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Keywords:

  • chromophobe renal cell carcinoma;
  • diagnosis;
  • electron microscopy;
  • histology;
  • molecular markers

Abstract

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

The recognition of chromophobe renal cell carcinoma (RCC) among other distinct types of renal cell tumors (RCT) based on light-microscopic features, such as cytoplasmic and nuclear characteristics, might pose a dilemma in some cases because of morphological pattern overlapping with renal oncocytoma or conventional RCC. The present article reviews chromophobe RCC with focus on aspects of its molecular pathology, which was shown using ancillary modern microarray-based technology that can distinguish it from its mimics and therefore be helpful for its correct diagnosis. Although the high resolution DNA-microarray analyses excluded with all certainty the occurrence of small specific alterations, the loss of entire chromosomes 2, 10, 13, 17 and 21 occurs exclusively in chromophobe RCC and therefore probes localized at these chromosomes might be used to establish the diagnosis of chromophobe RCC in cases with uncertain histology. The usefulness of proposed candidate genes selected by the global gene expression analyses in the diagnostic pathology is far below expectations. The conflicting staining patterns, together with the poor specificity of used antibodies, leads us to believe that these candidate immunomarkers might not help in the separation of chromophobe RCC, with the exception of CD82, which has recently been suggested to be used for routine histological diagnosis.


Prehistory and epidemiology

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Based on its morphological and electron-microscopic characteristics, chromophobe renal cell carcinoma (RCC) was delineated among experimental kidney tumors nitrosomorpholine-induced in rats.1 In 1985, Thoenes et al. described the first human renal carcinomas as an analogy to the cases in rodents showing similar morphology.2 They noticed a 4.6% incidence of the chromophobe variant in a series of 697 renal cell carcinoma cases. Initially, diagnosis of chromophobe RCC appeared to be straightforward as a result of its characteristic light microscopic features. The same authors 3 years later described an eosinophilic variant of chromophobe RCC, which can easily be confused with renal oncocytoma or “eosinophilic” clear cell carcinoma based on morphological characteristics.3 The latter discovery is also related to its close phenotypic similarity to “eosinophilic” clear cell carcinoma on the one hand and to oncocytoma on the other hand. Presently, chromophobe RCC is considered to account for approximately 5% of all cases of renal cell carcinoma. The mean age of incidence with chromophobe RCC in a series of 104 patients reported by Cindolo et al.4 was in the sixth decade (58 years), with a range in age of 27–86 years, and the number of men and women was roughly equal. Mortality was less than 10%.

Clinical symptoms, radiological findings and treatment

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Flank discomfort or pain, gross hematuria, flank mass and weight loss are observed as symptoms in patients with chromophobe RCC.5,6 On imaging, these are mostly large, solitary, (compact) solid masses without necrosis or calcifications.6,7 Nowadays, the diagnosis of small renal neoplasms might be suggested by computed tomography (CT), ultrasound examination (US) and magnetic resonance imaging (MRI). The correlation of double-phase helical CT appearances and pathological findings for different histological types of small renal cell tumors has been clearly shown.8

Macroscopy

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Depending on size, chromophobe RCCs are well-circumscribed solid neoplasms and consist of one or more solid tumor nodules with a slightly lobulate surface.9 In unfixed conditions, the cut surface appears homogeneously light brown or tan, turning light gray after formalin fixation.10 The uniform pale cut surface might be interspersed with a few hemorrhages. However, hemorrhagic or necrotic areas are rare (∼15%) and are generally centrally located. In general, the growth pattern is solid and compact, sometimes cribriform, associated with focal scarring and sclerosing, and associated with deposition of psammoma-like calcifications. Tumor infiltration by inflammatory cells is uncommon.

