Niklas Schultz - Academia.edu (original) (raw)

Papers by Niklas Schultz

bioRxiv (Cold Spring Harbor Laboratory), Sep 15, 2022

Cancer Research, Jun 15, 2022

Introduction: Defining the transition from benign to malignant tissue is fundamental to improve e... more Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumour areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in >120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer. Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundenberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2171.

Cancer Research, Jun 2, 2023

Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis... more Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. We provide an unsupervised approach (SpatialInferCNV) to study spatial genome integrity, in situ, to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in >120 000 spatial regions across multiple organs, tissues and tumors, including three whole axial prostates. We used this information to deduce clonal relationships between regions harboring 5-20 cells. We perform an in-depth spatial analysis of prostate cancer that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient. For these domains we inferred genome-wide information, which facilitated data driven clustering in a tissue wide fashion at high resolution. We observed that genome-wide somatic copy number alterations revealed distinct clonal patterns benign tissue and nearby tumours. In a tissue section from patient 1 (section H2_1), we observed that many CNVs occurred in clone C, most notably in chromosomes 8 and 10 encoding a MYC amplification and a PTEN deletion. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely diploid cells in clones A and B. The unobserved ancestor to clone C then gave rise to a further unobserved clone, and then clones E, F and G. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in prostate cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. We present the first comprehensive, large-scale, atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer. Citation Format: Andrew Erickson, Mengxiao He, Emelie Berglund, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Leire Alonso Galicia, Alia Shamikh, Elisa Basmaci, Teresita Díaz De Ståhl, Timothy Rajakumar, Dimitrios Doultsinos, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg. The spatial landscape of clonal somatic copy number alterations in benign and malignant prostate epithelia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A051.

Research Square (Research Square), Nov 19, 2021

Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in e... more Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in earlystage, localized tumors and their surrounding normal tissues is poorly characterized. To investigate this phenomenon, here we developed a method for spatially resolved single-cell CNA pro ling and applied it to characterize the CNA landscape in 10,007 nuclei extracted from 70 tumor and normal tissue regions (~125 mm3 tissue cubes) from prostatectomies performed in six patients with localized prostate cancer. We identi ed two distinct groups of cells with abnormal karyotype, one mainly consisting of sparse alterations ('pseudo-diploid' cells) and the other characterized by genome-wide karyotypic changes ('monster' cells). Pseudo-diploid cells displayed high clonal diversity and formed numerous small sized clones ranging from highly spatially localized to broadly spread clones, whereas monster cells were singular events detected throughout the prostate. We observed a remarkable correlation between the fraction of the genome affected by CNAs and the number of tissue regions in which pseudo-diploid cells were found. Highly localized pseudo-diploid clones were enriched in tumor regions and carried deletions of known or putative tumor suppressors, including APC, CDKN1B, FOXO1, FOXP1, and RB1. Spatially resolved targeted deep sequencing of 523 cancer genes detected non-synonymous mutations in both normal and tumor regions, including mutations in FOXA1, FOXP1, and SPOP genes previously implicated in prostate cancer. Strikingly, in two regions in which targeted deep sequencing detected a point mutation affecting the DNA-binding activity of the FOXA1 transcription factor, we also found a co-deletion of FOXO1 and FOXO3 genes in cells from two different pseudo-diploid clones, implicating combinatorial perturbations of Forkhead transcription factors as an early driver of prostate carcinogenesis. Our study reveals that CNAs and mutations are widespread across normal and tumor regions in the prostate glands of patients with localized prostate cancer and suggests that a subset of alterations-most likely small deletions causing the loss of key tumor suppressors-confer a tness advantage and channel cells towards tumorigenesis.

Nature Communications, Sep 17, 2022

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their preval... more Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we performed multi-region, single-cell DNA sequencing to characterize the SCNA landscape across multiple tumor-rich and normal tissue regions (~125 mm3 tissue cubes) obtained from prostatectomy performed in two patients with localized prostate cancer. We identified two distinct populations of cells with abnormal karyotypes, one marked by sparse deletions or amplifications (pseudo-diploid cells) and the second characterized by genome-wide copy number changes reminiscent of monster cells previously described in colorectal cancer. Pseudo-diploid cells formed numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones mainly featuring (sub-)chromosomal arm deletions. In contrast, monster cells harbored whole-chromosome gain...

Cancer Research, Jun 30, 2010

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at ... more Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or γH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. ©2010 AACR.

Nucleic Acids Research, Jun 28, 2012

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. Th... more Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase g (Polg), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

Cell Cycle, Jul 15, 2005

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the r... more Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumors. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumors.

Cancer Research, May 15, 2022

Introduction: Defining the transition from benign to malignant tissue is fundamental to improve e... more Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumor areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in >120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We additionally performed in silico assessment of spatial copy number inference. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumors and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Ludvig Bergenstråhle, Linda Kvastad, Alma Andersson, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR016.

Chemistry and Physics of Lipids, Aug 1, 2009

Clinical Cancer Research, Nov 1, 2021

Cytogenetic and Genome Research, 2007

The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes ... more The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.

