Henk B. Kal

Radiotherapy during pregnancy: fact and fiction

Radiotherapy during pregnancy might cause harm to the developing fetus. Generally, pregnant women with malignant diseases are advised to delay radiotherapy until after delivery. However, this advice is not based on knowledge of the risks of radiation to the unborn child. In general, the expected radiation effects, such as mental retardation and organ malformations probably only arise above a threshold dose of 0.1-0.2 Gy. This threshold dose is not generally reached with curative radiotherapy during pregnancy, provided that tumours are located sufficiently far from the fetus and that precautions have been taken to protect the unborn child against leakage radiation and collimator scatter of the teletherapy machine; such precautions also reduce the risk of radiation-induced childhood cancer and leukaemia in the unborn child.

Breast carcinoma during pregnancy. International recommendations from an expert meeting.

O ne of the recommendations from an expert meeting regarding breast carcinoma treatment during pregnancy was that radiation therapy should be delayed until after delivery. However, we believe the authors have overestimated the risks of radiation therapy. The risks of irradiation have been reviewed previously by the International Commission on Radiological Protection. In general, the expected effects are malformations, a decrease in intelligence, mental retardation (deterministic effects), and cancer induction. For deterministic effects, threshold doses of 0.2 gray (Gy) have been found. An estimate of the lifetime risk of radiation-induced fatal cancer at 0.01 Gy is approximately 0.06%. Maternal breast irradiation in the first 8 weeks of organogenesis will expose the fetus to 0.05–0.15 Gy (the reference dose is 50 Gy). Toward the end of pregnancy, the fetus lies closer to the radiation field and could receive >1 Gy for the same treatment course. However, the fetal dose due to leakage radiation from the tube head of the linear accelerator and scatter from collimator and blocks can be reduced with a factor 2 to 4 by proper shielding. Therefore, in the majority of cases, the radiation dose can be kept below the threshold dose for deterministic effects. The risk of radiation-induced cancer is low, and is negligible with a lifetime risk, without irradiation, of approximately 1 in 3. A review of successful radiation therapy for breast cancer (as well as Hodgkin disease) with supplemental shielding during pregnancy was published recently. In summary, the recommendation not to irradiate a pregnant patient until after birth is not tenable. Pregnancy is not a contraindication to radiotherapy in patients with breast cancer and other cancers that develop away from the pelvis.

DNA Double-Strand Breaks and γ-H2AX Signaling in the Testis

Abstract Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms γ-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These γ-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, γ-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit γ-H2AX foci but show homogeneous nuclear γ-H2AX staining, whereas in pachytene spermatocytes γ-H2AX is only present in the sex vesicle. In response to ionizing radiation, γ-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, γ-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear γ-H2AX staining in leptotene spermatocytes demonstrates a function for γ-H2AX during meiosis. γ-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of γ-H2AX foci at DNA double-strand breaks.

Involvement of the D-Type Cyclins in Germ Cell Proliferation and Differentiation in the Mouse

Abstract Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D1 is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D1 in spermatogonial proliferation, in particular during the G1/S phase transition. Cyclin D2 is first expressed at the start of spermatogenesis when gonocytes produce A1 spermatogonia. In the adult testis, cyclin D2 is expressed in spermatogonia around stage VIII of the seminiferous epithelium when Aal spermatogonia differentiate into A1 spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D2 during spermatogenesis, cyclin D2 expression was studied in vitamin A-deficient testis. Cyclin D2 was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A1 spermatogonia by administration of retinoic acid. Overall, cyclin D2 seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A1 spermatogonia. Cyclin D3 is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D3 expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D3 may control G1/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D1 and D3 seem to be involved in cell cycle regulation, whereas cyclin D2 likely has a role in spermatogonial differentiation.

Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells

Abstract Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5%% fetal calf serum, about 36%% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70%% decrease in expression of the cell cycle inhibitor CDKN1B (P27kip1) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25%% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70%% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be.

Dose–effect relation in stereotactic radiotherapy for brain metastases. A systematic review