Tumor location and size

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Chromophobe RCC is usually located in the renal cortex. The presence of cystic areas, as well as multifocality (10–12%), is rare. A propensity for a greater tumor size compared with clear cell or papillary tumors has been suggested.11

Histopathology

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

By light microscopy, chromophobe cells have slightly opaque or finely reticular cytoplasm when stained with hematoxylin–eosin (HE). There are two cytomorphological variants of chromophobe RCC, a classic form with clear cells and a so-called “eosinophilic” type. However, both cellular forms might occur within the same tumor. Furthermore, there is no strict border between typical chromophobe cells and eosinophilic form; the tumors in large series display a continuous spectrum of cellular variation. The typical chromophobe cell type (Fig. 1a) can be easily mistaken for conventional RCC, likewise showing tumor cells with a highly transparent cytoplasm, on routine histology. Usually, chromophobe cells are large polygonal cells, showing a fine reticular cytoplasm and central round to oval nucleus,3 and are arranged in compact broad alveoli with or without tubular or tubulocystic areas. The cytoplasm is generally denser in the periphery of the cell, making membranes appear thick. The characteristic pale “chromophobe” staining is a result of the presence of numerous cytoplasmic vesicles and the absence of mitochondria. These cells are commonly mixed with smaller cells with granular eosinophilic cytoplasm, which contain more pronounced acidophilia (eosinophila)/granularity of cytoplasm due to the higher number of mitochondria,3 and a central round to oval or irregular nucleus with a perinuclear “halo” as a result of cytoplasmic clearing (Fig. 1b). These eosinophilic cells mimic closely the cells seen in the eosinophilic variant of conventional RCC, the granular or eosinophilic variant of papillary RCC and in oncocytoma.

image

Figure 1. Histological sections of chromophobe renal cell carcinoma (RCC) showing large cells with (a) finely reticulated cytoplasm and (b) small cells having abundant eosinophilic cytoplasm with perinuclear clearing. (c) The polygonal chromophobe RCC cells have well-defined (thick) cytoplasmic membranes. The few mitochondria are localized near the cell membrane, whereas the cytoplasm is filled with small vesicles interspersed between glycogen and mitochondria. The latter might originate from the degradation of mitochondria (insert).

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Both cell types can occur in combination within a given tumor (Fig. 1b). The cells are usually arranged in characteristic solid nests (e.g. alveolar) or tubules. Another characteristic feature is the phenomenon of the lining up of huge, pale cells around blood vessels (Fig. 1a). Eccentrically placed nuclei in chromophobe RCC are round to oval, variable in size and shape and show slight pleomorphism and hyperchromasia with coarsely granular chromatin and irregular nuclear membranes. Mitotic figures are more common in chromophobe RCC than in conventional or papillary renal cell tumors (RCT). Binucleated and multinucleated cells are also very common.12,13 The nuclei are frequently small (“raisinoid”), irregular (in varying proportions) and wrinkled, probably because of loss of a large number of chromosomes.14 Clearly recognizable nucleoli are relatively rare and usually smaller than expected in most cases, but large nucleoli might appear in some cases.15

Ultrastructure

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

The most difficult problem is the differentiation of chromophobe RCC with prominent cytoplasmic granularity from renal oncocytoma, as well as chromophobe RCC from the granular variant of conventional RCC and the eosinophilic variant of papillary RCC in which, as implied, the Golgi apparatus and the rough endoplasmic reticulum are very poorly developed. Electron microscopically, the cytoplasm of typical chromophobe RCC cells is crowded by round to oval vesicles, mixed with scattered mitochondria, as well as glycogen and fat deposits in smaller numbers.16 The remaining cytoplasmic organelles are compressed to the periphery, giving rise to the variable degrees of eosinophilia and granularity observed by light microscopy and to the perinuclear clearing or “halo”.17 The vesicle structure showing “inner vesicles” or invaginations is a very characteristic feature of chromophobe RCC and similar to that observed in the intercalated cells of the cortical collecting ducts.18 The mitochondria show distinct abnormalities, including outer membrane outpouching (Fig. 1c), abundant tubulovesicular cristae, few circular cristae, and circular electron-dense structures with a less electron-dense amorphous inclusion within the matrix.19 Adjacent vesicles focally indent the mitochondrial membrane. In the eosinophilic cells, vesicles are less abundant and noted between numerous mitochondria with lamellar cristae, but without matrix density. The exact nature of invaginated (membrane-bound) vesicles measuring between 160 and 300 nm in diameter in chromophobe RCC is still obscure, although it has been suggested that they might be derived from outer membranes of mitochondria or represent altered endoplasmic reticulum.3,5,18,20