Human Molecular Genetics, Feb 27, 2006

The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk... more The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk of some cancers. XRCC3 is one out of five RAD51 paralogues and is involved in homologous recombination, as are the BRCA1 and BRCA2 proteins. However, in contrast to mutations in BRCA1 and BRCA2, the XRCC3 T241M protein is proficient in homologous recombination and reverts sensitivity to mitomycin C found in XRCC3-deficient cells, whereas XRCC3 D213N is defective in homologous recombination. Here, we report that both the XRCC3 D213N and T241M alleles are associated with an increase in centrosome number and binucleated cells. However, only the D213N allele gives an increase in spontaneous levels of apoptosis. We suggest that the inability of XRCC3 T241M to apoptotically eliminate aberrant cells with mitotic defects could increase cancer susceptibility in individuals carrying this variant. In contrast, cells carrying the XRCC3 D213N variant are able to eliminate aberrant cells by apoptosis, and consistent with this observation, this variant does not seem to be associated with cancer susceptibility.

Nature, Aug 10, 2022

Organ-wide clonal landscape in the prostate Next, we used a cross-section of an entire prostate o... more Organ-wide clonal landscape in the prostate Next, we used a cross-section of an entire prostate organ to explore the siCNV landscape of a commonly multifocal malignancy 12. The

Oncogene, Jan 26, 2004

Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss ... more Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53À/À mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53À/À MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53 þ / þ and p53À/À MEFs following treatments with HU. We found that HU induced DSBs only in p53À/À MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53À/À MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions.

Clinical Cancer Research, Mar 14, 2013

Figure S1 ADT induced Ku70 decrease correlates with a decrease of PSA in prostate tumours. A) Cor... more Figure S1 ADT induced Ku70 decrease correlates with a decrease of PSA in prostate tumours. A) Correlation between the changes induced of kirugical castration of Ku70 outside the nuclei versus days after castration. R 2 =0.76, P<0.0003. The values of Ku70 are given in % of value before castration. B) As in A, but patients with chemical castration. R 2 =0.16, P<0.003. The unfilled circle in A represent the value from patient 8 that has been considered as an outlier and are therefore not included in the calculation of R 2 and P.

International Journal of Radiation Biology, 2007

Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family memb... more Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (γ-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/γ-H2AX foci formation in relation to their co-localization, DSB repair and cell survival. Materials and methods: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci. Results: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of γ-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/γ-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair. Conclusions: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.

Nucleic Acids Research, Sep 1, 2003

Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister ch... more Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-de®cient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1 ±/± cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-speci®c DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, con-®rming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such.

bioRxiv (Cold Spring Harbor Laboratory), Sep 15, 2022

Cancer Research, Jun 15, 2022

Introduction: Defining the transition from benign to malignant tissue is fundamental to improve e... more Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumour areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in &gt;120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer. Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundenberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2171.

Cancer Research, Jun 2, 2023

Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis... more Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. We provide an unsupervised approach (SpatialInferCNV) to study spatial genome integrity, in situ, to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in &gt;120 000 spatial regions across multiple organs, tissues and tumors, including three whole axial prostates. We used this information to deduce clonal relationships between regions harboring 5-20 cells. We perform an in-depth spatial analysis of prostate cancer that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient. For these domains we inferred genome-wide information, which facilitated data driven clustering in a tissue wide fashion at high resolution. We observed that genome-wide somatic copy number alterations revealed distinct clonal patterns benign tissue and nearby tumours. In a tissue section from patient 1 (section H2_1), we observed that many CNVs occurred in clone C, most notably in chromosomes 8 and 10 encoding a MYC amplification and a PTEN deletion. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely diploid cells in clones A and B. The unobserved ancestor to clone C then gave rise to a further unobserved clone, and then clones E, F and G. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in prostate cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. We present the first comprehensive, large-scale, atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer. Citation Format: Andrew Erickson, Mengxiao He, Emelie Berglund, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Leire Alonso Galicia, Alia Shamikh, Elisa Basmaci, Teresita Díaz De Ståhl, Timothy Rajakumar, Dimitrios Doultsinos, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg. The spatial landscape of clonal somatic copy number alterations in benign and malignant prostate epithelia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A051.