PURPOSE Stereotactic radiotherapy (SRT) of brain metastases is considered effective when long-term local control is obtained. However, dose-effect data are scarce. We, therefore, performed a systematic literature search to assess the evidence concerning the relation of SRT dose and local control probability. METHODS AND MATERIALS A search was performed for papers describing patients treated with SRT for brain metastases, published from 1990 through 2009, in the electronic databases Medline (Pubmed) and Embase. We selected only papers reporting actuarial local control probability, in which a fixed dose had been prescribed and in which the size of the metastases was given. Series with SRT as a boost after whole brain irradiation (WBI) or with SRT after surgery were excluded. From the selected papers we extracted data on dose, local control rates and necrosis rates. Biological effective doses of the linear-quadratic-cubic model, using an α/β of 12Gy (BED(12)), were calculated and a dose-response curve was constructed. RESULTS Eleven papers fulfilled the selection criteria for further analysis. Six-month local control rates were higher than 80% in almost all the series irrespective of dose. Twelve-month local control rates, however, varied and were higher than 80%, higher than 60% and lower than 50% with single doses of ≥21Gy, ≥18Gy and ≤15Gy, respectively, and 70% or higher with fractionated SRT (FSRT). A BED(12) of at least 40Gy was associated with a twelve-month local control rate of 70% or more. CONCLUSION Local control after single fraction SRT is highly dependent upon dose and is high (>80%) after 21Gy or more, but low (<50%) after 15Gy or less. We conclude that SRT for brain metastases should preferably be applied with a BED(12) of at least 40Gy corresponding with a single fraction of 20Gy, two fractions of 11.6Gy or three fractions of 8.5Gy.

How low is the α/β ratio for prostate cancer?

Purpose: Recently, low / values of 1.2 and 1.5 Gy for prostate tumors have been derived from clinical results of external beam radiotherapy and of permanent implants of 125 I and 103 Pd. In the analyses the contributions of tumor repopulation, and edema as a result of inserting radioactive seeds in the prostate, have been ignored. In this paper we reanalyzed the clinical data and introduced the contribution of repopulation and edema. Methods and Materials: The linear quadratic– biologically effective dose model was used for reanalysis. In this model, the influence of repopulation and edema has been taken into account. The biologically effective dose was calculated as a function of / for 2 brachytherapy regimens with 125 I and 103 Pd and 2 fractionated treatments, and for different half-times for repair of sublethal damage for the brachytherapy regimens. Results: We have found a plausible / value of 3.1 to 3.9 Gy, an value of 0.1 to 0.15 Gy 1 , and a half-time of repair of about 0.5 h. Conclusions: It seems now that the / value is low, 3.1–3.9 Gy, but not as low as the 1.2 and 1.5 Gy reported earlier.

Apoptosis regulation in the testis: involvement of Bcl-2 family members.

Using immunohistochemical techniques and Western blot analysis, the possible role of Bcl‐2 family members Bax, Bcl‐2, Bcl‐xs, and Bcl‐xl in male germ cell density‐related apoptosis and DNA damage induced apoptosis was studied. The apoptosis inducer Bax was localized in all mouse and human testicular cell types, but despite the fact that irradiation induces its transcriptional activator, p53 in the human, Bax expression did not change after irradiation. The apoptosis inhibitor Bcl‐2 appeared to be present in late spermatocytes and spermatids and was up‐regulated in these cells after a dose of 4 Gy of X‐rays. Finally, Bcl‐x was expressed in both the mouse and human testis. The apoptosis inhibiting long transcripts of Bcl‐x, Bcl‐xl, were expressed in spermatogonia and spermatocytes and were up‐regulated after X‐irradiation. The apoptosis inducing shorter form of Bcl‐x, Bcl‐xs, was found to be expressed only in somatic cells, like peritubular and Leydig cells. While Bax is important in germ cell density regulation, Bax expression did not change after DNA damage inflicted by X‐radiation. Hence, spermatogonial apoptosis after X‐irradiation may not be induced via the apoptosis inducer Bax. Furthermore, as Bcl‐xl, but not Bcl‐2, is present in spermatogonia and spermatocytes, Bcl‐xl may regulate germ cell density, possibly in cooperation with Bax. As Bcl‐xl expression is enhanced after irradiation, this protein may also have a role in the response of spermatogonia and spermatocytes to irradiation. Mol. Reprod. Dev. 56:353–359, 2000.

Biologically effective doses of postoperative radiotherapy in the prevention of keloids: Dose-effect relationship

