Jump to comment:

• Author's reply to Parr et al.
  Antonio Alonso
  Published on: 15 February 2005
• MtDNA haplotyping of pathology specimens
  Ryan L. Parr
  Published on: 8 February 2005

• Published on: 15 February 2005

  Author's reply to Parr et al.

  • Antonio Alonso, Facultativo

  Dear Editor,

  Setting up standards when interpreting mtDNA CR sequence data from tumors.

  We agree with Parr et al.[1] on the importance to compare the mtDNA sequence data of our recent case report [2] (a divergence of 7 homoplasmic nucleotide positions within the 16024-16365 segment of the HV1 region between two morphologically different tissue sections found on the same slide) with the available...

  Show More

  Dear Editor,

  Setting up standards when interpreting mtDNA CR sequence data from tumors.

  We agree with Parr et al.[1] on the importance to compare the mtDNA sequence data of our recent case report [2] (a divergence of 7 homoplasmic nucleotide positions within the 16024-16365 segment of the HV1 region between two morphologically different tissue sections found on the same slide) with the available scientific data on mtDNA somatic mutations in tumors to better understand the significance of our findings. Indeed we performed such a comparison during case results interpretation and we found a rate and a pattern of mtDNA instability in different type of tumours similar to that reported by Tan et al. [3] (to use a paper cited by Parr et al. to challenge the hypothesis of Alonso et al.). Tan et al. reported 8 frozen tumor tissue samples (from a total of 19) displaying just one (7 samples) or two (1 sample) somatic mutations within the HV1 segment (16024-16365) analysed by Alonso et al. [2]. This discrete pattern of mtDNA instability is certainly not enough to explain the high divergence between two tissue sections obtained by Alonso et al.

  The results obtained by Chen et al. [4] are also cited by Parr et al. [1] to challenge the conclusions of Alonso et al. [2]. Chen et al. reported 3 prostate tumor samples (from a total of 16) with one (1 sample), five (1 sample), or eight (1 sample, all heteroplasmic positions) somatic mutations within the HV1 segment analysed by Alonso et al. Again the pattern of instability detected by Chen et al. with high incidence of heteroplasmy is different from the homoplasmic divergence reported by Alonso et al. Furthermore, Chen et al. used 45 PCR cycles (9 PCR cycles more than the standard used in forensic laboratories (5)) to amplify fragments around 600 bp from a reduced number of tumour cells obtained by laser microcapture from formalin-fixed tissue sections. Under these analytical conditions it is very difficult to guarantee (or to validate) the reproducibility of each single PCR reaction against background contamination and other artefacts. Especially if DNA degradation produced by formalin makes tumour DNA partially refractory to amplify 600 bp fragment sizes producing a situation of low copy number (LCN) [6,7] that could compromise seriously the reliability of the results. Specific DNA quantification is a recommended procedure of special interest when dealing with such a LCN DNA specimens[8]. Budowle et al. [9, 10] demonstrated high incidence of artifactual heteroplasmic results when using high number of PCR cycles to analyse the mtDNA CR sequence from hair samples.

  We do not share the arguments of Parr et al. to explain the significance of the mtDNA mutations reported by Alonso et al. First, because all the nucleotide positions reported by Alonso et al. (including 16293G) are polymorphic nucleotide positions in human populations (you can see that at http://www.genpat.uu.se/mtDB/ and http://www.fbi.gov/hq/lab/fsc/backissu/april2002/miller1.htm). Second, because the haplotypes detected by Alonso et al. were assigned to different haplogroups (haplogroups k & U5a1a) according to phylogenetic criteria. The lack of match after database searching was not only observed for the tumor haplotype (16256T, 16270T, 16293G) but also for the constitutional haplotype (16224C, 16234T, 16311C, 16356C) obtained from normal tissue. This is just reflecting that the human mtDNA diversity is still not fully represented in the reduced sample size of current mtDNA population databases.

  Finally, we would like to state that the conclusions of Alonso et al. [2] are based upon three different lines of evidence: (a) the lack of correspondence between the histological analysis of two different (a pre- and a post-surgical) biopsies, (b) the presence in the pre-surgical slide of two different (morphologically and histological) tissue sections that also present a different position pattern on the same slide (see Figure 1), and (c) the distinctive mtDNA sequence data (with phylogenetic consistency) obtained from each tissue section that showed a clear divergence on 7 nucleotide homoplasmic positions.