Tinctorial characteristics

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

The tinctorial characteristics of different renal epithelial neoplasms appear to be dependent on cytoplasmic contents, including various organelles. For example, the clear cells are primarily a result of abundant cytoplasmic glycogen and lipids in conventional RCC, and due to the characteristic microvesicles in chromophobe RCC, whereas cytoplasmic eosinophilia is, in general, a result of the abundance of nonribosomal cytoplasmic organelles; that is, mitochondria, lysosomes, neuroendocrine granules, cytofilamentes and smooth endoplasmic reticulum. Most RCT with prominent eosinophilic cytoplasmic granularity ultrastructurally show the presence of a large number of mitochondria. It has been hypothesized that differences in the location and size of mitochondria might partly be responsible for the differing tinctorial cytoplasmic characteristics among different granular renal epithelial neoplasms.21

One histochemical marker that has been proposed to help to differentiate chromophobe RCC from other types of RCT is Hale's colloidal iron.2,21–23 Reacting with mucopolysaccharides within the cytoplasmic microvesicles, it shows a characteristic strong/diffuse blue cytoplasmic (microvacuolated) staining pattern with peripheral accentuation in both types of chromophobe RCC. However, other subtypes of RCT (especially renal oncocytoma) also display positive reactions with variable intensities, because these characteristics are the result of the many vesicular structures inside the cytoplasm. The type of positivity differs between the two tumors, producing a microvesicular cytoplasmic pattern in chromophobe RCC and apical positivity in renal oncocytoma.23 The interpretive and technical challenges of Hale's colloidal iron staining limit its application.

Locus-specific and whole-genome analysis

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

The ploidy analysis generally carried out by flow cytometry showed that chromophobe RCC is a hypodiploid tumor.5,24 Endoreduplication or polyploidization of the hypodiploid tumor cells in chromophobe RCC,24,25 as well as pulverization of the chromosomes have also been observed.12,25 Using the standard technique for detection of telomerase activity, the TRAP assay based on the PCR, it was found less frequent in chromophobe RCC as compared with other histopathological subtypes, whereas the specimens of normal kidney did not show any evidence of it.26

Chromosomal alterations

The earliest report showed nonrandom losses of chromosomes 1, 2, 6, 10, 13, 17 and 21, leading to a low chromosome number in chromophobe RCC.12 The initial cytogenetic findings were further supported by results obtained by restriction fragment length polymorphism (RFLP),27 chromosomal CGH28 and microsatellite analyses.29–34 Other studies confirmed the specific genetic changes by karyotyping,32,33 fluorescence in situ hybridisation (FISH),34,35 as well as by array-based CGH.36 The loss of multiple chromosomes explains the hypodiploid DNA index of chromophobe RCC.37

Frequent loss of heterozygosity of all or part of a chromosome arm in chromophobe RCC might point to tumor suppressor gene inactivation. From the studies on the chromosome level, multiple genes appear to be involved in the tumorigenesis. Recently, oligonucleotide microarrays that query single-nucleotide polymorphisms (SNP) have been developed to globally analyze the genomes of tumors for genetic alterations without the need for paired normal samples. This technique enables the detection of small DNA copy number changes through the entire genome, which might mark the locus of putative tumor suppressor genes. Potential utility of the 10K SNP arrays in evaluation of renal epithelial tumors based on chromosomal abnormalities has been recently shown.38 However, only chromosomal copy number changes and loci of LOH were observed in formalin-fixed paraffin-embedded archived samples of nine chromophobe RCCs.39 Recently, using high-density whole-genome 250K SNP-based oligoarrays, no recurrent small deletions, which might mark the loci of genes involved in the development of chromophobe RCC, have been found.40 Comparing the genetic changes in 30 tumor cases, loss of chromosome 2, 10, 13, 17 and 21 occurred in 93%, 93%, 87%, 90% and 70% of chromophobe RCC, respectively. Based on the results obtained, any microsatellites or bacterial artificial chromosome clones localised at these chromosomes can be used to establish the diagnosis of chromophobe RCC in cases with uncertain histology. This can be achieved in most histopathological laboratories by applying microsatellite analysis or FISH to detect the specific genetic alterations.