Research Square (Research Square), Nov 19, 2021

Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in e... more Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in earlystage, localized tumors and their surrounding normal tissues is poorly characterized. To investigate this phenomenon, here we developed a method for spatially resolved single-cell CNA pro ling and applied it to characterize the CNA landscape in 10,007 nuclei extracted from 70 tumor and normal tissue regions (~125 mm3 tissue cubes) from prostatectomies performed in six patients with localized prostate cancer. We identi ed two distinct groups of cells with abnormal karyotype, one mainly consisting of sparse alterations ('pseudo-diploid' cells) and the other characterized by genome-wide karyotypic changes ('monster' cells). Pseudo-diploid cells displayed high clonal diversity and formed numerous small sized clones ranging from highly spatially localized to broadly spread clones, whereas monster cells were singular events detected throughout the prostate. We observed a remarkable correlation between the fraction of the genome affected by CNAs and the number of tissue regions in which pseudo-diploid cells were found. Highly localized pseudo-diploid clones were enriched in tumor regions and carried deletions of known or putative tumor suppressors, including APC, CDKN1B, FOXO1, FOXP1, and RB1. Spatially resolved targeted deep sequencing of 523 cancer genes detected non-synonymous mutations in both normal and tumor regions, including mutations in FOXA1, FOXP1, and SPOP genes previously implicated in prostate cancer. Strikingly, in two regions in which targeted deep sequencing detected a point mutation affecting the DNA-binding activity of the FOXA1 transcription factor, we also found a co-deletion of FOXO1 and FOXO3 genes in cells from two different pseudo-diploid clones, implicating combinatorial perturbations of Forkhead transcription factors as an early driver of prostate carcinogenesis. Our study reveals that CNAs and mutations are widespread across normal and tumor regions in the prostate glands of patients with localized prostate cancer and suggests that a subset of alterations-most likely small deletions causing the loss of key tumor suppressors-confer a tness advantage and channel cells towards tumorigenesis.

Nature Communications, Sep 17, 2022

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their preval... more Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we performed multi-region, single-cell DNA sequencing to characterize the SCNA landscape across multiple tumor-rich and normal tissue regions (~125 mm3 tissue cubes) obtained from prostatectomy performed in two patients with localized prostate cancer. We identified two distinct populations of cells with abnormal karyotypes, one marked by sparse deletions or amplifications (pseudo-diploid cells) and the second characterized by genome-wide copy number changes reminiscent of monster cells previously described in colorectal cancer. Pseudo-diploid cells formed numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones mainly featuring (sub-)chromosomal arm deletions. In contrast, monster cells harbored whole-chromosome gain...

Cancer Research, Jun 30, 2010

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at ... more Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or γH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. ©2010 AACR.

Nucleic Acids Research, Jun 28, 2012

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. Th... more Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase g (Polg), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

Cell Cycle, Jul 15, 2005

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the r... more Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumors. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumors.

Cancer Research, May 15, 2022

Introduction: Defining the transition from benign to malignant tissue is fundamental to improve e... more Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumor areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in &gt;120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We additionally performed in silico assessment of spatial copy number inference. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumors and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Ludvig Bergenstråhle, Linda Kvastad, Alma Andersson, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR016.

Chemistry and Physics of Lipids, Aug 1, 2009

Clinical Cancer Research, Nov 1, 2021

Cytogenetic and Genome Research, 2007

The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes ... more The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.

Human Molecular Genetics, Feb 27, 2006

The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk... more The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk of some cancers. XRCC3 is one out of five RAD51 paralogues and is involved in homologous recombination, as are the BRCA1 and BRCA2 proteins. However, in contrast to mutations in BRCA1 and BRCA2, the XRCC3 T241M protein is proficient in homologous recombination and reverts sensitivity to mitomycin C found in XRCC3-deficient cells, whereas XRCC3 D213N is defective in homologous recombination. Here, we report that both the XRCC3 D213N and T241M alleles are associated with an increase in centrosome number and binucleated cells. However, only the D213N allele gives an increase in spontaneous levels of apoptosis. We suggest that the inability of XRCC3 T241M to apoptotically eliminate aberrant cells with mitotic defects could increase cancer susceptibility in individuals carrying this variant. In contrast, cells carrying the XRCC3 D213N variant are able to eliminate aberrant cells by apoptosis, and consistent with this observation, this variant does not seem to be associated with cancer susceptibility.

Nature, Aug 10, 2022

Organ-wide clonal landscape in the prostate Next, we used a cross-section of an entire prostate o... more Organ-wide clonal landscape in the prostate Next, we used a cross-section of an entire prostate organ to explore the siCNV landscape of a commonly multifocal malignancy 12. The

Oncogene, Jan 26, 2004

Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss ... more Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53À/À mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53À/À MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53 þ / þ and p53À/À MEFs following treatments with HU. We found that HU induced DSBs only in p53À/À MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53À/À MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions.

Clinical Cancer Research, Mar 14, 2013

Figure S1 ADT induced Ku70 decrease correlates with a decrease of PSA in prostate tumours. A) Cor... more Figure S1 ADT induced Ku70 decrease correlates with a decrease of PSA in prostate tumours. A) Correlation between the changes induced of kirugical castration of Ku70 outside the nuclei versus days after castration. R 2 =0.76, P<0.0003. The values of Ku70 are given in % of value before castration. B) As in A, but patients with chemical castration. R 2 =0.16, P<0.003. The unfilled circle in A represent the value from patient 8 that has been considered as an outlier and are therefore not included in the calculation of R 2 and P.

International Journal of Radiation Biology, 2007

Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family memb... more Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (γ-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/γ-H2AX foci formation in relation to their co-localization, DSB repair and cell survival. Materials and methods: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci. Results: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of γ-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/γ-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair. Conclusions: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.

Nucleic Acids Research, Sep 1, 2003

Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister ch... more Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-de®cient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1 ±/± cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-speci®c DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, con-®rming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such.