  Anyway, the debate opened by Parr et al. is valid. A compilation of reliable data on cancer mtDNA instability would be desirable and very useful for both scientific communities. But if we like to perform an objective comparison of mtDNA sequence data across different labs we need to be sure of using comparable methods and interpretation standards. A certain degree of methodological standardization is crucial for this purpose. An important part of the standardization progress made in forensic mtDNA typing was accomplished by continuous inter-laboratory collaborative exercises organized by different scientific groups and societies (SWGDAM, EDNAP, ISFG-working groups…). A similar standardization progress is suggested for molecular oncology labs dealing with mtDNA instability in cancer.

  References

  (1) Ryan L. Parr, Gabriel D. Dakubo, Jennifer Maki MtDNA haplotyping of pathology specimens. JCP Online, eletter 31 Jan 2005.

  (2) Alonso, A. et al. Mitochondrial DNA haplotyping revealed the presence of mixed up benign and neoplastic tissue sections from two individuals on the same prostatic biopsy slide. J Clin Pathol 2005; 58, 83 -6.

  (3) Tan, D.J., Bai, R.K. & Wong, L.J. Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer. Cancer Res 2002; 62, 972-6.

  (4) Chen, J.Z., Gokden, N., Greene, G.F., Mukunyadzi, P. & Kadlubar, F.F. Extensive somatic mitochondrial mutations in primary prostate cancer using laser capture microdissection. Cancer Res 2002; 62, 6470-4.

  (5) Wilson, M.R., DiZinno, J.A., Polanskey, D., Replogle, J. and Budowle B (1995) Validation of mitochondrial DNA sequencing for forensic casework analysis. Int. J. Leg. Med., 10, 68-74.

  (6) Gill, P. Application of low copy number DNA profiling. Croat Med J 2001; 42, 229-232.

  (7) Budowle B, Hobson D, Smerick J, Smith J. Low copy number: consideration and caution. Proceedings from the Twelfth International Symposium on Human Identification 2001 (available at www.promega.com).

  (8) Alonso A, Martin P, Albarran C, Garcia P, Primorac D, Garcia O, Fernandez de Simon L, Garcia-Hirschfeld J, Sancho M, Fernandez-Piqueras J. Specific quantification of human genomes from low copy number DNA samples in forensic and ancient DNA studies. Croat Med J. 2003 Jun;44(3):273-80.

  (9) Budowle B, Allard MW, Wilson MR, Chakraborty R. Forensics and mitochondrial DNA: applications, debates, and foundations. Annu Rev Genomics Hum Genet. 2003;4:119-41. Review.

  (10) Budowle B, Allard MW, Wilson MR. Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair. Forensic Sci Int. 2002 Mar 28;126(1):30-3.

  Show Less

  Conflict of Interest:
  None declared.

  • Back to top
• Published on: 8 February 2005

  MtDNA haplotyping of pathology specimens

  • Ryan L. Parr, V. P. Research and Development
  • Other Contributors:
    • Gabriel D. Dakubo, Jennifer Maki

  Dear Editor

  Alonso et al [1] recommend the use of mitochondrial genetic typing to exclude the possibility of tissue carryover artifacts in situations where low DNA content and high degradation may compromise conventional short tandem repeat typing. They studied archived presurgical hematoxylin and eosin stained needle biopsy sections from the same slide to ascertain the authentic source of the malignant and...

  Show More

  Dear Editor

  Alonso et al [1] recommend the use of mitochondrial genetic typing to exclude the possibility of tissue carryover artifacts in situations where low DNA content and high degradation may compromise conventional short tandem repeat typing. They studied archived presurgical hematoxylin and eosin stained needle biopsy sections from the same slide to ascertain the authentic source of the malignant and benign biopsy specimen on the slide. To this end, they amplified the hypervariable region I (HVI) of the mitochondrial genome from DNA extracts from the malignant and normal prostate biopsy specimens, and available blood sample from the presumptive patient. Due to the high HVI sequence divergence between the malignant and normal samples, and the absence of heteroplasmy, they concluded that the samples were from two different individuals, and could not be explained by somatic mtDNA instability. Since the haplotype of the normal tissue section and blood were identical, the malignant biopsy on the slide was considered to be a contaminant from an unknown individual. Contending that in this particular case the tissue biopsy specimens were truly from two individuals, the general recommendation made by Alonso et al.[1] to use mtDNA sequence analysis for tissue typing will likely be fraught with erroneous conclusions in some cases.