Purpose:To review the recurrence rates of keloids after surgical excision followed by radiotherapy, and to answer the question whether after normalization of the dose, a dose-effect relationship could be derived.Material and Methods:A literature search was performed to identify studies dealing with the efficacy of various irradiation regimens for the prevention of keloids after surgery. Biologically effective doses (BEDs) of the various irradiation regimens were calculated using the linear-quadratic concept. A distinction between recurrence rates of keloids in the face and neck region and those in other parts of the body was made.Results:31 reports were identified with PubMed with the search terms keloids, surgery, radiation therapy, radiotherapy. 13 reports were excluded, because no link could be found between recurrence rate and dose, or if less than ten patients per dose group. The recurrence rate for surgery only was 50–80%. For BED values > 10 Gy the recurrence rate decreased as a function of BED. For BED values > 30 Gy the recurrence rate was < 10%. For a given dose, the recurrence rates of keloids in the sites with high stretch tension were not significantly higher than in sites without stretch tension.Conclusion:The results of this study indicate that for effectively treating keloids postoperatively, a relatively high dose must be applied in a short overall treatment time. The optimal treatment probably is an irradiation scheme resulting in a BED value of at least 30 Gy. A BED value of 30 Gy can be obtained with, for instance, a single acute dose of 13 Gy, two fractions of 8 Gy or three fractions of 6 Gy, or a single dose of 27 Gy at low dose rate. The radiation treatment should be administered within 2 days after surgery.Ziel:Auswertung der Rückfallraten von Keloiden nach chirurgischer Entferung und Bestrahlung und die Frage, ob sich aus den normalisierten Werten der Strahlendosis eine Dosis-Wirkungs-Beziehung ableiten lässt.Material und Methode:In einer Literaturrecherche wurden Studien zur Wirksamkeit verschiedener Bestrahlungsregime zur postoperativen Prävention von Keloiden gesucht. Biologisch effektive Dosen (BEDs) der verschiedenen Bestrahlungsregime wurden nach der linear-quadratischen Methode berechnet. Unterschieden wurde zwischen Keloid-Rückfallraten im Bereich von Gesicht und Hals und denen anderer Körperregionen.Ergebnisse:Mit den Begriffen „keloids“, „surgery“, „radiation therapy“, „radiotherapy“ wurden in PubMed 31 Publikationen gefunden. 13 Arbeiten wurden ausgeschlossen, wenn kein Bezug zwischen Rückfallrate und Strahlendosis zu finden war oder wenn die Dosierungsgruppen weniger als 10 Patienten umfassten. Bei alleiniger chirurgischer Behandlung lag die Rückfallrate bei 50–80%. Bei BED-Werten über 10 Gy verminderte sich die Rückfallrate in Abhängigkeit von der BED. Bei BED-Werten über 30 Gy lag die Rückfallrate unter 10%. Für eine bestimmte Dosis waren die Keloid-Rückfallraten in Bereichen hoher Streckbelastung nicht signifikant höher als in Bereichen ohne Streckbelastung.Schlussfolgerung:Die Ergebnisse dieser Untersuchung zeigen, dass für eine wirksame postoperative Behandlung von Keloiden eine relativ hohe Strahlendosis innerhalb einer kurzen Gesamtbehandlungszeit appliziert werden muss. Die optimale Behandlung ist wahrscheinlich ein Bestrahlungsschema, das zu einer BED von mindestens 30 Gy führt. Ein BED-Wert von 30 Gy kann beispielsweise mit einer Einzelakutdosis von 13 Gy, 2 Fraktionen von 8 Gy oder 3 Fraktionen von 6 Gy, oder mit einer Einzeldosis von 27 Gy mit geringer Dosisrate erzielt werden. Die Strahlentherapie sollte innerhalb von 2 Tagen nach der chirurgischen Behandlung durchgeführt werden.

Ataxia Telangiectasia Mutated Expression and Activation in the Testis

Abstract Ionizing radiation (IR) and consequent induction of DNA double-strand breaks (DSBs) causes activation of the protein ataxia telangiectasia mutated (ATM). Normally, ATM is present as inactive dimers; however, in response to DSBs, the ATM dimer partners cross-phosphorylate each other on serine 1981, and kinase active ATM monomers are subsequently released. We have studied the presence of both nonphosphorylated as well as active serine 1981 phosphorylated ATM (pS1981-ATM) in the mouse testis. In the nonirradiated testis, ATM was present in spermatogonia and spermatocytes until stage VII of the cycle of the seminiferous epithelium, whereas pS1981-ATM was found only to be present in the sex body of pachytene spermatocytes. In response to IR, ATM became activated by pS1981 cross-phosphorylation in spermatogonia and Sertoli cells. Despite the occurrence of endogenous programmed DSBs during the first meiotic prophase and the presence of ATM in both spermatogonia and spermatocytes, pS1981 phosphorylated ATM did not appear in spermatocytes after treatment with IR. These results show that spermatogonial ATM and ATM in the spermatocytes are differentially regulated. In the mitotically dividing spermatogonia, ATM is activated by cross-phosphorylation, whereas during meiosis nonphosphorylated ATM or differently phosphorylated ATM is already active. ATM has been shown to be present at the synapsed axes of the meiotic chromosomes, and in the ATM knock-out mice spermatogenesis stops at pachytene stage IV of the seminiferous epithelium, indicating that indeed nonphosphorylated ATM is functional during meiosis. Additionally, ATM is constitutively phosphorylated in the sex body where its continued presence remains an enigma.