Gene alterations

The genes affected by the specific chromosomal alterations in chromophobe RCC have not been identified yet. Loss of chromosome 17 was detected in 90% of chromophobe RCC, but the p53 tumor suppressor gene was mutated in only 27% of the cases.41 There is no mutation of the PTEN gene in chromophobe RCC, despite the frequent (86%) loss of chromosome 10.42 The germ line mutation of the folliculin gene is associated with Birt–Hogg–Dube syndrome and the development of so-called hybrid chromophobe-oncocytic RCT,43 but the folliculin is not mutated in sporadic chromophobe RCC.44

Mitochondrial alterations

Rearrangements in the mitochondrial DNA of the chromophobe RCC have been described, but the exact nature of these alterations is not yet known.45 Heteroplasmic mtDNA mutations in chromophobe RCC have also been identified by sequencing the entire mitochondrial genome.46 However, no correlation between the presence of mtDNA mutations and the number of mitochondria, for example, the staining characteristics of tumor cells, was found. Therefore, it is very likely that alterations of structural mitochondrial proteins, encoded in the nuclear DNA, are responsible for the morphological abnormalities and also the functional changes of mitochondria in cells of chromophobe RCC. Whether or not a lower mtDNA content or mtDNA mutations are associated with the lower abundance of mitochondrial transcripts in chromophobe RCC also remains to be clarified.

Immunoprofile

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Although chromophobe RCC have a distinct genotype, their morphological features can overlap with those of renal oncocytomas. The mRNA expression profiles obtained for approximately 40 chromophobe RCCs was highly similar to that of approximately 35 renal oncocytomas.47–56 Functional analyses suggest that many of the genes normally expressed in distal nephrons are retained in chromophobe RCC.54 The lack of expression of these genes in renal oncocytoma shows that two tumors are highly distinctive in their histogenesis. They either are derived from two different cell types, or represent two different stages of differentiation, with chromophobe RCC retaining more distal nephron markers than renal oncocytoma.57 Further studies are needed to elucidate the relationship between chromophobe RCC and renal oncocytoma.

Generally, the majority of transcripts downregulated in chromophobe RCC were located predominantly on chromosomes 1, 2, 6, 10, 13, 17 and 21, which are specifically lost from the karyotype of this type of RCT.40,58 After analyzing of hundreds genes by RT–PCR and the selected genes by immunohistochemistry (IHC), only very few proteins remained over and even these are not exclusively expressed in chromophobe RCC. The higher expression of CA2,50 CAV1,59 KRT7/CK7,59–65 CLDN7,52,66–68 EPCAM,65,69,70 EMA/MUC162,71 and MAL254 in chromophobe RCC compared with those observed by IHC in renal oncocytoma does not seem to be related to extra copies of chromosomal segments. Nor are the previously proposed negative markers, such as CD10/MME,72 CCND1,73 PAX264 and S100A1,56,57,74–76 sufficiently discriminatory for chromophobe RCC as well. For example, in a recent study using IHC on large series of RCT on conventional and tissue microarray sections (Fig. 2), the positive immunostaining for S100A1 observed in chromophobe RCC was more frequent (23%) compared with the literature data (0–6%).56 Summing up the data from the literature (Table 1), no reliable marker has been identified for the differential diagnosis of chromophobe RCC versus renal oncocytoma, with the exception of CD82/KAI1 (Fig. 3), which has recently been evaluated in two independent large-scale studies.77,78

image

Figure 2. Immunohistochemical analysis of S100A1 in normal and tumor kidney tissues.56 (a) In a normal kidney, the protein was expressed in proximal and distal tubular epithelial cells and collecting ducts. (b) In chromophobe renal cell carcinoma (RCC), a strong focal and weak diffuse membranous and also cytoplasmic staining was seen in two cases. (c) A strong diffuse cytoplasmic staining attenuated to the cell membrane was observed in two cases of chromophobe RCC. (d) In renal oncocytoma, a strong diffuse to focal membranous and also cytoplasmic staining was detected. (e) Six renal oncocytomas were immunonegative.

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Table 1.  Summary of the literature data on the immunohistochemical evaluation of the proposed candidate markers for chromophobe renal cell carcinoma
AntibodyFull name of the proteinchRCCROpRCCcRCCReference
+/−+/−+/−+/−
  1. cRCC, conventional renal cell carcinoma; chRCC, chromophobe RCC; pRCC, papillary RCC; RO, renal oncocytoma.