  Based on our findings we would like to propose an alternative explanation to that of Alonso et al.[1] Specifically, the mutations that were observed are not due to contamination but are in fact symptomatic of the malignancy. The mechanism by which somatic mutations accumulate during the pathogenesis of prostate adenocarcinoma is poorly understood. However, it is possible a burst of multiple mtDNA mutations occur in response to extreme cellular oxidative stress [2]. The displacement loop (which includes HVI) is a mutation hot spot and accumulates more mutations than the rest of the mitochondrial genome. Indeed the mitochondrial genetic signature of malignant tissues and in particular prostate adenocarcinoma is frequently different from the corresponding benign epithelial glands and blood [3]. Somatic mitochondrial mutations in cancers at polymorphic sites are not uncommon, and this can alter the haplotype of malignant tissues from the corresponding blood. We conducted a comprehensive study of prostate cancer mitochondrial genetics by having a certified pathologist laser capture pure malignant cells from biopsy specimens from several patients, and the D-loop sequences were compared with the corresponding blood. Several sequence variations (both homoplasmic and heteroplasmic somatic mutations) between blood and tumor specimens were observed. Mitochondrial DNA analysis of glioblastoma by Kirches et al.[4] revealed a D-loop sequence divergence between blood and matched tumors in 41% of the samples. Somatic mutations were identified in 74% of breast cancer samples with 81.5% of these being restricted to the D -loop [5]. Interestingly, one of the markers (16293G) detected in the prostate cancer specimen by Alonso et al.[1] is likely a disease specific marker of glioblastoma, breast cancer [4,5] and prostate cancer (our observation). Also the absence of heteroplasmic mutation in a specimen (as observed by Alonso et al.[1]) is not inconsistent with the malignant phenotype as homoplasmic mtDNA substitution mutations have been observed in many cancers including prostate adenocarcinoma [3]. Given that prostate needle biopsies of an individual are from different parts of the prostate gland, and that prostate cancer can be multi-focal, the data of Alonso et al.[1] can be consistent with all samples originating from the same individual. In this case the haplotype of the blood and normal samples will cluster (“wild type haplotype”) and may differ from the adenocarcinoma (“mutant haplotype”) as they observed. Indeed Alonso et al.[1] could not find any match when they searched two databases for these haplotypes. The lack of a population match suggests a somatic mutation process as attested to in the literature [6,7].

  The use of mtDNA sequences for unambiguous identification of tissue samples is highly reliable when using non-malignant samples, however malignant tissues contain mutations in mtDNA that may confound such determinations but never the less are not indications of sample cross- contamination.

  References

  (1) Alonso, A. et al. Mitochondrial DNA haplotyping revealed the presence of mixed up benign and neoplastic tissue sections from two individuals on the same prostatic biopsy slide. J Clin Pathol 2005; 58, 83 -6.

  (2) Chen, J.Z., Gokden, N., Greene, G.F., Green, B. & Kadlubar, F.F. Simultaneous generation of multiple mitochondrial DNA mutations in human prostate tumors suggests mitochondrial hyper-mutagenesis. Carcinogenesis 2003; 24, 1481-7

  (3) Chen, J.Z., Gokden, N., Greene, G.F., Mukunyadzi, P. & Kadlubar, F.F. Extensive somatic mitochondrial mutations in primary prostate cancer using laser capture microdissection. Cancer Res 2002; 62, 6470-4.

  (4) Kirches, E. et al. High frequency of mitochondrial DNA mutations in glioblastoma multiforme identified by direct sequence comparison to blood samples. Int J Cancer 2001; 93, 534-8.

  (5) Tan, D.J., Bai, R.K. & Wong, L.J. Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer. Cancer Res 2002; 62, 972-6.

  (6) Carew, J.S. & Huang, P. Mitochondrial defects in cancer. Mol Cancer 2002; 1, 9.

  (7) Abnet, C.C. et al. Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma. BMC Cancer 2004; 4, 30.

  Show Less

  Conflict of Interest:
  None declared.

  • Back to top