increases to ∼65-70% by age 10.40 There is often an upsurge in the growth rate of AMLs after 12 years of age.40 Alternative to surgery, these tumors can be treated with mTOR inhibitors such as sirolimus or everolimus.40 Although AMLs are considered a hallmark tumor of TSC, they are much more common in the sporadic setting than in patients with TSC and similarly harbor biallelic loss of TSC1/TSC2.1,47

Epithelioid AML which is now designated as epithelioid PEComa, are a less common variant in which it is composed of ≥80% epithelioid cells (Figure 4).4 They often show sheet-like growth of discohesive epithelioid cells which can display ganglion-like morphology and were mistaken for RCC in the past. A variety of studies evaluating features associated with more aggressive behavior have been performed but remain inadequately defined and include an association with TSC or concurrent AML, renal vein or extrarenal involvement, large tumor size (>7 cm), presence of coagulative tumor necrosis and elevated mitotic counts including atypical forms.48, 49, 50, 51 The relative risk of metastatic disease has varied, but a study with long clinical follow-up demonstrates it to be infrequent (5%).52 Molecular analysis has been done in a small subset of cases and have identified TSC2 alterations in the majority of cases (88%), as well as additional mutations in TP53, RB1 or ATRX.53,54 Similar to conventional AML, as an alternative to surgery, these tumors can be treated with mTOR inhibitors.54

The first series of renal cell carcinomas (RCC) associated with TSC, was published in 1996.3 Since that time, three larger contemporary series have been reported, and similarly show that TSC-associated RCCs occur at an earlier age at onset including children (mean age: ≤45 years), have a female predominance (7:3), and are frequently multifocal/bilateral.3,41,55,56 The true incidence of these TSC-associated renal epithelial tumors is unclear, but most report an occurrence of <5% in these patients, although it has been reported to comprise up to 37% of all renal epithelial neoplasia.3,41 Renal epithelial tumors associated with TSC are quite heterogeneous, which is different than other hereditary RCC syndromes in which there is often a predominant phenotype.1

In 2014, there were two groups that described a substantial series of TSC-associated RCCs, which laid the groundwork for the basis of the current classification. Yang and colleagues reported on 46 tumors from 19 TSC patients and classified them into 1)TSC-associated papillary RCC (PRCC)(52%), 2) hybrid oncocytic chromophobe tumors (HOCT) (33%) and unclassified RCCs (15%).55 The second by Guo et al, described 57 RCCs from 18 TSC patients and categorized them into 1) renal angiomyoadenomatous tumor (RAT)-like RCCs (30%), 2) chromophobe-like RCCs (59%) and 3) granular eosinophilic/macrocytic RCCs (11%).56 Despite the differences in designation, many displayed similar morphologic features and immunohistochemical profiles, leading to more uniform terminology and classification as supported by the World Health Organization (WHO).4

Currently, there are 4 main categories of tumors described in patients with TSC: 1) RCC with leiomyomatous stroma (RCC-LMS), 2) eosinophilic solid and cystic RCC (ESC-RCC), and 3) low-grade oncocytic tumor (LOT) and 4) eosinophilic vacuolated tumor (EVT) (Table 2).41, 55, 56, 57, 58 The remaining oncocytic tumors are either unclassified or display features of 1) oncocytoma, 2) chromophobe RCC (ChRCC) or 3) HOCT, the latter which was also termed TSC-associated oncocytic tumors.41

Renal tumors with clear cytoplasm have been described in patients with TSC using various terminology including TSC-associated PRCC, RCC-LMS or RCC with fibromyomatous stroma (RCC-FMS) and RAT-like RCC.41, 55, 56, 57 This is the most common subtype in these patients comprising ∼30-52% of all renal epithelial tumors.41,55,56 Despite the different designations, these tumors uniformly display similar morphologic and immunohistochemical features.

Histologically, these tumors exhibit 2 components. The first is an epithelial component displaying elongated tubuloalveolar to nested/solid, pseudopapillary or branching papillary architecture composed of cells with abundant voluminous clear to pale eosinophilic cytoplasm and well-defined cell membranes (Figure 5). The tumors also display a second component of prominent bands of fibromuscular/leiomyomatous stroma which is often present at the periphery and can surround and form nodules of tumor easily identified at low magnification.41, 55, 56, 57

Immunohistochemically, they are characteristically diffusely positive for CK7, show membranous and/or cup-shaped staining for CAIX, are often positive for CD10 and are absent of staining for CD117 and AMACR.41,43,55,56 Initially, SDHB was thought to be absent/deficient in these tumors, but later felt this was actually a false-negative result due to absent or weak staining relating to the presence of intracellular mitochondria which is commonly seen in the abundant clear cytoplasm that is characteristic of these neoplasms.55,59 Intermittently, these tumors can also display apical CK20 positivity.57

The tumors often exhibit features of either the International Society of Urology Pathology (ISUP)/World Health Organization (WHO) nuclear grade 2 (54%) or grade 3 (46%).55 Eosinophilic cytoplasmic hyaline globules have frequently been identified, imparting rhabdoid-like morphology, some which have interpreted this findings as ISUP/WHO Grade 4.41,55,60 However, most of these tumors behave indolently with rare occurence of metastatic disease.41,55,56,61,62 Interestingly, the few cases of metastatic disease reported in patients with TSC are best documented in this subtype of tumor with either a confirmed or documented genetic or clinical diagnosis of TSC.3,41,55,56, 63, 64, 65