CAIICarbonic anhydrase II10/05/51/93/750
Caveolin-1Caveolin-121/03/2359
CK7Cytokeratin711/01/2219/12/3564
19/30/175/4065
27/143/5263
21/025/159
27/32/2935/108/9462
12/010/0l1/1761
6/03/860
CLDN7Claudin74/22/44/150/3352
10/14/1368
24/1210/338/210/4267
19/18/310/196/17366
EPCAMEpithelial cell adhesion molecule9/30/311/927/12070
22/05/1215/3065
23/50/73/244/25269
EMAEpithelial membrane antigen21/04/1222/671
30/016/1530/4655/4762
KAI1 (CD82)CD82 molecule27/42/260/351/4777
69/190/900/1717/21378
MAL2Mal, T-cell differentiation protein 25/01/454
CD10/MMEMembrane metallo-endopeptidase0/193/613/158/472
CCND1Cyclin D12/139/95/1323/2273
PAX2Paired box 21/1020/317/331/664
S100A1S100 calcium binding protein A13/4837/330/230/1176
0/914/14/26/375
1/1513/210/2356/2574
21/5968/655/3373/5456
0/2423/257
image

Figure 3. Immunohistochemical analysis of CD82/KAI1 in normal and tumor kidney tissues.78 (a) In a normal kidney parenchyma, the protein was expressed preferentially in distal tubules. The expression was also seen at the basolateral membrane of outer medullary collecting ducts. (b) A strong diffuse to focal membranous and also weak cytoplasmic immunopositivity of tumor cells was observed in chromophobe renal cell carcinoma (RCC). (c) Immunoreactivity was also seen in some conventional RCC showing large “chromophobe-like” epithelial cells. (d) Several conventional RCC showed focal positivity in necrotic tumor areas or single necrotic cells. Immunopositivity was also seen in the infiltrating mononuclear cells in conventional RCC with a strong inflammatory reaction. Some areas of tumor cells adjacent to the inflammation were weakly positive. (e,f) A strong positivity was seen in the foamy cells and in the inflammatory cells infiltrating the papillary stalk.

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Prognosis and predictive factors

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References

Chromophobe RCC typically has a much better prognosis than of conventional RCC.3 The latest retrospective study based on an analysis of clinicopathological parameters and survival of patients showed that patients with nonmetastatic chromophobe RCC had a similar prognosis to nonmetastatic conventional RCC, whereas patients with metastatic chromophobe RCC actually had a worse prognosis than the clear-cell counterpart.79 The observation that over 90% of patients with chromophobe RCC are alive 5 years after surgery underlines the importance of precise diagnosis of this unique type of tumor.6,11,80 Excluding the report published by Cheville et al.,11 most series suggest that they have a better prognosis than papillary RCC as well. However, an aggressive clinical course has been reported for tumors larger than 8 cm in diameter.81 Although chromophobe RCC has a better prognosis than conventional or papillary RCC, it is a malignant tumor with a mortality rate of approximately 10%. It has been shown that metastatic chromophobe RCC progressed more indolently than other variants.82 Lymph node metastasis and infiltration of neighboring organs are rare, but nevertheless occur.6,83–85 The proclivity for hepatic metastases might distinguish chromophobe RCC from other subtypes of RCC, which tend to spread to bone or lungs.86

Sarcomatoid transformation, which historically was considered to be a distinctive histopathological entity, has been described in virtually every type of RCCs and represents a common pathway of transformation to a higher-grade malignancy. Chromophobe RCC with sarcomatoid transformation are uncommon, and the presence of sarcomatoid changes is associated with a more aggressive clinical course.79,83,87 Single reported cases showed large pleomorphic spindle or polygonal cells with large hyperchromatic nuclei and high mitotic activity. It has been suggested that multiple gains of chromosomes 1, 2, 6, 10 and 17 are important for the sarcomatoid transformation of chromophobe RCC, but not for its metastatic potential.88

References

  1. Top of page
  2. Abstract
  3. Prehistory and epidemiology
  4. Clinical symptoms, radiological findings and treatment
  5. Macroscopy
  6. Tumor location and size
  7. Histopathology
  8. Ultrastructure
  9. Tinctorial characteristics
  10. Locus-specific and whole-genome analysis
  11. Immunoprofile
  12. Prognosis and predictive factors
  13. Acknowledgment
  14. References