1. High resolution telomere FISH reveals anomalies in male germ cell
tumor subtypes and precursor lesion
Mohammed Talha Shekhani1, John R. Barber4, Stephania M. Bezerra1, Christopher M. Heaphy1,3, Nilda Gonzalez Roibon1, Leonardo O. Reis1, Gunes Guner1, Corinne E.
Joshu3,4, George J. Netto1,2,3, and Alan K. Meeker1,2,3
1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD; 2Department of Urology, James Buchanan Brady Urological Institute at Johns Hopkins, Baltimore, MD; 3Department of
Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; 4Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
Methodolgy
 Testicular germ cell tumor (TGCT) is the most common malignancy of young men between 15-40 years of age. It is biologically
unique, because it is in cells of non-somatic lineage and is an unusually curable cancer.
 We evaluated over 70 specimens (arranged on two TGCT tissue microarrays (TMAs)) using high resolution (single-cell) telomere-
specific fluorescent in situ hybridization (FISH) to delineate telomere length patterns in different testicular germ cell tumor
subtypes, as well as in their precursor lesion, intratubular germ cell neoplasia unclassified type (IGCNU). Telomere biology has
never previously been addressed in IGCNU. Oct4 is a highly specific histological marker of IGCNU, seminoma, and embryonal
carcinoma neoplastic cells. We used co-immunofluorescence against Oct4 to improve identification of cancer cells in said
lesions.
 Telomere lengths were scored as short (=1), medium (=2) or long (=3) by comparing the intensity of the telomere signals from
neoplastic cells to benign reference cell types. Reference cells are interstitial/stromal cells (short telomeres) and normal germ
cells (medium telomeres).
Abstract
Number:
3783
Summary of Key Findings
 Compared with normal germ cells, short telomeres were observed in intratubular germ cell neoplasia (IGCNU) lesions and in majority of pure seminomas
(p=0.006 and p=0.0005, respectively). In contrast, we noted longer telomere lengths in non-seminomas—cancer subtypes that also carry a worse prognosis.
Non-seminoma and seminoma components in mixed subtype demonstrated telomeres with slight variation from pure counterparts. Among the non-seminomas,
EC has the highest proportion of cases scored as having long telomeres.
 EC is the only subtype to display telomerase-independent alternative lengthening of telomeres (ALT) phenotype (43% of pure embryonal carcinomas). Loss of
ATRX or DAXX expression is strongly associated with ALT. However, neither protein anomalies existed in sub-group of EC evaluated by IHC—suggesting that there
may be (as yet undiscovered) genetic events associated with ALT in embryonal carcinoma.
 The pattern of telomere lengths among TGCT and their precursor, IGCNU, indicate that telomeres are affected early in carcinogenesis. Moreover, telomere
anomalies may contribute to tumor initiation and progression.
 Further investigation of telomeric aberration in TGCT may delineate new pathways for intervention and prognostication in this unique malignancy.
Table 2. Telomere length distributions for TGCT subtypes and IGCNU
precursor
Embryonal
Carcinoma
Seminoma Teratoma
Yolk Sac
Tumor
Mixed
Cancer cell
telomere lengths
Long (%) 42.9 14 0 33.3 N/A
Medium (%) 14.3 27.9 100 0 N/A
Short (%) 0 58.1 0 66.7 N/A
ALT Present (%) 42.9 0 0 0 15
IGCNU Present
(%)
71.4 58.1 0 33.3 70
IGCNU telomere
length
Long (%) 0 8 0 0 0
Medium (%) 0 8 0 0 7.1
Short (%) 100 84 0 100 92.9
ALT = alternative lengthening of telomeres; and IGCNU = intratubular
germ cell neoplasia of unclassified type
Table 3. Telomere lengths in mixed germ cell tumors
Embryonal
Carcinoma
Seminoma Teratoma
Yolk Sac
Tumor
N 18 9 12 9
Long (%) 38.9 66.7 0 0
Medium (%) 38.9 11.1 25 77.8
Short (%) 5.6 22.2 75 22.2
ALT (%) 16.7 0 0 0
ALT = alternative lengthening of telomeres
Figure 1. Example image a healthy seminiferous tubule and an adjacent
IGCNU/diseased tubule. (A) DAPI/Nuclear Stain/Blue and
Cy3/Telomere-FISH/Red channels. Telomeres are punctate red dots
within nuclei. Filled/solid arrow indicates neoplastic germ cell with
short telomeres (nearly absent FISH signal) in the IGCNU tubule. Open-
headed arrow indicates healthy spermatogonial stem cell’s nucleus for
comparison. (B) 3 channel (DAPI/Nuclear Stain/Blue, Cy3/Telomere-
FISH/Red and Oct4 immunostaining/Green) merged image. *(Asterisk)
= Region displaying non-specific autofluorescence (often associated
with red blood cell fragments from nearby capillaries); IGCNU =
Intratubular Germ Cell Neoplasia of Unclassified type; FISH =
fluorescence in situ hybridization; and DAPI = 4',6-diamidino-2-
phenylindole. Original magnification is 200X, scale bar is 100
micrometers.
Figure 2. Seminoma cells compared to normal healthy germ
cells from the same patient. (A) and (B) Display DAPI/Nuclear
Stain/Blue and Cy3/Telomere-FISH/Red channels. Telomeres
are punctate red dots within nuclei. (A) Shows region
containing seminoma cells and (B) shows contents of a
healthy unaffected seminiferous tubule. Insets demonstrate
enlarged cells for unequivocal telomere length comparisons.
(C) and (D) Display 3 channel (DAPI/Nuclear Stain/Blue,
Cy3/Telomere-FISH/Red and Oct4 immunostaining /Green)
merged images of (A) and (B), respectively. *(Asterisk) =
Region displaying non-specific autofluorescence; FISH =
fluorescence in situ hybridization; and DAPI = 4',6-diamidino-
2-phenylindole. Original magnification is 400X, scale bar is 50
micrometers.
Figure 3. Comparison of an embryonal carcinoma (A, top) with another
EC displaying ALT phenotype (B, below). Both panels demonstrate 3
channel (DAPI/Nuclear Stain/Blue, Cy3/Telomere-FISH/Red and Oct4
immunostaining/Green) merged image. (A) This EC shows long
telomeres in the cancer cells compared to the adjacent stroma. Inset
delineates dramatic telomere length difference between EC and a
stromal cell. (B) This ALT positive EC shows ultra-bright telomeric DNA
foci (arrows) and intracellular telomere length heterogeneity that is
characteristic. Note short telomeres in surrounding interstitium.
*(Asterisk) = Region displaying non-specific autofluorescence; ALT =
alternative lengthening of telomeres; EC = embryonal carcinoma; FISH =
fluorescence in situ hybridization; and DAPI = 4',6-diamidino-2-
phenylindole. Original magnification is 400X, scale bar is 50
micrometers.