Figure 1.
Pattern of mtDNA mutation in patient 1.
The patient was operated twice on 2002 and 2004 with removal of tumors from the right upper (T1) and left lower lobe (T2) respectively. Five bronchoscopically abnormal airway mucosal biopsies were obtained following second surgery (T2) of this patient from main carina (M1), right upper lobe (M2 and M3) and left lower lobe (M3 and M4) surrounding both the tumors as depicted (Panel A–E). The mucosal biopsies exhibited a range of 3–11 mtDNA mutations as represented in different color (Panel A–E). Identical mutations are shown in the same color in different biopsies and the tumor. A different color code was also used for each mucosa to indicate the clonal progression of the lesions on both the lungs with accumulated mtDNA mutations. Representative histological photomicrograph of the mucosa and the tumor has been shown in panel A and F. Both the tumors T1 and T2 exhibited twenty identical mtDNA mutations (E–F, Bottom rectangle) including all the 16 mutations exhibited by the five mucosal biopsies (Matched color). The additional 4 mtDNA mutations detected in the tumors are represented underlined in black color in the bottom rectangle (Panel E–F). Two mtDNA mutations (G8836C and A8341C) were present in 4/5 and 3 other mutations (A2249C, A3742C and T15982G) were present in 3/5 mucosal biopsies. Tumors are encircled to indicate their development from the clonal mucosal patches. T1: Tumor from the right upper lobe; T2: Tumor from the left lower lobe; M: Mucosa.
Figure 2.
Pattern of mtDNA mutation in patient 2.
The patient was operated on 2005 for surgical removal of tumor from the right upper lobe. Five bronchoscopically abnormal airway mucosal biopsies were obtained from main carina (M1), right lower lobe (M2 and M3) and right upper lobe (M3 and M4) surrounding the tumors as depicted (Panel A–E). The mucosal biopsies exhibited a range of 2–5 mtDNA mutations as represented in different color (Panel A–E). Identical mutations are shown in same color in different biopsies and the tumor. A different color code was also used for each mucosa to indicate the clonal progression of the lesions with accumulated mtDNA mutations. Representative histological photomicrograph of the tumor and normal mucosa are shown in Panel A and D. The tumor exhibited 9 mtDNA mutations (Represented in the Bottom rectangle) including all the 8 mutations exhibited by the five mucosal biopsies (Matched color). An additional mtDNA mutation (T7001C) detected in the tumor is underlined in black in the bottom rectangle. Tumor was encircled to indicate its development from the clonal mucosal patches. M: Mucosa.
Table 1.
Pattern of mtDNA mutation in patient 3.
Table 2.
Pattern of somatic mtDNA mutation in the 8 lung cancer patients.
Table 3.
The pattern of germ line mtDNA sequence variants in the lung cancer patients.
Figure 3.
MtDNA content in the lung cancer patients.
mtDNA content was measured by multiplex real-time PCR using nuclear encoded β-actin and mitochondria encoded COI gene. A ratio of COI/β -actin corresponds to the fold difference compared to control. MtDNA content increased significantly (P<0.001) in the mucosal biopsies and corresponding tumors compared to the normal control in both the patients as indicated. N: Normal lymph node; M: Mucosa; T: Tumor. *P value <0.05 compared to normal lymph node.
Figure 4.
mtDNA content index in patient 3.
mtDNA content increased significantly (P<0.05) in the mucosa (M), adjacent normal (NT) and in the metaplastic margin (MT) compared to normal lymph node used a s control.
Figure 5.
MtDNA genetic mutation tree in patient 1.
Possible clonal evolution of the lung tumors has been depicted. Each mucosal biopsy (Circled, M1-M5) shared some identical mtDNA mutations (Matched color) along with additional mutations in the heterogeneous mucosal field. The fittest clones emerged in the primary tumors (T1-T2) with the more selective mtDNA mutations (Total 20 mtDNA mutations, 16 were shared between M1-M5 as in Figure 1).
Table 4.
History of the 8 lung cancer patients additionally sequenced for mtDNA mutation.