Sporadic RCC-LMS have been found to harbor either TSC1/TSC2 or mTOR alterations.60, 61, 62,65 Additionally, a subset of morphologically and immunohistochemically analogous cases have been discovered to contain ELOC mutations and have been identified as a separate molecularly defined subtype of RCC in the WHO.60,61, 65, 66, 67, 68

These tumors have been initially described as granular eosinophilic/macrocystic RCC or unclassified RCC and is now termed eosinophilic solid and cystic RCC (ESC-RCC) and comprise ∼7-11% of all TSC-associated renal epithelial neoplasia.41,56 Histologically, these tumors are composed of solid sheets/nests and variably sized cysts of cells with abundant eosinophilic granular cytoplasm that can display basophilic cytoplasmic stippling imparting a leishmaniasis-like morphology (Figure 6).41,55,56 The cysts are often lined by a single layer of hobnail cells and may be associated with inflammation including foamy macrophages and psammomatous calcifications.57,69

Immunohistochemical studies reveal these tumors are often positive for CK20, at least focally in the majority of cases (Figure 7). Both CD117 and CK7 are typically negative, but when the latter is positive, it is often patchy/focal and to a less extent than the CK20. Additionally, they can be positive for vimentin as well as cathepsin K and Melan-A in a subset of cases.

Sporadic ESC-RCCs occur more frequently in females and similarly harbor alterations in TSC1/TSC2 (somatic bi-allelic loss of function mutations).69, 70, 71, 72 Most of the ESC-RCCs behave indolently, but there have been a few reports of metastatic disease, mainly in the sporadic setting or without confirmed documented history of TSC or TSC1/TSC2 gene alterations except for one which had a confirmed likely pathogenic TSC2 gene mutation.43,57, 73, 74, 75 Recently, Argani et al. reviewed oncocytoid RCCs after neuroblastoma or other childhood cancers and noted these neoplasms displayed comparable morphology to ESC-RCC and were also found to harbor TSC1/TSC2 gene alterations.76

A subset of oncocytic tumors were described as displaying chromophobe-like, HOCT or unclassified RCC features, and consistently demonstrated staining for CK7 with absence of staining for CD117, comprising up to 27% of all TSC-associated renal epithelial neoplasia.41, 55, 56, 57 Overall, these tumors uniformly display solid growth with low-grade oncocytic cells containing round to oval nuclei, eosinophilic cytoplasm and frequent perinuclear clearing (Figure 8). These tumors typically lack the significant nuclear irregularities seen in ChRCC and can contain intermixed cords or islands of edematous stroma.77, 78, 79, 80 In addition to the hallmark immunoprofile of these tumors (CK7+; CD117-), they are additionally positive for GATA3 and typically absent of staining for cathepsin K, CK20, vimentin and FOXI1.78, 81, 82, 83 All of the cases reported to date have behaved in a benign fashion, without evidence of metastatic disease, supporting this is a tumor as the name implies.66 Sporadic LOTs are more commonly identified in females and harbor alterations in TS1/TSC2, RHEB or mTOR, the latter being most common, resulting in mTOR pathway activation.70,81,83

EVT was first described in 2018 as “high-grade oncocytic tumor (HOT)” by He and colleagues and later as “sporadic RCC with eosinophilic and vacuolated cytoplasm” in 2019 by Chen et al., from an assembly of tumors that displayed similar features to ChRCC and RO.84,85 Akin to the other tumors discussed, it was noted that these neoplasms displayed similar morphologic features and subsequently molecular alterations, and were finally designated as EVTs.66, 85, 86, 87

In the contemporary series of TSC-Associated RCCs, rare (<5%) tumors displaying solid nests and compact tubulocystic lobules of cells with eosinophilic cytoplasm containing pronounced vacuolization, enlarged nuclei and prominent nucleoli were identified, analogous to EVTs.41,56,57,88 These tumors are well-circumscribed but unencapsulated with thick-walled vessels at the periphery.84 Occasionally, they can contain loose intermixed stroma.84

These tumors are typically positive for CD117, CD10 and cathepsin K and negative or with only rare scattered cells staining for CK7 and CK20.41,84 Despite the nuclear grade being equivalent to what would be considered high-grade (WHO/ISUP Grade 3 or 4), they behave indolently, supporting the designation as a tumor rather than carcinoma.66,84,85 Sporadic EVTs, similar to LOTs, more frequently occur in females and reliably harbor mutually exclusive mutations in TSC1, TSC2 or mTOR resulting in activation of the mTOR pathway.85,86,89

The remaining tumors that do not fit in the LOT or EVT category, and display characteristic features of renal oncyctoma (RO), chromophobe RCC (ChRCC) and HOCT comprise the final group of oncocytic tumors. HOCT is a term reserved for tumors that display morphologic characteristics of RO and ChRCC in varying amounts and are therefore called hybrid tumors.55,90 For instance, most of the oncocytic tumors in one of the larger series of TSC-associated RCC were described as HOCT, with all tumors tested in this category being CD117 positive. Many of these HOCTs displayed either a pattern in which it was composed of polygonal eosinophilic cells with central round nuclei (RO-like) which also displayed perinuclear clearing/halos (ChRCC-like) or had an admixing of scattered nests of ChRCC-like cells in the background of typical RO (Figure 9). Additionally, HOCT is a designation that should only be reserved for tumors arising in patients with a hereditary syndrome, and not in a sporadic setting.66 The term “oncocytic renal neoplasm of low malignant potential, not further classified” has been recommended for these hybrid tumors that occur sporadically.66 Immunohistochemically, they are often positive for CD117, but with variable staining for CK7.55,90